**The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods and Tools**

Slavko Arsovski, Miladin Stefanović, Danijela Tadić and Ivan Savović

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53154

## **1. Introduction**

A number of trends and challenges such as increased competition, new technologies and regulations, quality and new consumer trends have been forcing the food industry to change [1, 2, 3]. In response to these new challenges, food companies are improving compet‐ itiveness by restructuring, redesigning existing processes, and intensifying the fight for mar‐ ket share through product differentiation and/or the development of new food products [1]. In order to improve, companies in the food industry must adopt: Restructure of their organi‐ zations and redesign their processes; Automation of production and other processes to de‐ crease dependence on human resources and transfer activities to self-service facilities for customers and partners; Optimization of logistical infrastructure and systems; Energy sav‐ ing measures through new technology and materials, new production methods and goodpractice implementation; Political and regulatory developments (food safety and other regulations); Technological changes (biotechnology, ICT and RFID, robotics, sensors, e-busi‐ ness); Understand globalization, market developments and customer trends.

In this chapter process development is analyzed because of its impact on food quality, safety and sustainability [4, 5, 6]. A redesign of process development could be accomplished through many different approaches, techniques and tools [7, 8, 9, 10]. In this chapter Busi‐ ness Process Management (BPM) is used with accompanied quality engineering methods and tools. A number of important questions will be addressed concerning the redesign of process development in food production organizations using quality engineering tools and methods.

© 2013 Arsovski et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The first question that will be raised is the quality of processes in the food industry. The analysis will start from a typical process map for companies in the food processing industry in Serbia. After that the production process, as one of the most important processes in food processing will be decomposed. For each sub process appropriate metrics will be defined as the road of evaluation for the quality of the sub processes and quality of the goal of the proc‐ ess itself. Redesign of process development will be analyzed by comparing different ap‐ proaches and by consideration of process redesign as the process. Different quality engineering methods and tools in the food industry will be compared according to the fre‐ quency of their implementation in the Serbian food industry as well as a correlation between the application of different quality engineering methods and tools, and profit in the compa‐ nies. As an extension of the general ranking idea presented on the ranking and definition of goals in the production process, a fuzzy approach for evaluation of the importance of enti‐ ties in supply chains in the food industry is presented. The general idea is to present an ap‐ plication of a mathematical tool in a situation that is very common in the food industry where conditions have been constantly changing so the observed values could not be sto‐ chastically described and where there is not a sufficient amount of data for statistical analy‐ sis. In other words, the application of fuzzy sets on evaluation of the importance of entities in the supply chain will be presented. A strategic map as a strategic part of the BSC (Bal‐ anced Score Card) framework is presented as one of the quality engineering methods. The presented strategic map started from the Kaplan – Norton model but it was adjusted in or‐ der to meet the needs of food processing companies in Serbia. Relations between entities (from all four perspectives) are defined as the result of research among Serbian companies. In the final part of the chapter the process framework for food processing companies is pre‐ sented. The questionnaire used for the research is presented as well as gathered data from 53 Serbian companies. The gathered data was the input in modeling and evaluations pre‐ sented in previously discussed issues.

analysis, and redesign and improvement of the process the first step is an analysis of the process map of a typical organization in the food industry. A typical process map for an or‐

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

KP0 Strategic management

KP3

KP4 Sales and marketing

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441

SP5 Human Resources Management

Delivery

SP3

Maintenance

Besides strategic management, each component process is functioning at a tactical and oper‐ ative level and has quality metrics. In this chapter the major focus will be placed on the pro‐ duction process (KP2), all other decompositions could be performed using the same pattern, with appropriate quality metrics. In further analysis the production process could be de‐

The production process (according to figure 2) consists of four sub processes: Logistic proc‐ ess, Process realization, Process planning and scheduling and Production process control.

In further analysis the logistic sub process (KP 2.1) could be decomposed into the following sub processes: Definitive logistic strategy; Plan inbound material flow; Operate outbound

SP4

Finance

KP2

Production

SP2 Quality management

**Figure 1.** Typical process map for organizations in the food industry

composed into the sub processes presented in figure 2.

warehousing; Operate transportation and Manage reverse logistics.

ganization in the food industry is presented in figure 1.

KP1

Purchasing

Key processes

Supporting processes

SP1 Development and design

The main idea of the chapter is to provide an overview of the redesign of process develop‐ ment, starting from analysis (decomposition of processes), redesign, implementation of modern quality engineering tools and methods (frequency of usage and impact of different tools and methods and implantation of some of them) as well as theoretical and mathemati‐ cal tools on ranking of quality goals (theory of fuzzy sets) and finally providing a process framework and, at the same time, the keeping a connection and solid ground in data gath‐ ered from the food industry.

## **2. Quality of processes in the food industry**

The food industry contains a number of completely different processes which require a wide range of measuring instruments. On the one hand, the quality of processes has market goals in brand development, demand management and new product introduction, while embrac‐ ing food security and quality requirements. On the other hand, quality of processes in the food industry depends on many factors such as customer demands, key performance indica‐ tors, the process map, technology level, management level etc. In order to provide quality analysis, and redesign and improvement of the process the first step is an analysis of the process map of a typical organization in the food industry. A typical process map for an or‐ ganization in the food industry is presented in figure 1.

**Figure 1.** Typical process map for organizations in the food industry

The first question that will be raised is the quality of processes in the food industry. The analysis will start from a typical process map for companies in the food processing industry in Serbia. After that the production process, as one of the most important processes in food processing will be decomposed. For each sub process appropriate metrics will be defined as the road of evaluation for the quality of the sub processes and quality of the goal of the proc‐ ess itself. Redesign of process development will be analyzed by comparing different ap‐ proaches and by consideration of process redesign as the process. Different quality engineering methods and tools in the food industry will be compared according to the fre‐ quency of their implementation in the Serbian food industry as well as a correlation between the application of different quality engineering methods and tools, and profit in the compa‐ nies. As an extension of the general ranking idea presented on the ranking and definition of goals in the production process, a fuzzy approach for evaluation of the importance of enti‐ ties in supply chains in the food industry is presented. The general idea is to present an ap‐ plication of a mathematical tool in a situation that is very common in the food industry where conditions have been constantly changing so the observed values could not be sto‐ chastically described and where there is not a sufficient amount of data for statistical analy‐ sis. In other words, the application of fuzzy sets on evaluation of the importance of entities in the supply chain will be presented. A strategic map as a strategic part of the BSC (Bal‐ anced Score Card) framework is presented as one of the quality engineering methods. The presented strategic map started from the Kaplan – Norton model but it was adjusted in or‐ der to meet the needs of food processing companies in Serbia. Relations between entities (from all four perspectives) are defined as the result of research among Serbian companies. In the final part of the chapter the process framework for food processing companies is pre‐ sented. The questionnaire used for the research is presented as well as gathered data from 53 Serbian companies. The gathered data was the input in modeling and evaluations pre‐

The main idea of the chapter is to provide an overview of the redesign of process develop‐ ment, starting from analysis (decomposition of processes), redesign, implementation of modern quality engineering tools and methods (frequency of usage and impact of different tools and methods and implantation of some of them) as well as theoretical and mathemati‐ cal tools on ranking of quality goals (theory of fuzzy sets) and finally providing a process framework and, at the same time, the keeping a connection and solid ground in data gath‐

The food industry contains a number of completely different processes which require a wide range of measuring instruments. On the one hand, the quality of processes has market goals in brand development, demand management and new product introduction, while embrac‐ ing food security and quality requirements. On the other hand, quality of processes in the food industry depends on many factors such as customer demands, key performance indica‐ tors, the process map, technology level, management level etc. In order to provide quality

sented in previously discussed issues.

**2. Quality of processes in the food industry**

ered from the food industry.

440 Food Industry

Besides strategic management, each component process is functioning at a tactical and oper‐ ative level and has quality metrics. In this chapter the major focus will be placed on the pro‐ duction process (KP2), all other decompositions could be performed using the same pattern, with appropriate quality metrics. In further analysis the production process could be de‐ composed into the sub processes presented in figure 2.

The production process (according to figure 2) consists of four sub processes: Logistic proc‐ ess, Process realization, Process planning and scheduling and Production process control.

In further analysis the logistic sub process (KP 2.1) could be decomposed into the following sub processes: Definitive logistic strategy; Plan inbound material flow; Operate outbound warehousing; Operate transportation and Manage reverse logistics.

**Logistic strategy realization %**

**Level of preparing workers**

**Realization of inbound plan of material flow**

**Table 1.** Quality metrics of "Logistic process" KP 2.1

**Flexibility of working plans** **Costs of**

**\*100**

0.25 0.25 0.25 0.25 weight

**Quantitative Waste %**

**Level of plan fulfillment \*100%**

10 10 >100 <0.5 <0.5 10

 9 90-100 0.5-1.0 0.5-1.0 9 8 80-90 1.0-2.0 1.0-2.0 8 7 70-80 2.0-3.0 2.0-3.0 7 6 60-70 3.0-4.0 3.0-4.0 6 5 50-60 4.0-5.0 4.0-5.0 5 4 40-50 5.0-6.0 5.0-6.0 4 3 30-40 6.0-7.0 6.0-7.0 3 2 20-30 7.0-8.0 7.0-8.0 2 1 <20 >8.0 >8.0 1

0.2 0.1 0.2 0.25 0.25 weight

**Table 2.** Quality metrics of "Production process realization" KP 2.2

95-100 >100 <50 >100 10 85-95 90-100 50-60 90-100 9 75-85 80-90 60-70 80-90 8 65-75 70-80 70-80 70-80 7 55-65 60-70 80-90 60-70 6 45-55 50-60 90-100 50-60 5 35-45 40-50 100-110 40-50 4 25-35 30-40 110-120 30-40 3 <25 <30 >120 <30 2

**warehousing /plan**

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

**Realization of plan of outbound transportation**

**Value of waste % Score**

**Score**

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443

**Figure 2.** Decomposition of production process

The next important step is the definition of quality metrics. According to the definition a metric is a verifiable measure stated in either quantitative or qualitative terms. Quality met‐ ric data may be used to: spot trends in performance, compare alternatives and predict per‐ formance. Organizations need to collect information for a particular quality metric in order to evaluate and improve their processes. For further analysis of the logistic process the fol‐ lowing quality metrics are presented in Table 1.

The second sub process of KP2, Production process realization (KP 2.2) is decomposed into sub processes: Preparing workers for obligatory measures; Preparing working places; Reali‐ zation of working activities and Work reporting. The accompanied quality metrics are pre‐ sented in table 2.

Production process planning and scheduling (KP 2.3) is decomposed into the following sub processes: Manage demand for products; Create material requirement plan (MRP) and Schedule production. The accompanied quality metrics are presented in table 3.

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods... http://dx.doi.org/10.5772/53154 443


**Table 1.** Quality metrics of "Logistic process" KP 2.1

KP2.2

0

The next important step is the definition of quality metrics. According to the definition a metric is a verifiable measure stated in either quantitative or qualitative terms. Quality met‐ ric data may be used to: spot trends in performance, compare alternatives and predict per‐ formance. Organizations need to collect information for a particular quality metric in order to evaluate and improve their processes. For further analysis of the logistic process the fol‐

The second sub process of KP2, Production process realization (KP 2.2) is decomposed into sub processes: Preparing workers for obligatory measures; Preparing working places; Reali‐ zation of working activities and Work reporting. The accompanied quality metrics are pre‐

Production process planning and scheduling (KP 2.3) is decomposed into the following sub processes: Manage demand for products; Create material requirement plan (MRP) and

Schedule production. The accompanied quality metrics are presented in table 3.

KP2.1

Logistic process

KP2.3

Process planing and scheduling

**Figure 2.** Decomposition of production process

sented in table 2.

442 Food Industry

lowing quality metrics are presented in Table 1.

Production

KP2

Process realization

KP2.4

Production process control


**Table 2.** Quality metrics of "Production process realization" KP 2.2


Production process control (KP 2.4) could be decomposed into the following sub process‐ es: Control of inputs; Process control, Control of outputs, Non conformance product con‐ trol and Control of measurement devices. The accompanied quality metrics are presented

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

Quality systems focus on the quality of what the organization in the food industry produces, the factors which will cause the organization to achieve its goals, the factors which might prevent it satisfying customers and the factors which might prevent it from being produc‐ tive, innovative and profitable. To control, assure and improve quality there is a need to fo‐ cus on certain goals, in the case of goals of the production process (KP2) of a typical company in the food industry we can define goals as the constituent of the previous sub

> Quality goal of production process

0.3 0.25

process realisation Quality gool of production

By decomposition of the production process, analysis of sub processes and definition of metrics for each sub process it is possible to control, assure and improve the quality of a process in companies from the food industry sector. Definition of scores and weights in the metrics of each sub process is performed according to the authors' experience and available literature. In order to clearly demonstrate the idea all values are presented as deterministic ones. Of course some of them could be expressed by linguistic expressions rather than precious numbers but that issue will be elaborated in further text. The key processes in the food industry were analyzed and compared, and the results of research

planing and scheduling

0.25

Quality gool of production process control

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445

in table 4.

process goals (figure 3).

Quality gool of logistic production process

0.2

**Figure 3.** Quality goals of Production process KP2

Quality gool of production

**Table 3.** Quality metrics of "Production process planning and scheduling" KP 2.3


**Table 4.** Quality metrics of "Production process control" KP 2.4

Production process control (KP 2.4) could be decomposed into the following sub process‐ es: Control of inputs; Process control, Control of outputs, Non conformance product con‐ trol and Control of measurement devices. The accompanied quality metrics are presented in table 4.

Quality systems focus on the quality of what the organization in the food industry produces, the factors which will cause the organization to achieve its goals, the factors which might prevent it satisfying customers and the factors which might prevent it from being produc‐ tive, innovative and profitable. To control, assure and improve quality there is a need to fo‐ cus on certain goals, in the case of goals of the production process (KP2) of a typical company in the food industry we can define goals as the constituent of the previous sub process goals (figure 3).

**Figure 3.** Quality goals of Production process KP2

**Accuracy of demand**

**Effectiveness of control of inputs %** **Accuracy of MRP**

**Work in progress/ production**

**On time delivery**

**Score**

**(OTD) %**

**Level of control**

**of measurement devices %**

**Score**

**%**

85-95 85-95 5-10 90-95 9 75-85 75-85 10-15 85-90 8 65-75 65-75 15-20 80-85 7 55-65 55-65 20-25 75-80 6 45-55 45-55 25-30 70-75 5 35-45 35-45 30-35 65-70 4 25-35 25-35 35-40 60-65 3 15-25 15-25 40-45 55-60 2 <15 <15 >45 <55 1

0.15 0.25 0.3 0.3 weight

**Effectiveness of control of outputs %**

>95 >95 >95 10 10

85-95 85-95 90-95 9 9 75-85 75-85 85-90 8 8 65-75 65-75 80-85 7 7 55-65 55-65 75-80 6 6 45-55 45-55 70-75 5 5 35-45 35-45 65-70 4 4 25-35 25-35 60-65 3 3 15-25 15-25 55-60 2 2 <15 <15 <55 1 1

0.25 0.25 0.3 0.2 weight

**Table 3.** Quality metrics of "Production process planning and scheduling" KP 2.3

**Effectiveness of process control %**

**Table 4.** Quality metrics of "Production process control" KP 2.4

**%**

**%**

444 Food Industry

By decomposition of the production process, analysis of sub processes and definition of metrics for each sub process it is possible to control, assure and improve the quality of a process in companies from the food industry sector. Definition of scores and weights in the metrics of each sub process is performed according to the authors' experience and available literature. In order to clearly demonstrate the idea all values are presented as deterministic ones. Of course some of them could be expressed by linguistic expressions rather than precious numbers but that issue will be elaborated in further text. The key processes in the food industry were analyzed and compared, and the results of research are presented in figure 4., depicting the existence of the gap between the quality of devel‐ opment and design process, and other processes. The analysis was performed on selected companies from Serbia.

**No. Change**

2 Process

3 Continuous process improvement

**methodology**

improvement

1 BPR & lean -reduction more

**Amount of change**

than 50% of time, costs and quality



**Table 5.** Comparison of change methodologies (adapted from [11])

chosen solution with approval of senior management.

evaluate measurements for continuous improvement.

**•** Meeting with affected process managers and employees.

There are five phases in this model:

solution with staff and customers.

The first step covers:

**Score of change**

or


The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

functional teams



**•** Analysis Phase — Identify areas of opportunity and target specific problems.

**•** Design Phase — Generate solutions and identify the required resources to implement the

**•** Development Phase — Formulate a detailed procedure for implementing the approved

**•** Implementation Phase — Execution of the solution and implementation of the redesign.

**•** Evaluation Phase — Build metrics, measurement tools, monitor implementation, and

Process redesign as a process could be presented in the 10 steps, according to figure 5. The first five steps are common for most companies and the other steps could be defined accord‐ ing to the specific problem and in some cases using different quality methods and tools.

**•** Meeting with senior management for the purpose of discussing barriers to process,

**•** Improvement, problem of eventual job losses and crafting of the kick-off-speech,

**Used tools**




**of success**

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**Applicable in**

yes

yes

yes

**food industry**

447

**Figure 4.** Quality of development and design process compared to quality of key processes

The gap between quality of the development and design process and other processes indi‐ cates that the quality of the development and design process is lower than the quality of the other processes so the focus in process redesign and improvement should be on the redesign of process development and design.

## **3. Redesign of process development**

#### **3.1. Redesign of process development: Different approaches**

The redesign of process development is connected to different methodologies of process change. In the redesign of process development and in the process of business process rede‐ sign itself there are a number of methodologies which cover the different amount of changes, results, used tools and probability of success. According to [11], and presented in table 5 a comparison of possible methodologies indicating whether they are applicable in the food industry.

According to table 5 it is clear that Continuous Process Improvement (CPI) as a never end‐ ing effort to discover, and eliminate the main causes of problems, is most likely to succeed in companies from the food industry.

Process improvement and BPR & lean are less likely to succeed but they cover a larger num‐ ber of changes in the companies. Beside a redesign of process development can be viewed as a process. According to [11], the redesign of process development is divided into 5 phases (4 different entities) (Fig.5) with 10 steps.

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods... http://dx.doi.org/10.5772/53154 447


**Table 5.** Comparison of change methodologies (adapted from [11])

There are five phases in this model:

are presented in figure 4., depicting the existence of the gap between the quality of devel‐ opment and design process, and other processes. The analysis was performed on selected

The gap between quality of the development and design process and other processes indi‐ cates that the quality of the development and design process is lower than the quality of the other processes so the focus in process redesign and improvement should be on the redesign

The redesign of process development is connected to different methodologies of process change. In the redesign of process development and in the process of business process rede‐ sign itself there are a number of methodologies which cover the different amount of changes, results, used tools and probability of success. According to [11], and presented in table 5 a comparison of possible methodologies indicating whether they are applicable in the

According to table 5 it is clear that Continuous Process Improvement (CPI) as a never end‐ ing effort to discover, and eliminate the main causes of problems, is most likely to succeed in

Process improvement and BPR & lean are less likely to succeed but they cover a larger num‐ ber of changes in the companies. Beside a redesign of process development can be viewed as a process. According to [11], the redesign of process development is divided into 5 phases (4

**Figure 4.** Quality of development and design process compared to quality of key processes

companies from Serbia.

446 Food Industry

of process development and design.

companies from the food industry.

different entities) (Fig.5) with 10 steps.

food industry.

**3. Redesign of process development**

**3.1. Redesign of process development: Different approaches**


Process redesign as a process could be presented in the 10 steps, according to figure 5. The first five steps are common for most companies and the other steps could be defined accord‐ ing to the specific problem and in some cases using different quality methods and tools.

The first step covers:


The second step is very important for success of the complete process. It is realized accord‐ ing to the team engineering approach [12, 13] with specified roles of team members: project manager, project principal, process improvement team, facilitator, and expert in ICT.

Senior management has the important task of directing team work in the first meetings and by the definition of statements and roles which will define the procedures for the team

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449

The third step is performed by team work, starting from analysis of the existing state of

The fourth step covers the interviews with customers, according to: Customer request and needs; Ranking the criteria; Needed performance according to each criteria and Competitor

The use of benchmarks and the development of new forms of benchmarking for best prac‐ tice is well established in the food industry, although less so in other areas of the food chain. Benchmarking and best practice analysis is performed in step five through analysis of food industry competitions, using information from: industry trade associations (trade chambers etc.), industry studies, consultants' reviews, distributors, former employees, competitors themselves, published documents, indirect information sources from competitors, govern‐

Other steps are performed according to appropriate redesign methodology adapted for each specific organization from the food industry. In addition, different quality engineering tools and methods could be employed in the following steps. It is important to emphasize that ICT could be used as a support during process redesign on the one hand, and on the other hand ICT and Business Process Management cover various aspects such as process control

Quality engineering methods and tools have an important role in the food industry because customers and markets demand proven high quality products and protection against low quality and unsafe products. On the other hand, the food industry needs to attain the quali‐ ty that meets international standards for quality products. As it was mentioned the food in‐ dustry needs to cope with the challenge of modern technological production methods, know them and assimilate them in quality assurance areas using new innovative hi-tech sensory and measurement instruments, supervise the production process, so that the designated quality level is always met. In order to perform all of these tasks and in order to meet all of the existing challenges the food industry has a number of engineering methods and tools

We analyzed companies from Serbia (listed in the following text) and compared results with world practice. The following quality engineering methods and tools used by the food in‐ dustry have been analyzed according to their frequency and success rate: (1) Ichicawa's sev‐ en basic tools for quality (flow chart, check sheet, histograms, scatter plots, control chart, cause-effect diagram, Pareto analysis); (2) The seven new tools for improvement (affinity di‐ agram, interrelationship diagram, tree diagram, prioritization grid, matrix diagram, process decision program chart, activity network diagram) ; (3) Cost of Quality (CoQ) ; (4) Project management; (5) Simultaneous engineering; (6) Statistical process control; (7) Reliability and

processes, best practice and creating the as-is state of processes.

ment sources, customers, supplies, and reverse – engineering products.

**3.2. Quality engineering methods and tools in the food industry**

position according to ranking and criteria.

and supply chain management.

available.

work.

**Figure 5.** Process redesign as process

Senior management has the important task of directing team work in the first meetings and by the definition of statements and roles which will define the procedures for the team work.

The second step is very important for success of the complete process. It is realized accord‐ ing to the team engineering approach [12, 13] with specified roles of team members: project

> 2 Creating the team

3 Creating the asis process map

0

10 Installing the metrics and continuous improvement

6 Creating the ideal (desired) process

5 Benchmarking and best practices analysis

4 Customer interview

manager, project principal, process improvement team, facilitator, and expert in ICT.

1 Introduction to process redesign

7.2 Review by senior management

7.1 Presenting the redesigned process to senior management

8 Charing the redesign with staff and customer

Customer

Senior management

448 Food Industry

team

process

9 Implementing the redesign

**Figure 5.** Process redesign as process

The third step is performed by team work, starting from analysis of the existing state of processes, best practice and creating the as-is state of processes.

The fourth step covers the interviews with customers, according to: Customer request and needs; Ranking the criteria; Needed performance according to each criteria and Competitor position according to ranking and criteria.

The use of benchmarks and the development of new forms of benchmarking for best prac‐ tice is well established in the food industry, although less so in other areas of the food chain. Benchmarking and best practice analysis is performed in step five through analysis of food industry competitions, using information from: industry trade associations (trade chambers etc.), industry studies, consultants' reviews, distributors, former employees, competitors themselves, published documents, indirect information sources from competitors, govern‐ ment sources, customers, supplies, and reverse – engineering products.

Other steps are performed according to appropriate redesign methodology adapted for each specific organization from the food industry. In addition, different quality engineering tools and methods could be employed in the following steps. It is important to emphasize that ICT could be used as a support during process redesign on the one hand, and on the other hand ICT and Business Process Management cover various aspects such as process control and supply chain management.

### **3.2. Quality engineering methods and tools in the food industry**

Quality engineering methods and tools have an important role in the food industry because customers and markets demand proven high quality products and protection against low quality and unsafe products. On the other hand, the food industry needs to attain the quali‐ ty that meets international standards for quality products. As it was mentioned the food in‐ dustry needs to cope with the challenge of modern technological production methods, know them and assimilate them in quality assurance areas using new innovative hi-tech sensory and measurement instruments, supervise the production process, so that the designated quality level is always met. In order to perform all of these tasks and in order to meet all of the existing challenges the food industry has a number of engineering methods and tools available.

We analyzed companies from Serbia (listed in the following text) and compared results with world practice. The following quality engineering methods and tools used by the food in‐ dustry have been analyzed according to their frequency and success rate: (1) Ichicawa's sev‐ en basic tools for quality (flow chart, check sheet, histograms, scatter plots, control chart, cause-effect diagram, Pareto analysis); (2) The seven new tools for improvement (affinity di‐ agram, interrelationship diagram, tree diagram, prioritization grid, matrix diagram, process decision program chart, activity network diagram) ; (3) Cost of Quality (CoQ) ; (4) Project management; (5) Simultaneous engineering; (6) Statistical process control; (7) Reliability and risk engineering; (8) World class manufacturing (WCM) ; (9) Six-sigma; (10) Lean six-sigma; (11) Taguchi method; (12) Zero defect (ZD) ; (13) Design of experiments; (14) Quality Func‐ tion Deployment (QFD) ; (15) FMEA; (16) FMECA; (17) Just in Time (JiT) ; (18) Business Process Reengineering; (19) Balance Score Cards (BSC) ; and (20) other.

quality are the most commonly used but their contribution to profit increase is the lowest compared with other frequently used quality methods and tools. Another result is that the seven new tools do not contribute more significantly to the increase of profit due to the low‐

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

level of application of quality tools and methods

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3 4 6 14 15 16 18

er level of knowledge of employees connected with the new approaches.

without tools 7 new

increases in the analyzed organizations.

**Figure 7.** Impact of level of application of quality tools and methods on profit/employee

tools

Finally, although rather "old" QFD proved itself as a very useful and efficient tool. General‐ ly, with an increase of the level of application of quality tools and methods profit/employees

**3.3. Fuzzy approach for evaluation of the importance of entities in supply chains in the**

The process of logistics and the food supply chain is very important for all companies from the food industry. In food supply chains, definition of weight / importance of different enti‐ ties (processes, sub processes and goals) in the presence of uncertainties is one of the impor‐ tant goals for the management team. A similar problem has been met and emphasized in the definition of quality metrics and evaluation of scores and weights in section 2. The solution

Level of profit/ employs

200.000

100.000



**food industry**

0

The results of the usage and frequency of the specific quality engineering methods and tools listed above, in the case of the Serbian food industry, is presented in Figure 6.

**Figure 6.** Distribution of quality methods' & tools' application

According to the results of the research, presented in figure 6, the dominant method or tool in food companies in Serbia position have Ichicawa's seven basic tools for quality (flow chart, check sheet, histograms, scatter plots, control chart, cause-effect diagram, Pareto anal‐ ysis). It is also obvious that Serbian companies in general do not employ (at sufficient level) more advanced, new methods needed for higher quality process improvement.

Project management and FMEA are also popular in Serbian food processing companies. The second question is an analysis of profit compared with the quality tools and methods. Ac‐ cording to research there is a high positive correlation between an increase of profit and ap‐ plication of quality tools and methods and it is presented in figure 7.

According to figure 7 the level of profit (per employee) increases with implementation of different quality methods and tools. The largest increase in profit can be seen in the applica‐ tion of: Cost of Quality (CoQ), Statistical process control, Business Process Reengineering. The increase in the profit with the application of seven new tools is larger than with the ap‐ plication of the basic seven tools for quality. It is interesting that the basic seven tools for quality are the most commonly used but their contribution to profit increase is the lowest compared with other frequently used quality methods and tools. Another result is that the seven new tools do not contribute more significantly to the increase of profit due to the low‐ er level of knowledge of employees connected with the new approaches.

risk engineering; (8) World class manufacturing (WCM) ; (9) Six-sigma; (10) Lean six-sigma; (11) Taguchi method; (12) Zero defect (ZD) ; (13) Design of experiments; (14) Quality Func‐ tion Deployment (QFD) ; (15) FMEA; (16) FMECA; (17) Just in Time (JiT) ; (18) Business

The results of the usage and frequency of the specific quality engineering methods and tools

According to the results of the research, presented in figure 6, the dominant method or tool in food companies in Serbia position have Ichicawa's seven basic tools for quality (flow chart, check sheet, histograms, scatter plots, control chart, cause-effect diagram, Pareto anal‐ ysis). It is also obvious that Serbian companies in general do not employ (at sufficient level)

Project management and FMEA are also popular in Serbian food processing companies. The second question is an analysis of profit compared with the quality tools and methods. Ac‐ cording to research there is a high positive correlation between an increase of profit and ap‐

According to figure 7 the level of profit (per employee) increases with implementation of different quality methods and tools. The largest increase in profit can be seen in the applica‐ tion of: Cost of Quality (CoQ), Statistical process control, Business Process Reengineering. The increase in the profit with the application of seven new tools is larger than with the ap‐ plication of the basic seven tools for quality. It is interesting that the basic seven tools for

more advanced, new methods needed for higher quality process improvement.

plication of quality tools and methods and it is presented in figure 7.

Process Reengineering; (19) Balance Score Cards (BSC) ; and (20) other.

450 Food Industry

**Figure 6.** Distribution of quality methods' & tools' application

listed above, in the case of the Serbian food industry, is presented in Figure 6.

**Figure 7.** Impact of level of application of quality tools and methods on profit/employee

Finally, although rather "old" QFD proved itself as a very useful and efficient tool. General‐ ly, with an increase of the level of application of quality tools and methods profit/employees increases in the analyzed organizations.

## **3.3. Fuzzy approach for evaluation of the importance of entities in supply chains in the food industry**

The process of logistics and the food supply chain is very important for all companies from the food industry. In food supply chains, definition of weight / importance of different enti‐ ties (processes, sub processes and goals) in the presence of uncertainties is one of the impor‐ tant goals for the management team. A similar problem has been met and emphasized in the definition of quality metrics and evaluation of scores and weights in section 2. The solution to this problem must be placed on the whole of the supply chain because of its critical effect on efficiency.

The importance of the goals which are defined on the level of each sub process are defined by direct estimation made by a management team. According to literature, it can be con‐ cluded that this approach of estimation of importance of an entity is justified when the num‐

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

bed using five linguistic expressions which are modeled by triangular fuzzy numbers

*ijk* , *i* =1, .., *I*; *j* =1, .., *Ji*

dure of linear normalization is used [16]. Normalized values of goals weights are marked as

For the management team carrying out the analysis, the following tasks are important: (1) to determine the rank of the process in a company (2) to determine the rank of a sub process on the process level in a company, (3) to determine the rank of sub processes with respect to the importance of goals and the importance of the considered sub process (4) to determine the rank of processes with respect to the importance of the goals, the relative importance of subprocesses of process i, i=1,..Im and the relative importance of process i, i=1,.,I, and (5) calcu‐ late the degree of belief that the sub process, or process which is on second place in the rank could be on first place; answers to these questions are given by comparing triangular fuzzy

, respectively.

The algorithm for analysis of the relative importance of processes, sub-processes and goals

=1, .., *I*; *i* ≠*i* '

each process i, i=1,..,I by placing on the first place the sub process with the highest value *wij*

Step 4. Transform all linguistic expressions which are modeled by triangular fuzzy numbers

*ijk* =(*z*; *L ijk* , *Mijk* , *Uijk* )by applying linear normalization procedure [16].

Step 5. Calculate the weighted normalized aggregated relative importance of sub-process j:

; *p* =1, ..., *P*

, ..., *wI* ); rank the processes by placing on first

, ..., *wiJ <sup>i</sup>*); rank sub processes on the level of

.

*ijk* , *mijk* , *uijk* ). The total number of goals on the level of each sub process is denoted

is considered.

. The value of domains is defined on a standard measurement scale [18]. These triangu‐ lar fuzzy numbers are defined in the following way: very low value-(*y*; 1, 1, 3), low value- (*y*; 1, 3, 5), medium value-(*y*; 3, 5, 7), high value-(*y*; 5, 7, 9) and very high value- (*y*; 8, 9, 9). Since the goals could be beneficial or costly it is necessary to perform

on the level of sub process j, j=1,.., *Ji*

; *k* =1, .., *Kij*

. In further analysis only one goal with a critical effect

could be descri‐

453

http://dx.doi.org/10.5772/53154

. In this case the proce‐

ber of entities is less than five.

normalization of fuzzy values *v*

on the management of sub-process j, j=1,.., *Ji*

*<sup>i</sup>* , *i* =1, .., *I*; *j* =1, .., *Ji*

~ = [*w* ~ *ii* ' ], *i*, *i* '

.

in a company p ∈ P is formally given as follows.

Step 2. Calculate weight vector*W* =(*w*1, .., *wi*

Step 3. Calculate weight vector*Wi* =(*wi*1, .., *wij*

*ijk* , *i* =1, .., *I*; *j* =1, .., *J <sup>i</sup>*

~

, *k* =1, .., *Kij*

*v* ~

*r* ~

numbers *c*

*v* ~

*ijk* into *r* ~ ~ *ij*, *d* ~

Step 1. Input fuzzy matrix *W*

place the process with highest *wi*

as *Kij*

*ijk* =(*y*; *l*

The importance of each goal k, k=1,,.,*Kij*

It is realistic to assume, that decision makers express their evaluations more easy and more precisely by using linguistic expressions than numbers. The number and the type of linguis‐ tic expression used for a description of importance are defined by the management team and depend on the size of supply chain in food companies. Different mathematical ap‐ proaches such as probability theory, fuzzy sets, rough set theory and others enable quantifi‐ cation of linguistic expressions. The development of fuzzy set theory enables the elimination of uncertainties and imprecision caused by lack of good evidence. In the fuzzy approach the uncertainties and imprecision caused are described by linguistic variables. They can be modeled by fuzzy sets with a different shape (triangle, trapezoid, but in some cases with Gaussian distribution, discrete fuzzy numbers) of membership functions.

The fuzzy approach has been used in cases: (1) where conditions have been constantly changing so the observed value could not be stochastically described (2) where there is no sufficient amount of data for statistical analysis. In other words, fuzzy sets theory could sim‐ ulate human way of thinking in the process of decision making under imprecise, approxi‐ mative and unclear data.

According to [14] the advantages of fuzzy sets theory can be presented in the following: it is conceptually clear, flexible, covers different non-linear functions of different complexity; tol‐ erant on imprecise data; includes expert opinions and viewpoints; based on natural lan‐ guage; enables better communication between experts and managers.

Estimation of the relative importance of the processes and sub processes on the level of the sup‐ ply chain p ∈ P is defined by a pair-wise comparison matrix whose elements are defined as the relative importance of process/sub-process i/j compared to process/sub-process *i* ' / *j* ' ' , *i*, *i*, '=1, .., *I*; *j*, *j* ' =1, .., *Ji* where I and, *Ji* are the total number of analyzed processes and sub processes i, i (1,..,I, respectively). A number of authors consider this approach better compared to direct estimation. Elements of the pair-wise comparison matrix are linguistic ex‐ pressions which are modeled by triangular fuzzy numbers [14, 15]. The domains of these fuz‐ zy numbers are defined on interval [1-5]. Value 1, or value 5 define that analyzed entity i/j compared to entity *i* ' / *j* ' ' , *i*, *i*, '=1, .., *I*; *j*, *j* ' =1, .., *Ji* has the same or extremely higher impor‐ tance retrospectively. These triangular fuzzy numbers are defined as: Very low importance - *R* ~ <sup>1</sup> =(*x*; 1, 1, 2) Low importance - *R* ~ <sup>2</sup> =(*x*; 1, 1, 3), Medium importance – *R* ~ <sup>3</sup> =(*x*; 1, 3, 5), High importance -*R* ~ <sup>4</sup> =(*x*; 3, 5, 5) and Very high importance-*R* ~ <sup>5</sup> =(*x*; 4, 5 , 5).

The weights vector can be calculated by applying fuzzy extent analysis [16]. The weights vector of processes is denoted as *W* =(*w*1, .., *wi* , ..., *wI* )and sub processes *Wi* =(*wi*1, .., *wij* , ..., *wiJ <sup>i</sup>*). The relative importance of processes *wi* and sub-processes *w*are or‐ dinal numbers. In literature, there are many papers in which the weights vector is given by applying extent analysis [17].

The importance of the goals which are defined on the level of each sub process are defined by direct estimation made by a management team. According to literature, it can be con‐ cluded that this approach of estimation of importance of an entity is justified when the num‐ ber of entities is less than five.

to this problem must be placed on the whole of the supply chain because of its critical effect

It is realistic to assume, that decision makers express their evaluations more easy and more precisely by using linguistic expressions than numbers. The number and the type of linguis‐ tic expression used for a description of importance are defined by the management team and depend on the size of supply chain in food companies. Different mathematical ap‐ proaches such as probability theory, fuzzy sets, rough set theory and others enable quantifi‐ cation of linguistic expressions. The development of fuzzy set theory enables the elimination of uncertainties and imprecision caused by lack of good evidence. In the fuzzy approach the uncertainties and imprecision caused are described by linguistic variables. They can be modeled by fuzzy sets with a different shape (triangle, trapezoid, but in some cases with

The fuzzy approach has been used in cases: (1) where conditions have been constantly changing so the observed value could not be stochastically described (2) where there is no sufficient amount of data for statistical analysis. In other words, fuzzy sets theory could sim‐ ulate human way of thinking in the process of decision making under imprecise, approxi‐

According to [14] the advantages of fuzzy sets theory can be presented in the following: it is conceptually clear, flexible, covers different non-linear functions of different complexity; tol‐ erant on imprecise data; includes expert opinions and viewpoints; based on natural lan‐

Estimation of the relative importance of the processes and sub processes on the level of the sup‐ ply chain p ∈ P is defined by a pair-wise comparison matrix whose elements are defined as the relative importance of process/sub-process i/j compared to process/sub-process

and sub processes i, i (1,..,I, respectively). A number of authors consider this approach better compared to direct estimation. Elements of the pair-wise comparison matrix are linguistic ex‐ pressions which are modeled by triangular fuzzy numbers [14, 15]. The domains of these fuz‐ zy numbers are defined on interval [1-5]. Value 1, or value 5 define that analyzed entity i/j

=1, .., *Ji*

<sup>2</sup> =(*x*; 1, 1, 3), Medium importance – *R*

, ..., *wiJ <sup>i</sup>*). The relative importance of processes *wi* and sub-processes *w*are or‐

~

<sup>5</sup> =(*x*; 4, 5 , 5).

tance retrospectively. These triangular fuzzy numbers are defined as: Very low importance -

The weights vector can be calculated by applying fuzzy extent analysis [16]. The weights

dinal numbers. In literature, there are many papers in which the weights vector is given by

are the total number of analyzed processes

has the same or extremely higher impor‐

~

, ..., *wI* )and sub processes

<sup>3</sup> =(*x*; 1, 3, 5), High

where I and, *Ji*

Gaussian distribution, discrete fuzzy numbers) of membership functions.

guage; enables better communication between experts and managers.

, *i*, *i*, '=1, .., *I*; *j*, *j* '

~

<sup>4</sup> =(*x*; 3, 5, 5) and Very high importance-*R*

vector of processes is denoted as *W* =(*w*1, .., *wi*

=1, .., *Ji*

/ *j* ' '

<sup>1</sup> =(*x*; 1, 1, 2) Low importance - *R*

~

applying extent analysis [17].

on efficiency.

452 Food Industry

mative and unclear data.

, *i*, *i*, '=1, .., *I*; *j*, *j* '

compared to entity *i* '

importance -*R*

*Wi* =(*wi*1, .., *wij*

*i* ' / *j* ' '

*R* ~

The importance of each goal k, k=1,,.,*Kij* on the level of sub process j, j=1,.., *Ji* could be descri‐ bed using five linguistic expressions which are modeled by triangular fuzzy numbers *v* ~ *ijk* =(*y*; *l ijk* , *mijk* , *uijk* ). The total number of goals on the level of each sub process is denoted as *Kij* . The value of domains is defined on a standard measurement scale [18]. These triangu‐ lar fuzzy numbers are defined in the following way: very low value-(*y*; 1, 1, 3), low value- (*y*; 1, 3, 5), medium value-(*y*; 3, 5, 7), high value-(*y*; 5, 7, 9) and very high value- (*y*; 8, 9, 9). Since the goals could be beneficial or costly it is necessary to perform normalization of fuzzy values *v* ~ *ijk* , *i* =1, .., *I*; *j* =1, .., *Ji* ; *k* =1, .., *Kij* . In this case the proce‐ dure of linear normalization is used [16]. Normalized values of goals weights are marked as *r* ~ *ijk* , *i* =1, .., *I*; *j* =1, .., *J <sup>i</sup>* , *k* =1, .., *Kij* . In further analysis only one goal with a critical effect on the management of sub-process j, j=1,.., *Ji* is considered.

For the management team carrying out the analysis, the following tasks are important: (1) to determine the rank of the process in a company (2) to determine the rank of a sub process on the process level in a company, (3) to determine the rank of sub processes with respect to the importance of goals and the importance of the considered sub process (4) to determine the rank of processes with respect to the importance of the goals, the relative importance of subprocesses of process i, i=1,..Im and the relative importance of process i, i=1,.,I, and (5) calcu‐ late the degree of belief that the sub process, or process which is on second place in the rank could be on first place; answers to these questions are given by comparing triangular fuzzy numbers *c* ~ *ij*, *d* ~ *<sup>i</sup>* , *i* =1, .., *I*; *j* =1, .., *Ji* , respectively.

The algorithm for analysis of the relative importance of processes, sub-processes and goals in a company p ∈ P is formally given as follows.

Step 1. Input fuzzy matrix *W* ~ = [*w* ~ *ii* ' ], *i*, *i* ' =1, .., *I*; *i* ≠*i* ' ; *p* =1, ..., *P*

Step 2. Calculate weight vector*W* =(*w*1, .., *wi* , ..., *wI* ); rank the processes by placing on first place the process with highest *wi* .

Step 3. Calculate weight vector*Wi* =(*wi*1, .., *wij* , ..., *wiJ <sup>i</sup>*); rank sub processes on the level of each process i, i=1,..,I by placing on the first place the sub process with the highest value *wij* .

Step 4. Transform all linguistic expressions which are modeled by triangular fuzzy numbers *v* ~ *ijk* into *r* ~ *ijk* =(*z*; *L ijk* , *Mijk* , *Uijk* )by applying linear normalization procedure [16].

Step 5. Calculate the weighted normalized aggregated relative importance of sub-process j:

$$\bar{\mathbf{c}}\_{ij} = \frac{1}{\mathbf{K}\_{ij}} \cdot \sum\_{k=1}^{K\_{il}} w\_{ij} \cdot \bar{r}\_{ijk} \text{ i } i = 1, \dots, I; \ j = 1, \dots, J \text{ } ^i, \ k = 1, \dots, K\_{ij}.$$

Step 6. Calculate the weighted normalized aggregated relative importance of process i:

$$\bar{c}\_{Ci} = \frac{1}{J\_i} \cdot \sum\_{j=1}^{L} w\_i \cdot \bar{c}\_{ij\prime\prime} \quad i = 1, \dots, I; \ j = 1, \dots, J^{(L)}$$

Step 7. Rank sub-processes and processes according to decreasing order, *c* ~ *ij* and *d* ~ *i*, respec‐ tively and define level of belief that sub-process j, j=1,...,*Ji* , or process i, i=1,..,I could have the highest importance with respect to the importance of all goals and the importance of sub process j, j=1,...,*Ji* , or process i, i=1,..,I [19].

The developed procedure is illustrated with an example with real-life data from the authors' research.

The pair-wise comparison matrix of relative importance of processes is:

$$
\begin{bmatrix}
\mathbf{1},\mathbf{1},\mathbf{1} & \mathbf{1}/\mathbf{\bar{R}}\_3 & \mathbf{\bar{R}}\_2 \\
\mathbf{\bar{R}}\_3 & \mathbf{1},\mathbf{1},\mathbf{1} & \mathbf{\bar{R}}\_3 \\
\mathbf{1}/\mathbf{\bar{R}}\_2 & \mathbf{1}/\mathbf{\bar{R}}\_3 & \mathbf{1},\mathbf{1},\mathbf{1}
\end{bmatrix}
\tag{1}
$$

The weight vector of sub-processes on level process i=1 is:

The pairwise comparison matrix of relative importance of subprocesses under process i=2 is:

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

~~~~ 2232 ~ ~ ~~ 2 2 23 ~ ~ ~~ 2 2 23 ~ ~ ~ ~ ~~ 3 2 2 2 34 ~ ~ ~~ 2 2 23 ~~~~ ~ 2232 2

*RRRR R R RR R R RR R R R R RR R R RR*

1,1,1 1,1,1 1 / 1,1,1 1,1,1 1,1,1 1,1,1

1,1,1 1,1,1 1 / 1,1,1 1,1,1 1 / 1 / 1 / 1 / 1,1,1

1,1,1 1 / 1 / 1 / 1 / 1,1,1 1,1,1

é ù ê ú

1,1,1

ë û

*RRRR R*

The pairwise comparison matrix of relative importance of subprocesses under process i=3 is:

~ ~~ 2 33 ~ ~~ 2 22

*R RR*

1 / 1 / 1,1,1 1,1,1 1 / 1 / 1,1,1 1,1,1

ë û

*R RR*

é ù ê ú

~ 3 2 1 / 1,1,1 *R* (3)

455

http://dx.doi.org/10.5772/53154

(4)

~~~~~ 1334

*RRRRR*

1/ 1/ 1/ 1/ 1/

Sub process under process is "Level of leadership transformation" (j=4).

1,1,1

~ ~ 3 2 ~ ~ 3 2

*R R R R*

1 / 1,1,1

The weight vector of sub-processes on level process i=2 is:

The weight vector of sub-processes on level process i=3 is:

Subprocess under process is "Roles and responsibilities" (j=1).

*W*<sup>3</sup> =(0.39 0.275 0.168 0.168)

*W*<sup>2</sup> =(0.095 0.165 0.178 0.229 0.165 0.107 0.061)

Sub process under process is "Strategic choice" (j=2).

*W*<sup>1</sup> =(0.295 0.497 0.208)

The weight vector of processes is:

*W* =(0.015 0.072 0.114 0.114 0.171 0.171 0.171 0.171)

The most important processes in considered food company are: Project execution (i=5), Proc‐ ess execution (i=6), Quality assurance (i=7) and Support processes (i=8).

The pairwise comparison matrix of relative importance of subprocesses under process i=1 is:

$$\begin{bmatrix} 1,1,1 & 1 \ \bar{R}\_1 & 1 \ \bar{R}\_3 & 1 \ \bar{R}\_3 & 1 \ \bar{R}\_4 & 1 \ \bar{R}\_4 & 1 \ \bar{R}\_4 & 1 \ \bar{R}\_4 \\ \bar{R}\_1 & 1,1,1 & 1 \ \bar{R}\_2 & 1 \ \bar{R}\_2 & 1 \ \bar{R}\_3 & 1 \ \bar{R}\_3 & 1 \ \bar{R}\_3 & 1 \ \bar{R}\_3 \end{bmatrix}$$

$$\begin{bmatrix} \bar{R}\_1 & \bar{R}\_2 & 1,1,1 & 1,1,1 & 1/\bar{R}\_3 & 1/\bar{R}\_3 & 1/\bar{R}\_3 & 1/\bar{R}\_3\\ \bar{R}\_3 & \bar{R}\_2 & 1,1,1 & 1,1/\bar{R}\_1 & 1/\bar{R}\_3 & 1/\bar{R}\_3 & 1/\bar{R}\_3 \end{bmatrix} \tag{2}$$

$$\begin{bmatrix} \bar{R}\_4 & \bar{R}\_3 & \bar{R}\_3 & \bar{R}\_3 & 1,1,1 & 1,1,1 & 1,1,1\\ \bar{R}\_4 & \bar{R}\_3 & \bar{R}\_3 & \bar{R}\_3 & 1,1,1 & 1,1,1 & 1,1,1\\ \bar{R}\_4 & \bar{R}\_3 & \bar{R}\_3 & \bar{R}\_3 & 1,1,1 & 1,1,1 & 1,1,1 & 1,1\\ \bar{R}\_4 & \bar{R}\_3 & \bar{R}\_3 & \bar{R}\_3 & 1,1,1,1 & 1,1,1,1 & 1,1,1\\ \bar{R}\_4 & \bar{R}\_3 & \bar{R}\_3 & \bar{R}\_3 & 1,1,1,1 & 1,1,1,1 & 1,1,1 \end{bmatrix}$$

The weight vector of sub-processes on level process i=1 is:

*W*<sup>1</sup> =(0.295 0.497 0.208)

*c* ~ *ij* <sup>=</sup> <sup>1</sup> *Kij* ⋅∑ *k*=1 *Kij*

454 Food Industry

*c* ~ *<sup>i</sup>* <sup>=</sup> <sup>1</sup> *Ji* ⋅∑ *j*=1 *Ji wi* ⋅ *c* ~

process j, j=1,...,*Ji*

The weight vector of processes is:

~ 1

*R*

research.

*wij* ⋅ *r* ~

*ijk i* =1, .., *I*; *j* =1, .., *J <sup>i</sup>*

*ij*, *i* =1, .., *I*; *j* =1, .., *J <sup>i</sup>*

tively and define level of belief that sub-process j, j=1,...,*Ji*

, or process i, i=1,..,I [19].

*W* =(0.015 0.072 0.114 0.114 0.171 0.171 0.171 0.171)

~~ ~ ~ 43 3 3 ~~ ~ ~ 43 3 3

*RR R R RR R R* ~~ ~ ~ 43 3 3 ~~ ~ ~ 43 3 3

*RR R R RR R R*

ess execution (i=6), Quality assurance (i=7) and Support processes (i=8).

, *k* =1, .., *Kij*

highest importance with respect to the importance of all goals and the importance of sub

The developed procedure is illustrated with an example with real-life data from the authors'

~ ~ 3 2

*R R*

The most important processes in considered food company are: Project execution (i=5), Proc‐

The pairwise comparison matrix of relative importance of subprocesses under process i=1 is:

~~~~~~~ 1334444 ~~~~~~ 223333

*RRRRRRR*

1,1,1 1,1,1 1 / 1 / 1 / 1 / 1,1,1 1,1,1 1 / 1 / 1 / 1 /

*RRRRRR*

1,1,1 1

1,1,1 1,1,1 1,1,1 1,1,1

1,1,1 1,1,1 1,1,1 1,1,1 1,1,1 1,1,1 1,1,1 1,1,1

,1,1 1,1,1 1,1,1

~ ~ ~~~~ 3 2 3333 ~ ~ ~~~~ 3 2 3333

*R R RRRR R R RRRR*

ë û

1,1,1 1 / 1 / 1 / 1 / 1 / 1 /

1,1,1 1 / 1 / 1 / 1 / 1 / 1 / 1 /

é ù ê ú

~ ~ 3 3 ~ ~ 2 3

é ù ê ú

> *R R R R*

ë û

1,1,1 1 / 1 / 1,1,1

1,1,1 1 /

Step 6. Calculate the weighted normalized aggregated relative importance of process i:

Step 7. Rank sub-processes and processes according to decreasing order, *c*

The pair-wise comparison matrix of relative importance of processes is:

,

~ *ij* and *d* ~

, or process i, i=1,..,I could have the

*i*, respec‐

(1)

(2)

Sub process under process is "Strategic choice" (j=2).

The pairwise comparison matrix of relative importance of subprocesses under process i=2 is:


The weight vector of sub-processes on level process i=2 is:

*W*<sup>2</sup> =(0.095 0.165 0.178 0.229 0.165 0.107 0.061)

Sub process under process is "Level of leadership transformation" (j=4).

The pairwise comparison matrix of relative importance of subprocesses under process i=3 is:

$$\begin{bmatrix} 1,1,1 & \bar{R}\_2 & \bar{R}\_3 & \bar{R}\_3 \\ \bar{1}/\bar{R}\_2 & 1,1,1 & \bar{R}\_2 & \bar{R}\_2 \\ 1/\bar{R}\_3 & 1/\bar{R}\_2 & 1,1,1 & 1,1 \\ \vdots & \vdots & \vdots & \vdots \\ 1/\bar{R}\_3 & 1/\bar{R}\_2 & 1,1,1 & 1,1 \end{bmatrix} \tag{4}$$

The weight vector of sub-processes on level process i=3 is:

*W*<sup>3</sup> =(0.39 0.275 0.168 0.168)

Subprocess under process is "Roles and responsibilities" (j=1).

The pairwise comparison matrix of relative importance of subprocesses under process i=4 is:

$$\begin{bmatrix} 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1 \ /\bar{R}\_3 \\ \bar{R}\_2 & 1,1,1 & \bar{R}\_2 & 1,1,1 & \bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 \\ 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1 \ /\bar{R}\_3 \\ \bar{R}\_2 & 1 \ /\bar{R}\_2 & \bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 \\ 1,1,1 & 1 \ /\bar{R}\_2 & 1,1,1 & \bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 & 1 \ /\bar{R}\_3 \\ \bar{R}\_2 & 1,1,1 & \bar{R}\_2 & 1,1,1 & \bar{R}\_2 & 1,1,1 & 1 \ /\bar{R}\_2 \\ \bar{R}\_3 & \bar{R}\_2 & \bar{R}\_3 & \bar{R}\_2 & \bar{R}\_3 & \bar{R}\_2 & 1,1,1 \end{bmatrix} \tag{5}$$

processes and the rank of the processes with respect to the relative importance of the sub processes and the relative importance of processes is determined. The calculated ranks are

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

According to the calculated values of importance of the sub processes with respect to their relative importance and the relative importance of the goals of each sub process (see Table 2) the following analysis can be made: The sub process which has the highest importance: (1) Strategic alignment (i=1) is Strategic choice (j=2), (2) Process development (i=4) is Process sig‐ nificance (j=7) and (3) Quality assurance of goals (j=3) and documentation of the process (j=4). Based on the calculated degree of belief it is clear that the other sub processes, i=1, i=4

*ijk* ijk, i=1,..,I; j=1,...,*Ji*

 *low value* (0.11,0.11,0.33) 511 *high value* (0.56,0.78,1) *medium value* (0.33,0.56,0.78) 522 *high value* (0.56,0.78,1) *very low value* (0.11,0.11,0.33) 533 *high value* (0.56,0.78,1) *medium value* (0.33,0.56,0.78) 544 *high value* (0.56,0.78,1) *high value* (0.56,0.78,1) 611 *high value* (0.56,0.78,1) *medium value* (0.33,0.56,0.78) 622 *very high value* (0.89,1,1) *medium value* (0.33,0.56,0.78) 633 *very high value* (0.89,1,1) *high value* (0.56,0.78,1) 644 *very high value* (0.89,1,1) *medium value* (0.33,0.56,0.78) 711 *high value* (0.56,0.78,1) *medium value* (0.33,0.56,0.78) 722 *very high value* (0.89,1,1) *medium value* (0.33,0.56,0.78) 733 *very high value* (0.89,1,1) *high value* (0.56,0.78,1) 744 *very high value* (0.89,1,1) *very high value* (0.89,1,1) 755 *very high value* (0.11,0.11,0.12) *high value* (0.56,0.78,1) 811 *high value* (0.56,0.78,1) *high value* (0.56,0.78,1) 822 *high value* (0.56,0.78,1) *very high value* (0.89,1,1) 833 *high value* (0.56,0.78,1) *high value* (0.56,0.78,1) 844 *high value* (0.56,0.78,1) *high value* (0.56,0.78,1) 855 *high value* (0.56,0.78,1) *high value* (0.56,0.78,1) 866 *medium value* (0.33,0.56,0.78) *very high value* (0.89,1,1) 877 *very high value* (0.89,1,1)

**Table 6.** Importance of goals on the level of each sub process with the critical effect on management of those sub

k=1,.., *Kij*

*v* ~

*ijk r*

~ *ijk*

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457

and i=7 have very low importance compared to the first ranked sub process.

presented in Table 7 and Table 8.

*v* ~

*ijk r*

~

ijk, i=1,..,I; j=1,...,*Ji*

k=1,.., *Kij*

processes

The weight vector of sub-processes on level process i=4 is:

## *W*<sup>4</sup> =(0.1 0.155 0.1 0.144 0.123 0.155 0.222)

Sub-process under process i=4 is "Process significance". (j=7).

Processes i=5 and i=6 could be decomposed on four sub processes each. The relative impor‐ tance of sub processes under process i=5 and i=6 are equal. Such that *w*<sup>5</sup> *<sup>j</sup>* =*w*<sup>6</sup> *<sup>j</sup>* =0.25, *j* =1, 2, 3, 4.

The pairwise comparison matrix of relative importance of subprocesses under process i=7 is:

$$
\begin{bmatrix}
1,1,1 & 1/\bar{R}\_3 & 1/\bar{R}\_4 & 1/\bar{R}\_4 & 1/\bar{R}\_3 \\
\bar{R}\_3 & 1,1,1 & 1/\bar{R}\_3 & 1/\bar{R}\_3 & 1,1,1 \\
\bar{R}\_4 & \bar{R}\_3 & 1,1,1 & 1,1,1 & \bar{R}\_2 \\
\bar{R}\_4 & \bar{R}\_3 & 1,1,1 & 1,1,1 & \bar{R}\_2 \\
\bar{R}\_4 & \bar{R}\_3 & 1,1,1 & 1,1,1 & \bar{R}\_2 \\
\bar{R}\_3 & 1,1,1 & 1/\bar{R}\_2 & 1/\bar{R}\_2 & 1,1,1
\end{bmatrix}
\tag{6}
$$

The weight vector of sub-processes on level process i=7 is:

*W*<sup>7</sup> =(0.027 0.186 0.29 0.29 0.205)

Sub process under process i=7 is "Level of accomplishment of quality goals".(j=3).

Process i=8 could be decomposed on eight sub processes. According to the fuzzy rating of the management team, all sub processes have equal of the relative importance, so that *w*<sup>8</sup> *<sup>j</sup>* =0.143, *j* =1, ..., 8.

Estimation of the importance of goals with critical effect management of the sub processes are given in Table 6. By applying the Algorithm (Step 5 to Step 7) rank of sub processes with respect to the relative importance of the defined goals and the relative importance of the sub processes and the rank of the processes with respect to the relative importance of the sub processes and the relative importance of processes is determined. The calculated ranks are presented in Table 7 and Table 8.

The pairwise comparison matrix of relative importance of subprocesses under process i=4 is:

~ ~ ~~ 2 2 23

*R R RR*

*R R RR*

*R R RR*

~~~ 32 3 2 *RRR* 1,1,1

Processes i=5 and i=6 could be decomposed on four sub processes each. The relative impor‐ tance of sub processes under process i=5 and i=6 are equal. Such that

The pairwise comparison matrix of relative importance of subprocesses under process i=7 is:

~~~~ 3443

*RRRR*

1,1,1 1 / 1 / 1,1,1 1,1,1 1,1,1 1,1,1 1,1,1 1,1,1 1 / 1 / 1,1,1

~ ~ ~ 3 3 3 ~ ~ ~ 4 3 2 ~ ~ ~ 4 3 2 ~ ~ ~ 3 2 2

*R RR*

*R RR*

Sub process under process i=7 is "Level of accomplishment of quality goals".(j=3).

Process i=8 could be decomposed on eight sub processes. According to the fuzzy rating of the management team, all sub processes have equal of the relative importance, so that

Estimation of the importance of goals with critical effect management of the sub processes are given in Table 6. By applying the Algorithm (Step 5 to Step 7) rank of sub processes with respect to the relative importance of the defined goals and the relative importance of the sub

1,1,1 1 / 1 / 1 / 1 /

é ù ê ú

*R R R R R R*

ë û

(5)

(6)

~~ ~ ~ 22 2 2 ~ ~ ~~ 2 2 23

é ù ê ú

*RR R R*

1,1,1 1,1,1 1,1,1 1 /

1,1,1 1 / 1,1,1 1 / 1,1,1 1 / 1 /

1,1,1 1 / 1,1,1 1 / 1,1,1 1 / 1 /

1,1,1 1 / 1,1,1 1,1,1 1 / 1 /

~ ~~ ~ ~ 2 22 2 2 ~ ~ ~ ~ 2 2 2 3

*R RR R R*

1 / 1,1,1 1 / 1,1,1 1 /

1,1,1 1,1,1 1,1,1 1 /

~~ ~ ~ 22 2 2

*RR R R*

ë û

~~~ 3 2

*RRR*

Sub-process under process i=4 is "Process significance". (j=7).

The weight vector of sub-processes on level process i=7 is:

*W*<sup>7</sup> =(0.027 0.186 0.29 0.29 0.205)

*w*<sup>8</sup> *<sup>j</sup>* =0.143, *j* =1, ..., 8.

The weight vector of sub-processes on level process i=4 is:

*W*<sup>4</sup> =(0.1 0.155 0.1 0.144 0.123 0.155 0.222)

*w*<sup>5</sup> *<sup>j</sup>* =*w*<sup>6</sup> *<sup>j</sup>* =0.25, *j* =1, 2, 3, 4.

456 Food Industry

According to the calculated values of importance of the sub processes with respect to their relative importance and the relative importance of the goals of each sub process (see Table 2) the following analysis can be made: The sub process which has the highest importance: (1) Strategic alignment (i=1) is Strategic choice (j=2), (2) Process development (i=4) is Process sig‐ nificance (j=7) and (3) Quality assurance of goals (j=3) and documentation of the process (j=4). Based on the calculated degree of belief it is clear that the other sub processes, i=1, i=4 and i=7 have very low importance compared to the first ranked sub process.


**Table 6.** Importance of goals on the level of each sub process with the critical effect on management of those sub processes


Process execution is the most important process in the analyzed food processing companies according to analysis of the importance of all sub processes, the importance of the defined goals of the sub process and the importance of the process. The degree of belief that the process which is denoted as Project execution has the highest importance of 0.8. According to the calculated result, the mentioned process has high importance for the specific food company, so the management team must have this in mind in making strategic decisions.

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

5 (0.024,0.033,0.043) 2 0.8

**Table 8.** Rank of process with respect to its importance and importance of goals of the sub-process of each process

ables calculation of the importance of each goal, process and sub process [20, 21].

The presented model is used for the development of a very usable software solution that en‐

A strategy map describes how an organization can create sustained value for its sharehold‐

The strategy map is developed based on the Kaplan and Norton model. A Strategy map de‐ scribes how the organization creates value by connecting strategy objectives in an explicit cause and effect relationship in the four BSC objectives (financial, customer, processes, learning and growth). Strategy map is a strategic part of the BSC (Balanced Score Card)

**•** Internal perspective's eight processes: (P1) strategy alignment, (P2) process leadership, (P3) process governance, (P4) process development, (P5) project execution, (P6) process

**Rank Degree of belief that process has the highest**

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**importance**

i i=1,..,I *d* ~ *i*

**3.4. Strategy map**

ers, customers and communities.

framework to describe strategies for value creation.

**•** Financial perspective is recognized in competitiveness.

**•** Customer perspective is identified: (1) quality, (2) safety, and (3) image.

execution, (P7) quality assurance process, and (P8) supporting processes.

**•** Perspective or growth and learning : (1) technology and (2) people capability.

 (0.001,0.0.002,0.003) 4 (0.004,0.006,0.009) 7 (0.013,0.019,0.024) 5 (0.011,0.014,0.016) 6

6 (0.1015,0.04,0.043) 1 7 (0.025,0.028,0.028) 3 8 (0.014,0.019,0.024) 5

**Table 7.** Rank of sub-process with respect to the importance and goals of the sub process

Under the following processes, the sub processes which have the most importance are: (1) Process governance (i=3)- Rules and responsibilities (j=1), (2) Process execution (i=6)- Plan‐ ned and achieved goals (j=2), Resource utilization (j=3), and Human resources (j=4), and (3) Support processes (i=8)- ICT support (j=7). Based on the degree of beliefs, the management team can conclude that the sub processes which are placed on second place in the rank un‐ der treated processes have less importance compared to the sub processes which are placed on first place. However, in making operational decisions, the importance of the sub process‐ es which are on second place should be considered. The most important sub-process under Process leadership (i=2) is Level of leadership transformation (j=5). Since the degree of belief for the sub process Level of trust and communication in an organization (j=4) is 0.99, it is clear that these two sub-processes have equal relative importance.

Process execution is the most important process in the analyzed food processing companies according to analysis of the importance of all sub processes, the importance of the defined goals of the sub process and the importance of the process. The degree of belief that the process which is denoted as Project execution has the highest importance of 0.8. According to the calculated result, the mentioned process has high importance for the specific food company, so the management team must have this in mind in making strategic decisions.


**Table 8.** Rank of process with respect to its importance and importance of goals of the sub-process of each process

The presented model is used for the development of a very usable software solution that en‐ ables calculation of the importance of each goal, process and sub process [20, 21].

#### **3.4. Strategy map**

ij, i=1,..,I; j=1,.*Ji*

458 Food Industry

*c* ~ *ij*

47 (0.196,0.22,0.23) **1**

Rank

Degree of belief

 (0.032,0.097,0.165) **2** 0.005 51 (0.14,0.195,0.25) **1** (0.164,0.278,0.388) **1** 52 (0.14,0.195,0.25) **1** (0.023,0.023,0.068) **3** 53 (0.14,0.195,0.25) **1** (0.031,0.053,0.074) **5** 54 (0.14,0.195,0.25) **1**

 (0.059,0.1,0.139) **3** 62 (0.222,0.25,0.25) **1** (0.076,0.128,0.179) **2** 0.99 63 (0.222,0.25,0.25) **1** (0.092,0.129,0.165) **1** 64 (0.222,0.25,0.25) **1** (0.035,0.05,0.083) **4** 71 (0.015,0.021,0.027) **4**

31 (0.129,0.218,0.304) **1** 73 (0.258,0.29,0.29) **1** 32 (0.091,0.154,0.214) **3** 74 (0.258,0.29,0.29) **1** 33 (0.094,0.131,0.168) **4** 75 (0.023,0.023,0.026) **3**

45 (0.069,0.096,0.123) **5** 86 (0.055,0.08,0.112) **3** 46 (0.087,0.121,0.155) **2** 0.00 87 (0.127,0.143,0.143) **1**

**Table 7.** Rank of sub-process with respect to the importance and goals of the sub process

clear that these two sub-processes have equal relative importance.

Under the following processes, the sub processes which have the most importance are: (1) Process governance (i=3)- Rules and responsibilities (j=1), (2) Process execution (i=6)- Plan‐ ned and achieved goals (j=2), Resource utilization (j=3), and Human resources (j=4), and (3) Support processes (i=8)- ICT support (j=7). Based on the degree of beliefs, the management team can conclude that the sub processes which are placed on second place in the rank un‐ der treated processes have less importance compared to the sub processes which are placed on first place. However, in making operational decisions, the importance of the sub process‐ es which are on second place should be considered. The most important sub-process under Process leadership (i=2) is Level of leadership transformation (j=5). Since the degree of belief for the sub process Level of trust and communication in an organization (j=4) is 0.99, it is

22 (0.092,0.129,0.165) **1** 61 (0.14,0.195,0.25) **2** 0.34

27 (0.02,0.034,0.048) **6** 72 (0.165,0.185,0.185) **2** 0.00

 (0.15,0.168,0.168) **2** 0.44 81 (0.08,0.112,0.143) **2** 0.34 (0.056,0.078,0.1) **6** 82 (0.08,0.112,0.143) **2** 0.34 (0.087,0.121,0.155) **2** 83 (0.08,0.112,0.143) **2** 0.34 (0.089,0.1,0.1) **4** 84 (0.08,0.112,0.143) **2** 0.34 (0.081,0.112,0.144) **3** 85 (0.08,0.112,0.143) **2** 0.34

ij, i=1,..,I; j=1,..*Ji*

*c* ~ *ij* Rank

Degree of belief

A strategy map describes how an organization can create sustained value for its sharehold‐ ers, customers and communities.

The strategy map is developed based on the Kaplan and Norton model. A Strategy map de‐ scribes how the organization creates value by connecting strategy objectives in an explicit cause and effect relationship in the four BSC objectives (financial, customer, processes, learning and growth). Strategy map is a strategic part of the BSC (Balanced Score Card) framework to describe strategies for value creation.


Process

Pi

**Estimation of process importance** **Estimation of importance of sub process in the**

P1 5 7 8 6 5 6 4

P3 7 8 7 6 6 6 6 7 8

P5 8 7 7 7 7 7 7 7 7 P6 8 7 6 7 6 7 8 8 8 P7 8 7 8 9 9 8 7 8 9 9 8

**Estimation of importance of sub process goals**

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Project execution P5

Process governance P3 Process leadership

Core processes

Process execution P6

people capability tehnology project results

ICT support Maintenance

Quality management

P8 – support processes

Other management systems

PP1 PP2 PP3 PP4 PP5 PP6 PP7 PP1 PP2 PP3 PP4 PP5 PP6 PP7

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

P2 6 6 7 7 8 7 6 5 5 6 7 6 6 7 6

P4 7 6 7 6 7 6 7 8 7 7 8 7 7 7 8

P8 8 5 6 6 6 5 5 6 7 7 7 7 7 6 8

Strategy alignment

Process leadership

Human Resouces Management

Each process presented in figure 9 has its own importance as a whole. It is clear that each

Process performance

New projects

**Table 9.** Cross reference of processes, their importance and importance of their sub processes and goals

P1

P2

Process Development P4

> Quality assurance process

Management

Marketing Sales Finance

process could be decomposed on the accompanied sub processes.

P7

CRM Supply

**Figure 9.** Process framework for companies in the food industry

customer

Needs & requests

**frame of specific process**

**Figure 8.** Strategy map

A strategy map for the food industry is presented in figure 8. The level of each component process is identified using a questioner for companies in the Serbian food industry. Rela‐ tions among processes are defined using a method with 3 iterations.

A strategic map should describe a strategy and present the strategy to management and em‐ ployees, and in the same way connect stakeholders, customer management, process man‐ agement, quality management, core capabilities, innovations, human resources, ICT, organizational design / redesign and learning.

## **4. Research results: Case study of the serbian food industry**

#### **4.1. Proposed model: Process framework for companies in the food industry**

In this section we will provide the process framework for companies from the food industry sector. All processes are divided into the following categories: leadership processes, core processes and support processes. As it was shown in the section above "Project execution" process (P5) has the highest importance.

The support processes contain the following: CRM (Customer Relation Management), Sup‐ ply management, Human resources management, ICT support, Maintenance, Marketing, Sales, Finances, Quality management and Other management systems.

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**Table 9.** Cross reference of processes, their importance and importance of their sub processes and goals

C / Competitivity

Financial perspective

460 Food Industry

Customer perspective

Internal perspective

> Perspective of growth and learning

organizational design / redesign and learning.

process (P5) has the highest importance.

**Figure 8.** Strategy map

Q / Quality S / Food safety I / Image

P4 P5 P6 P7

T/Technology P/People capability

A strategy map for the food industry is presented in figure 8. The level of each component process is identified using a questioner for companies in the Serbian food industry. Rela‐

A strategic map should describe a strategy and present the strategy to management and em‐ ployees, and in the same way connect stakeholders, customer management, process man‐ agement, quality management, core capabilities, innovations, human resources, ICT,

In this section we will provide the process framework for companies from the food industry sector. All processes are divided into the following categories: leadership processes, core processes and support processes. As it was shown in the section above "Project execution"

The support processes contain the following: CRM (Customer Relation Management), Sup‐ ply management, Human resources management, ICT support, Maintenance, Marketing,

tions among processes are defined using a method with 3 iterations.

**4. Research results: Case study of the serbian food industry**

Sales, Finances, Quality management and Other management systems.

**4.1. Proposed model: Process framework for companies in the food industry**

P1 P2 P3 P8

**Figure 9.** Process framework for companies in the food industry

Each process presented in figure 9 has its own importance as a whole. It is clear that each process could be decomposed on the accompanied sub processes.


**Questionnaire**

2 Evaluation of relevance of process goals and indicators;

3 Evaluation of achievement of the

4 Evaluation of documentation of

10 Level of inclusion and complexity of

2 Evaluation of establishment of project

3 Evaluation of portfolio management

4 Evaluation of project management

Evaluation of technology level of

Evaluation of implementation of system and process approach (reengineering, time, electivity)

Evaluation of business decision making, business intelligence

Evaluation of level of quality approach (WCM, Lean, 6 sigma, IMS)

process demands .

organization;

and control;

frameworks.

production

process goals;

processes;

**Questionnaire M Questionnaire** M

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

5 Evaluation of processes investigation; 5 Level of process coast/planned process

6 Evaluation of analysis of processes; 8 Evaluation of support processes

8 Evaluation of process flexibility; 2 Evaluation of sale;.

5 Questionnaire for Project execution 5 Evaluation of finances;

7 Evaluation of process significance; 1 Evaluation of marketing process;

9 Evaluation of Process agility; 3 Evaluation of customer relations

1 Evaluation of ,,right" projects; 6 Evaluation of human resources

Evaluation – Technology 10 Evaluation of other management

2 Level of quality of inputs in process

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3 Level of quality of working procedures

4 Level of achieving the quality goals

management (CRM);

4 Evaluation of supply chain management (SCM);

management;

7 Evaluation of ICT support;

8 Evaluation of implementation of quality management;

9 Evaluation of maintenance;

systems

perspective

9 Evaluation of entities in customers

1 Evaluation of product quality

3 Evaluation of product safety

2 Evaluation of quality of organization

costs

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**Questionnaire**

462 Food Industry

1 Estimation of strategic alignment of

4 Level of trust and communication in

1 Estimation of roles selection and

3 Estimation of evaluation and control of management including estimation of

contemporary methods and tools in

responsibilities;

the risk of process;

business processes.

process;

4 Evaluation of implementation of

1 Evaluation of concept of desired

organization;

processes;

**Questionnaire M Questionnaire** M

2 Estimation of strategic choices; 2 Gap between planned and achieved

3 Estimation of process architecture. 3 Level of resource utilization (for process

5 Level of business process awareness; Evaluation of increase of process

6 Level of process innovation; Evaluation of definition and

7 Level of promotion of manager success. Evaluation of understanding or

2 Estimation of roles selection; Evaluation of management of the

3 Questionnaire for Process governance Evaluation of process metrics;

4 Questionnaire for Process development 7 Questionnaire for Quality assurance of

1 Level of achieving of process goals

6.1 Questionnaire for process performance

establishment of rewards;

Evaluation of continuous

Evaluation of communications.

1 Level of effectiveness of processes – On time Delivery of products

responsibilities for the process;

Evaluation of performance monitor;

goals

needs):

awareness;

process;

processes

improvement;

1 Questionnaire for strategy alignment 6 Questionnaire for process execution

2 Questionnaire for Process Leadership 4 Human;

2 Level of transactional leadership; 6 ICT;

1 Level of transformational leadership; 5 Equipment;

3 Level of trust in leadership; 7 Knowledge.



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**Table 10.** Questionnaire for Serbian companies

Total number of 53 companies were analyzed and results are presented in table 10.

Each sub process has importance in the frame of the specific process as well as the impor‐ tance of its goal. The overall data gathered as the result of research in Serbian companies in the food industry is presented in table 9. The presented data could be combined with lin‐ guistically expressed opinion and used for ranking and simulation of quality goals accord‐ ing to the approach presented using fuzzy sets. Data were gathered using the questionnaire presented as table 10.


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**Questionnaire**

464 Food Industry

Evaluation of Center of Business

Evaluation of CBI engagement model.

Innovation (CBI)

**Table 10.** Questionnaire for Serbian companies

presented as table 10.

**Questionnaire M Questionnaire** M

Total number of 53 companies were analyzed and results are presented in table 10.

Each sub process has importance in the frame of the specific process as well as the impor‐ tance of its goal. The overall data gathered as the result of research in Serbian companies in the food industry is presented in table 9. The presented data could be combined with lin‐ guistically expressed opinion and used for ranking and simulation of quality goals accord‐ ing to the approach presented using fuzzy sets. Data were gathered using the questionnaire

**No. Company Stand. 1 2 3 4 5 5.1 5.2 6 6.1 7 8 9 10** Company 01 22000 8 6 7 7 5 8 8 1 7 9 8 6 9 Company 02 22000 5 8 5 4 7 6 4 1 6 7 7 6 5 Company 03 22000 7 6 7 5 8 6 4 1 7 5 9 7 2 Company 04 22000 7 6 7 4 5 6 5 1 6 6 7 5 6 Company 05 22000 6 8 5 5 7 8 7 1 7 6 9 8 6 Company 06 CAC 4 7 2 2 5 5 1 1 5 2 6 4 3 Company 07 CAC 5 7 4 4 6 8 4 1 6 5 8 8 3 Company 08 CAC 7 8 6 4 6 8 5 1 8 7 9 7 5 Company 09 CAC 3 6 2 2 6 4 1 1 3 2 4 4 2 Company 10 CAC 5 6 5 3 5 4 4 1 4 4 6 4 5 Company 11 CAC 6 7 6 5 5 6 6 1 5 7 7 5 8 Company 12 CAC 4 7 4 3 5 6 2 1 5 5 5 5 4 Company 13 CAC 4 5 4 2 5 6 5 1 5 4 6 6 4 Company 14 CAC 4 5 3 3 7 5 4 1 6 6 6 6 2 Company 15 CAC 5 6 5 3 6 8 1 1 5 5 6 7 4 Company 16 CAC 4 4 3 3 4 4 3 1 5 4 4 3 4

Evaluation of human resources 4 Evaluation of the brand, image of

Evaluation of internal capability. 10 Evaluation of entities in financial perspective

organization

EU market

1 Level of competitiveness compared to



Legend: CAC – Codex Alimentarius – Food Hygiene, Recommended International Code of Practice General Principles of Food Hygiene CAC/RCP – 1969, Rev. 4-2003.

#### **Table 11.** Gathered data from Serbian companies

In table 11, the numbers in the columns correspond to the Questionnaire presented in table 10 (The names of companies are left and replaced by "company x" due to protection of com‐ panies data.). Structure of the sample is: 25 organizations with less than 10 employees, 21 organizations with 10-50 employees, 3 organizations with 50-250 employees, 3 organizations with 250-500 employees and 1 organization with more than 500 employees.

Based on the expert opinion of consultants working with organizations in the sample the rela‐ tion among processes and other entities are determined. The form of relation is: RI, O where I goal (performance) of entity and O – goal (performance source entity ) of destination entity


Starting values of constants are determined by investigation of the organization in the sam‐ ple. Presented relations describe the importance of relations presented in the strategy map.

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The food industry is a sector that is involved in rapid, multidimensional changes. At the same time this is an industrial sector that is emerging and increasing its importance in rela‐ tion to different trends and challenges: growth of population, increased need for healthy

It is completely clear that the food industry must respond in many different directions in or‐ der to avoid challenges, reduce threats and explore its strengths using available opportuni‐ ties. Some of the changes in the food industry will go in the direction of automation of the production process and technological changes, employing the full potential of biotechnolo‐ gy, information and communication technology, RFID, robotics, sensors and even e-busi‐ ness. Other changes will be directed in the optimization of logistic infrastructure and energy savings. But companies in the food industry will also need to understand globalization trends, market developments and chanciness and swings in customers' needs. All these changes will increase the success of the food industry on the global markets. Although all of these changes could have only limited, partial results that will not fulfill the complete poten‐ tial of the suggested changes if companies do not restructure their organizations and rede‐ sign their processes. Changing and adopting the changes of organization as well as restructure and redesign of processes are a pre-condition for all other changes. On the other hand, companies usually do not pay much attention to organizational challenges and proc‐ ess redesign, compared with other directions of changes such as the implementation of new

In this chapter we addressed different questions and issues in the redesign of process devel‐ opment in food production organizations using quality engineering tools and methods. The first step is analysis of existing processes, its decomposition and introduction of quality met‐ rics for evaluation of the quality of processes and quality of goals. A typical process map for a company in the food processing industry in Serbia is presented and the production proc‐ ess is decomposed on sub processes. All indicators for quality metrics were proposed as nu‐

After decomposition another step is redesign of process development. Different changing methodologies are compared according to the amount of change, score of change, used tools, probability of success and their applicability in food industry. The concept of continu‐ ous process improvement is presented and redesign of the process has 5 phases with 10 steps. Different quality engineering methods and tools used in the food industry were com‐ pared according to the frequency of their usage and correlation to increase of profit in Serbi‐ an companies. The general conclusion was that companies are using older, established methods and tools even if they do not have that large impact on profit increase compared to the modern tools and methods. A total number of 20 different quality engineering methods

food, food safety regulations, and different customers' demands.

meric values in order to clearly demonstrate the concept.

**5. Conclusion**

and emerging technologies.

Starting values of constants are determined by investigation of the organization in the sam‐ ple. Presented relations describe the importance of relations presented in the strategy map.

## **5. Conclusion**

**No. Company Stand. 1 2 3 4 5 5.1 5.2 6 6.1 7 8 9 10** Company 50 6 5 6 6 5 6 7 1 6 5 7 8 5 Company 51 5 7 7 7 7 7 7 1 7 6 7 7 4 Company 52 4 6 7 7 6 6 5 1 7 6 8 7 4 Company 53 6 4 6 6 5 7 3 0 5 5 8 8 7

Legend: CAC – Codex Alimentarius – Food Hygiene, Recommended International Code of Practice General Principles

In table 11, the numbers in the columns correspond to the Questionnaire presented in table 10 (The names of companies are left and replaced by "company x" due to protection of com‐ panies data.). Structure of the sample is: 25 organizations with less than 10 employees, 21 organizations with 10-50 employees, 3 organizations with 50-250 employees, 3 organizations

Based on the expert opinion of consultants working with organizations in the sample the rela‐ tion among processes and other entities are determined. The form of relation is: RI, O where I goal (performance) of entity and O – goal (performance source entity ) of destination entity

> RP1, S: GS= GS01 + 0.03\*DP1 RP2, S: GS= GS02 + 0.04\*DP2 RP3, S: GS= GS03 + 0.08\*DP3 RP4, S: GS= GS04 + 0.18\*DP4 RP6, S: GS= GS06 + 0.28\*DP6 RP7, S: GS= GS07 + 0.15\*DP7 RP8, S: GS= GS08 + 0.20\*DP8

R S, I: GI= GS0 + 0.38\*DI RP6, I: GI= GI06 + 0.32\*DP6 RP7, I: GI= GI07 + 0.30\*DP7 RP8, I: GI= GI08 + 0.35\*DP8

RQ, C: GC= GQ0 + 0.39\*DQ RS, C : GC= GS0 + 0.28\*DS RI, C : GC= GI0 + 0.15\*DI

with 250-500 employees and 1 organization with more than 500 employees.

of Food Hygiene CAC/RCP – 1969, Rev. 4-2003.

466 Food Industry

**Table 11.** Gathered data from Serbian companies

RT, P4: GP4= GPot + 0.11\*DT RT, P6: GP6= GPop + 0.15\*DP RP1, P: GP1= GP01 + 0.25\*DP RP2, P: GP2= GP02 + 0.28\*DP RP3, P: GP3= GP03 + 0.24\*DP RP4, P: GP4= GP04 + 0.27\*DP RP5, P: GP5= GP05 + 0.18\*DP RP6, P: GP6= GP06 + 0.16\*DP

RP1, Q: GQ= GQ01 + 0.30\*DP1 RP2, Q: GQ= GQ02 + 0.32\*DP2 RP3, Q: GQ= GQ03 + 0.29\*DP3 RP4, Q: GQ= GQ04 + 0.35\*DP4 RP5, Q: GQ= GQ05 + 0.18\*DP5 RP6, Q: GQ= GQ06 + 0.17\*DP6 RP7, Q: GQ= GQ07 + 0.24\*DP7 RP8, Q: GQ= GQ08 + 0.17\*DP8 The food industry is a sector that is involved in rapid, multidimensional changes. At the same time this is an industrial sector that is emerging and increasing its importance in rela‐ tion to different trends and challenges: growth of population, increased need for healthy food, food safety regulations, and different customers' demands.

It is completely clear that the food industry must respond in many different directions in or‐ der to avoid challenges, reduce threats and explore its strengths using available opportuni‐ ties. Some of the changes in the food industry will go in the direction of automation of the production process and technological changes, employing the full potential of biotechnolo‐ gy, information and communication technology, RFID, robotics, sensors and even e-busi‐ ness. Other changes will be directed in the optimization of logistic infrastructure and energy savings. But companies in the food industry will also need to understand globalization trends, market developments and chanciness and swings in customers' needs. All these changes will increase the success of the food industry on the global markets. Although all of these changes could have only limited, partial results that will not fulfill the complete poten‐ tial of the suggested changes if companies do not restructure their organizations and rede‐ sign their processes. Changing and adopting the changes of organization as well as restructure and redesign of processes are a pre-condition for all other changes. On the other hand, companies usually do not pay much attention to organizational challenges and proc‐ ess redesign, compared with other directions of changes such as the implementation of new and emerging technologies.

In this chapter we addressed different questions and issues in the redesign of process devel‐ opment in food production organizations using quality engineering tools and methods. The first step is analysis of existing processes, its decomposition and introduction of quality met‐ rics for evaluation of the quality of processes and quality of goals. A typical process map for a company in the food processing industry in Serbia is presented and the production proc‐ ess is decomposed on sub processes. All indicators for quality metrics were proposed as nu‐ meric values in order to clearly demonstrate the concept.

After decomposition another step is redesign of process development. Different changing methodologies are compared according to the amount of change, score of change, used tools, probability of success and their applicability in food industry. The concept of continu‐ ous process improvement is presented and redesign of the process has 5 phases with 10 steps. Different quality engineering methods and tools used in the food industry were com‐ pared according to the frequency of their usage and correlation to increase of profit in Serbi‐ an companies. The general conclusion was that companies are using older, established methods and tools even if they do not have that large impact on profit increase compared to the modern tools and methods. A total number of 20 different quality engineering methods and tools were analyzed among 53 Serbian companies from the food industry sector. With many different problems, starting from the ranking of importance of quality goals, ranking of importance of processes or entities, up to ranking the methods or tools, there is a need for an approach that will solve these issues. All these problems could be solved by usage of the fuzzy approach. As an extension of the general ranking idea presented on the ranking and definition of goals in the production process, the fuzzy approach for evaluation of the im‐ portance of entities in supply chains in the food industry is presented. A strategic map as a strategic part of the BSC (Balanced Score Card) framework is presented as the role model for food processing companies. The special contributions of this map are the relations between entities (from all four perspectives) that are defined as the result of the research in Serbian companies. In addition, the framework of processes for the companies from the food indus‐ try was presented. Finally, the general contribution of all the presented issues, decomposi‐ tion, redesign, evaluation of quality tools and methods, fuzzy ranking and the strategic map, is based on results of research in 53 Serbian companies. The questionnaire for Serbian com‐ panies is presented as well as the results of research. A very important contribution present‐ ed is the fact that all decompositions, redesigns, modeling, simulations and calculations were performed using real life data acquired from Serbian companies.

[2] Nikhil Daxini, Use BPM to Assist in New Product Development, TRIZ Journal, http://

The Redesign of Processes' Development in Food Production Organizations Using Quality Engineering Methods...

http://dx.doi.org/10.5772/53154

469

[3] Aikens, H., Quality Inspired Management: The Key to Sustainability, Prentice Hall,

[4] Hur, M., The influence of total quality management practices on the transformation of how organizations works, Total Management and Business Intelligence, Vol. 20,

[5] Foster, T., Managing Quality: Integrating the supply chain, Prentice Hall, ISBN:

[6] Summers, D., Quality Management: Creating and Sustaining Organizational Effec‐

[7] Foster, T., Managing Quality: An Integrative Approach, Pearson & Prentice Hall,

[9] Rao et all., Total Quality Management: A Cross Functional Perspective, John Willey

[10] Goetch, D., Davis, S., Introduction to Total Quality, Prentice Hall, Upper Saddle Riv‐

[12] Born, G., Process Management to Quality Improvement, John Willey & Sons, New

[13] Davenport T.H., Process Innovation Reengineering Work Through Information Tech‐

[14] Zimmermann, H.J., Fuzzy set Theory and its applications. Kluwer Nijhoff Publising:

[15] Klir, G.J., Folger, T.A., Fuzzy Sets, Uncertainty and Information, Pretence-Hall, Eng‐

[16] Chang, D.Y., Applications of the extent analysis method on fuzzy AHP, European

[17] Tadić, D., Milanović, D., Misita, M., Tadić, B., New integrated approach to the prob‐ lem of ranking and supplier selection under uncertainties, Proceedings of the Institu‐ tion of Mechanical Engineers. Part B: Journal of Engineering manufacture, doi:

[18] Saaty, T.L., How to make a decision: The Analytic Hierarchy Process, European Jour‐

[11] Jeston, J., Nelis, J., Business Process Management, Elsevier, Amsterdam, 2008.

[8] Juran, J., Managerial Breakthrough, Mc Grow Hill, New York, 1995.

www.triz-journal.com/content/c081201a.asp

Numbers 7-8, p. 847 – 863, ISSN: 1478 – 3363, 2009.

tiveness, Pearson International Edition, 2009.

2011.

978-0-13-507819-8, 2010.

2004, ISBN: 0-13-123018-3

& Sons, New York, 1996.

nology, Boston, MA, 1993.

lewood, Cliffs, NJ., 1988.

10.1243/09544054JEM2105.

Journal of Operational Research, 95, 649-655, 1996.

nal of Operational Research, 48, 9-26, 1990.

er, New Jersey, 1997.

York, 1996.

Boston, 1996.

A possible and very useful extension of this research would be a comparison of data with data from the EU but according to the literature the variation would not be significant.

## **Acknowledgements**

The research presented in this paper was supported by The Ministry of Science and Techno‐ logical Development of The Republic of Serbia, Grant III-44010, Title: Intelligent Systems for Software Product Development and Business Support based on Models.

## **Author details**

Slavko Arsovski, Miladin Stefanović\* , Danijela Tadić and Ivan Savović

\*Address all correspondence to: miladin@kg.ac.rs

Center for Quality, Faculty of Engineering, University of Kragujevac, Serbia

## **References**

[1] Trends and drivers of change in the food and beverage industry in Europe: Mapping report, www.eurofound.eu.int

[2] Nikhil Daxini, Use BPM to Assist in New Product Development, TRIZ Journal, http:// www.triz-journal.com/content/c081201a.asp

and tools were analyzed among 53 Serbian companies from the food industry sector. With many different problems, starting from the ranking of importance of quality goals, ranking of importance of processes or entities, up to ranking the methods or tools, there is a need for an approach that will solve these issues. All these problems could be solved by usage of the fuzzy approach. As an extension of the general ranking idea presented on the ranking and definition of goals in the production process, the fuzzy approach for evaluation of the im‐ portance of entities in supply chains in the food industry is presented. A strategic map as a strategic part of the BSC (Balanced Score Card) framework is presented as the role model for food processing companies. The special contributions of this map are the relations between entities (from all four perspectives) that are defined as the result of the research in Serbian companies. In addition, the framework of processes for the companies from the food indus‐ try was presented. Finally, the general contribution of all the presented issues, decomposi‐ tion, redesign, evaluation of quality tools and methods, fuzzy ranking and the strategic map, is based on results of research in 53 Serbian companies. The questionnaire for Serbian com‐ panies is presented as well as the results of research. A very important contribution present‐ ed is the fact that all decompositions, redesigns, modeling, simulations and calculations

A possible and very useful extension of this research would be a comparison of data with data from the EU but according to the literature the variation would not be significant.

The research presented in this paper was supported by The Ministry of Science and Techno‐ logical Development of The Republic of Serbia, Grant III-44010, Title: Intelligent Systems for

, Danijela Tadić and Ivan Savović

[1] Trends and drivers of change in the food and beverage industry in Europe: Mapping

were performed using real life data acquired from Serbian companies.

Software Product Development and Business Support based on Models.

Center for Quality, Faculty of Engineering, University of Kragujevac, Serbia

**Acknowledgements**

468 Food Industry

**Author details**

**References**

Slavko Arsovski, Miladin Stefanović\*

\*Address all correspondence to: miladin@kg.ac.rs

report, www.eurofound.eu.int


[19] Shih, H.S., Shyur, H.J., Lee, E.S., 2007. An extension of TOPSIS for group decision making. Mathematical and Computer Modelling 45 (7/8), 801-813.

**Chapter 21**

**Calculus Elements for Mechanical Presses in Oil**

Oil products industry produces edible and inedible oils. About 2/3 of total oil products are the edible oils, which are used directly in foods or in the manufacture of margarine, mayonnaise, bakery and pastry products, cooking fats, preserves etc. The remaining 1/3 of the total volume of produced oil are the technical oils, used in the production of various products, such as: de‐ tergents, paint, glycerin, fatty acids, varnish, pharmaceuticals or cosmetics (Banu, 1999).

Vegetable oils are one of the oldest classes of known chemical compounds. There are multiple references and clues on the use of these oils during Stone Age and Bronze Age (Willems, 2007). Raw material for vegetable oil industry are oilseeds, an important component of modern ag‐ riculture. Oilseeds provide easily highly nutritious human food and oil crops and their products represent one of the most important commerce commodity. Vegetable oils are a source of vitamins, calories and essential fatty acids for human diet, at a relatively low cost. After the processing of oilseeds remains the cake, or the solid part which is a valuable

Fats are found in plant and animal tissues, and in secretions of animal body glands (i.e. in milk). Fatty matter of plants is concentrated only in seeds, kernels, germs, fruits and tubers, and they reserve substances that the plant uses as energy source. Fat content in these parts

There are a wide range of raw materials for oils industry. In the vegetable reign for example are more than 100 oleaginous plants, but only 40 of them can be are used for oil expression.

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sorin-Stefan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Industry**

Valentin Vladut

**1. Introduction**

http://dx.doi.org/10.5772/53167

Biris Sorin-Stefan, Mariana Ionescu,

Gheorghe Voicu, Nicoleta Ungureanu and

Additional information is available at the end of the chapter

source of protein for animal feeds (Bargale, 1997).

of the plant is highly variable (below 5% in most plants).


## **Calculus Elements for Mechanical Presses in Oil Industry**

[19] Shih, H.S., Shyur, H.J., Lee, E.S., 2007. An extension of TOPSIS for group decision

[20] Marić, A, Arsovski, S., Mastilović, J.: Contribution to the improvement of products quality in baking industry, International Journal for Quality research, Vol.3, No. 3,

[21] D. Tadić, M. Stefanović, D. Milanović, Fuzzy Approach in Evaluation of Operations in Food Production, International Journal for Quality Research, Vol1, no 2, pp 97 -

making. Mathematical and Computer Modelling 45 (7/8), 801-813.

pp. 209-216, 2009

104, 2007.

470 Food Industry

Biris Sorin-Stefan, Mariana Ionescu, Gheorghe Voicu, Nicoleta Ungureanu and Valentin Vladut

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53167

## **1. Introduction**

Oil products industry produces edible and inedible oils. About 2/3 of total oil products are the edible oils, which are used directly in foods or in the manufacture of margarine, mayonnaise, bakery and pastry products, cooking fats, preserves etc. The remaining 1/3 of the total volume of produced oil are the technical oils, used in the production of various products, such as: de‐ tergents, paint, glycerin, fatty acids, varnish, pharmaceuticals or cosmetics (Banu, 1999).

Vegetable oils are one of the oldest classes of known chemical compounds. There are multiple references and clues on the use of these oils during Stone Age and Bronze Age (Willems, 2007).

Raw material for vegetable oil industry are oilseeds, an important component of modern ag‐ riculture. Oilseeds provide easily highly nutritious human food and oil crops and their products represent one of the most important commerce commodity. Vegetable oils are a source of vitamins, calories and essential fatty acids for human diet, at a relatively low cost. After the processing of oilseeds remains the cake, or the solid part which is a valuable source of protein for animal feeds (Bargale, 1997).

Fats are found in plant and animal tissues, and in secretions of animal body glands (i.e. in milk). Fatty matter of plants is concentrated only in seeds, kernels, germs, fruits and tubers, and they reserve substances that the plant uses as energy source. Fat content in these parts of the plant is highly variable (below 5% in most plants).

There are a wide range of raw materials for oils industry. In the vegetable reign for example are more than 100 oleaginous plants, but only 40 of them can be are used for oil expression.

© 2013 Sorin-Stefan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The other plants are unprofitable, as they have low oil content of the seeds or as they require a difficult expression process. The most important oleaginous plants are: sunflower, soya, rape, cotton, poppy, almond, sesame, nut, palm, coconut, olive, flax, castor (Banu, 1999).

Steam distillation is the method used for the extraction of 93% from the essential oils, the rest of 7% being extracted with other method (Masango, 2005). Hot steam releases the aro‐ matic molecules from the plant material, by forcing the open of the pockets in which the oils are kept in the plant material. The molecules of these volatile oils will exit from the plant material and evaporate into the steam. The steam mixed with the essential oil is furter passed through a cooling system to condense the steam, which forms a liquid from which

Calculus Elements for Mechanical Presses in Oil Industry

http://dx.doi.org/10.5772/53167

473

The mechanical process is another method for oil extraction. Mechanical expression of oil re‐ quires the application of pressure to force oil out of the oil bearing material (Ogunsina et al., 2008). Various types of machines can be used for compression: screw presses, hydraulic presses, roll presses and mills, collapsible-plate and frame-filter presses, disc mills, inter‐ locking-finger juice extractors, juice reamers (Khan & Hanna, 1983). From this variety of ma‐ chines, there are available for expression processing two of them, the hydraulic press and screw press mechanisms. Hydraulic press, based on the principle of the hydraulic ram, orig‐ inates in England and it was first patented in 1795 by Joseph Bromah (Dunning, 1953). The first screw press was developed by V.D. Anderson in the United States, in 1900. Due to the advantages it presents (continuous operation, high working capacity, run without high shocks and vibrations, working pressures which can be easily adjusted, etc.) screw presses quickly replaced hydraulic presses (Biris et al., 2009/b). In the figure below is presented a model of screw press developed by De Smet Rosedowns company, from United Kingdom.

**Figure 2.** De Smet Rosedowns screw oil press 1 – gearbox; 2 – thrust housing; 3 – worm assembly; 4 – drainage cage; 5

the essential oil and water is then separated.

– discharge end bearing; 6 – frame; 7 – feeder.

Separation of oil from oilseeds is an important processing operation. The process employed has a direct effect on the quality and quantity of protein and oil obtained from oilseeds (Bar‐ gale, 1997). The terms "expression" and "extraction" are used frequently when discussing about vegetable oil separation. Expression is the process of mechanically pressing liquid out of liquid-containing solids. Extraction is the process of separating a liquid from a liquid-sol‐ id system with the use of a solvent (Khan & Hanna, 1983). There has been some confusion in the literature between the operations of "expression" and "extraction". The latter word has been used quite loosely to designate either operation (Gurnham & Mason, 1946). This ten‐ dency has been so extensive that the distinction between the two terms appears to be disap‐ pearing from the literature. The term "extraction" is also used for mechanical oil expression (Biris et al., 2009/a).

Worldwide, for extraction of oil from seeds, fruits and nuts, four basic methods are used, as shown in the following figure.

**Figure 1.** Oil extraction methods (Sari, 2006)

Chemical extraction method is based on the use of enzymes or solvent to extract the oil from the raw material. Solvent extraction method uses a solvent (which is, in generally, a hexane, meaning a petroleum distillate) mixed with ground seeds. Seeds are grounded to maximize the contact area of the seed with the solvent; thus the oil yield is higher. After the mixing process, the obtained mixture is heated up to 100°C to separate the oil from the solvent. The other chemical extraction, enzymatic extraction, is adopted by powerfull vegetable oil com‐ panies, as the process produces many high value products. For this extraction methods, seeds are cooked and put into water. Next, enzymes are added as they digest the solid mate‐ rial. The basic difference of this type of extraction method from the solvent type is that the residual enzymes in the oil are separated by the use of a liquid-liquid centrifuge (Sari, 2006).

The extraction using high pressure carbon dioxide (i.e. supercritical fluid extraction, SFE) embodies several features of conventional solvent extraction, but it has important features of its own (Bulley et al., 1984). This extraction method consists in seeds mixing with a liquid form of high pressure carbon dioxide. Oil is dissolved in the carbon dioxide and when the pressure is released, the carbon dioxide becomes a gas, leaving the oil behind.

Steam distillation is the method used for the extraction of 93% from the essential oils, the rest of 7% being extracted with other method (Masango, 2005). Hot steam releases the aro‐ matic molecules from the plant material, by forcing the open of the pockets in which the oils are kept in the plant material. The molecules of these volatile oils will exit from the plant material and evaporate into the steam. The steam mixed with the essential oil is furter passed through a cooling system to condense the steam, which forms a liquid from which the essential oil and water is then separated.

The other plants are unprofitable, as they have low oil content of the seeds or as they require a difficult expression process. The most important oleaginous plants are: sunflower, soya, rape, cotton, poppy, almond, sesame, nut, palm, coconut, olive, flax, castor (Banu, 1999).

Separation of oil from oilseeds is an important processing operation. The process employed has a direct effect on the quality and quantity of protein and oil obtained from oilseeds (Bar‐ gale, 1997). The terms "expression" and "extraction" are used frequently when discussing about vegetable oil separation. Expression is the process of mechanically pressing liquid out of liquid-containing solids. Extraction is the process of separating a liquid from a liquid-sol‐ id system with the use of a solvent (Khan & Hanna, 1983). There has been some confusion in the literature between the operations of "expression" and "extraction". The latter word has been used quite loosely to designate either operation (Gurnham & Mason, 1946). This ten‐ dency has been so extensive that the distinction between the two terms appears to be disap‐ pearing from the literature. The term "extraction" is also used for mechanical oil expression

Worldwide, for extraction of oil from seeds, fruits and nuts, four basic methods are used, as

Chemical extraction method is based on the use of enzymes or solvent to extract the oil from the raw material. Solvent extraction method uses a solvent (which is, in generally, a hexane, meaning a petroleum distillate) mixed with ground seeds. Seeds are grounded to maximize the contact area of the seed with the solvent; thus the oil yield is higher. After the mixing process, the obtained mixture is heated up to 100°C to separate the oil from the solvent. The other chemical extraction, enzymatic extraction, is adopted by powerfull vegetable oil com‐ panies, as the process produces many high value products. For this extraction methods, seeds are cooked and put into water. Next, enzymes are added as they digest the solid mate‐ rial. The basic difference of this type of extraction method from the solvent type is that the residual enzymes in the oil are separated by the use of a liquid-liquid centrifuge (Sari, 2006). The extraction using high pressure carbon dioxide (i.e. supercritical fluid extraction, SFE) embodies several features of conventional solvent extraction, but it has important features of its own (Bulley et al., 1984). This extraction method consists in seeds mixing with a liquid form of high pressure carbon dioxide. Oil is dissolved in the carbon dioxide and when the

pressure is released, the carbon dioxide becomes a gas, leaving the oil behind.

(Biris et al., 2009/a).

472 Food Industry

shown in the following figure.

**Figure 1.** Oil extraction methods (Sari, 2006)

The mechanical process is another method for oil extraction. Mechanical expression of oil re‐ quires the application of pressure to force oil out of the oil bearing material (Ogunsina et al., 2008). Various types of machines can be used for compression: screw presses, hydraulic presses, roll presses and mills, collapsible-plate and frame-filter presses, disc mills, inter‐ locking-finger juice extractors, juice reamers (Khan & Hanna, 1983). From this variety of ma‐ chines, there are available for expression processing two of them, the hydraulic press and screw press mechanisms. Hydraulic press, based on the principle of the hydraulic ram, orig‐ inates in England and it was first patented in 1795 by Joseph Bromah (Dunning, 1953). The first screw press was developed by V.D. Anderson in the United States, in 1900. Due to the advantages it presents (continuous operation, high working capacity, run without high shocks and vibrations, working pressures which can be easily adjusted, etc.) screw presses quickly replaced hydraulic presses (Biris et al., 2009/b). In the figure below is presented a model of screw press developed by De Smet Rosedowns company, from United Kingdom.

**Figure 2.** De Smet Rosedowns screw oil press 1 – gearbox; 2 – thrust housing; 3 – worm assembly; 4 – drainage cage; 5 – discharge end bearing; 6 – frame; 7 – feeder.

Mechanical pressing and solvent extraction are the most commonly used methods for com‐ mercial oil extraction. Oils obtained by mechanical pressing has high quality, but it can be recovered up to 90-95% of available oil. Solvent extraction has the advantage of the high yield that can be obtained (up to 99%), and as for disadvantages, oil quality is lower (Karaj & Müller, 2009). This quality reduction is produced by the extensive solvent recovery proc‐ esses that are necessary and the fact that the solvent co-extracts undesired components from the cell walls (Willems et al., 2008).

Pressing technology of oleaginous material meal occurs under the influence of compression forces in mechanical presses. First, the oil from particles surface is separated – retained by surface forces of the molecular field, by the channels formed between particles. For a certain pressure, deformation and strong compression of particles begin, producing the elimination of oil from the capilars of particles. At a certain point, the space between particles gets so small that the oil film is subjected to the retaining forces exerted by particles surfaces, the oil can not be removed, the particle breaks in several places, the particle surfaces are in contact, and the briquetting begins, namely the forming of broken (cakes).

Increasing the pressure on meal particles must be done gradually, as for the harsh increase, fine meal particles will block the outler of oil from the capilaries, thus reducing the general pressing yield.

Pressing process cand be assimilated to the process of capillary filtration (fluid flow throuth capillary), expressed by the equation:

$$V = \frac{\pi \cdot p \cdot d \cdot t}{128 \cdot \eta \cdot l} \left[ \text{m}^3 \right] \tag{1}$$

Pressing time can be determined as the sum of the pressing times in each section (compres‐

<sup>=</sup> å (2)

Calculus Elements for Mechanical Presses in Oil Industry

http://dx.doi.org/10.5772/53167

475

<sup>=</sup> - (3)

]; *cs* – pressing degree of meal in

/s]; *βs* – correction

1

where: *n* is the number of pressing stages, and the pressing time in a certain section is given

(1 ) *ls s*

coefficient related to the quantity of meal removed from the press with the oil, until the ana‐

Pressing time also depends on the design and functional characteristics of the press and it ranges between 40-200 seconds. Pressing time depends on the shaft speed, cake (brocken) thickness at the press exit, and physico-chemical characteristics of the meal. Pressing time is inversely proportional to shaft speed and to cake thichness. As cake thickness is greater, the pressure in pressing chamber decreases, and pressing time decreases due to the fact that the material passes easier through the pressing chamber. At high pressure presses, by reducing the thickness of sunflower brocken from 11 mm to 4 mm the pressing time increases from 93

**•** capillars lenght *l* can be lowered by advanced destruction of cellular structure by grind‐ ing and, partially, during roasting, as well as by the reduction of passing distance of oil to the outlet hole from the pressing chamber (material layer in the pressing chamber must

In the technological diagram of pressing followed by solvent extraction, the presses can be

**•** for moderate preliminary pressing, which ensure the separation of 75-80 % oil and 18-22

**•** for advanced preliminary pressing, which ensure 12-14 % remaining oil in brocken.

b

*v s V c*

*s*

the respective section ; *Qv* – volumic feed flow of the press with meal, [m3

Parameters *η*, *l* and *d* are influenced by the preparation oparations of meal thus:

**•** oil viscosity *η* decreases by meal heating in roasting operation;

To obtain oil just by pressing, without solvent extraction, there are:

*Q*

*t*

where: *Vls* – volume of free space in the pressing section, [m3

*n si i t t* =

sion stage) of the press:

by:

lyzed section.

s to 106 s.

have small thickness).

% remaining oil in brocken;

of various types:

where: *V* – volume of separated liquid (passing through capillaries), [m3]; *p* – applied pres‐ sure, [N/m2 ]; *d* – diameter of cappilary channel, [m]; *η* - dinamic viscosity of oil, [Pa s]; *l* – lenght of capillary channel which must be passed by the separated oil, [m]; *t* – time of ap‐ plied pressure, [s].

From the above equation it results that the process of oil separation can be positively influ‐ enced if the values of *p*, *d* and *t* are increasing and if values of *l* and η will decrease.

Pressure *p* in mechanical presses is created by an helical conveyer (worm) which rotates in a closed space (pressing chamber). Gradual increase of pressure is done by the decrease of free volume of pressing chamber from one stage to another (by increasing the shaft diameter and decreasing the chamber diameter) and by reducing the pitch of worm. Pressing force is influenced by the resistance at the exit of material from the pressing chamber, as the amteri‐ al is forced to pass through a space with variable section.

Pressing time *t* must be high enough to allow the proper oil flow. A prolongation of the pressing time does not lead to significant increase of oil extraction efficiency, but leads to the sensitive decrease of press productivity.

Pressing time can be determined as the sum of the pressing times in each section (compres‐ sion stage) of the press:

Mechanical pressing and solvent extraction are the most commonly used methods for com‐ mercial oil extraction. Oils obtained by mechanical pressing has high quality, but it can be recovered up to 90-95% of available oil. Solvent extraction has the advantage of the high yield that can be obtained (up to 99%), and as for disadvantages, oil quality is lower (Karaj & Müller, 2009). This quality reduction is produced by the extensive solvent recovery proc‐ esses that are necessary and the fact that the solvent co-extracts undesired components from

Pressing technology of oleaginous material meal occurs under the influence of compression forces in mechanical presses. First, the oil from particles surface is separated – retained by surface forces of the molecular field, by the channels formed between particles. For a certain pressure, deformation and strong compression of particles begin, producing the elimination of oil from the capilars of particles. At a certain point, the space between particles gets so small that the oil film is subjected to the retaining forces exerted by particles surfaces, the oil can not be removed, the particle breaks in several places, the particle surfaces are in contact,

Increasing the pressure on meal particles must be done gradually, as for the harsh increase, fine meal particles will block the outler of oil from the capilaries, thus reducing the general

Pressing process cand be assimilated to the process of capillary filtration (fluid flow throuth

where: *V* – volume of separated liquid (passing through capillaries), [m3]; *p* – applied pres‐

lenght of capillary channel which must be passed by the separated oil, [m]; *t* – time of ap‐

From the above equation it results that the process of oil separation can be positively influ‐

Pressure *p* in mechanical presses is created by an helical conveyer (worm) which rotates in a closed space (pressing chamber). Gradual increase of pressure is done by the decrease of free volume of pressing chamber from one stage to another (by increasing the shaft diameter and decreasing the chamber diameter) and by reducing the pitch of worm. Pressing force is influenced by the resistance at the exit of material from the pressing chamber, as the amteri‐

Pressing time *t* must be high enough to allow the proper oil flow. A prolongation of the pressing time does not lead to significant increase of oil extraction efficiency, but leads to the

enced if the values of *p*, *d* and *t* are increasing and if values of *l* and η will decrease.

al is forced to pass through a space with variable section.

sensitive decrease of press productivity.

128 <sup>m</sup> *pdt <sup>V</sup> l*

h

<sup>=</sup> é ù

p

3

]; *d* – diameter of cappilary channel, [m]; *η* - dinamic viscosity of oil, [Pa s]; *l* –

ë û (1)

and the briquetting begins, namely the forming of broken (cakes).

the cell walls (Willems et al., 2008).

capillary), expressed by the equation:

pressing yield.

474 Food Industry

sure, [N/m2

plied pressure, [s].

$$t = \sum\_{i=1}^{n} t\_{si} \tag{2}$$

where: *n* is the number of pressing stages, and the pressing time in a certain section is given by:

$$t\_s = \frac{V\_{ls} \cdot c\_s}{Q\_v \cdot (1 - \beta\_s)}\tag{3}$$

where: *Vls* – volume of free space in the pressing section, [m3 ]; *cs* – pressing degree of meal in the respective section ; *Qv* – volumic feed flow of the press with meal, [m3 /s]; *βs* – correction coefficient related to the quantity of meal removed from the press with the oil, until the ana‐ lyzed section.

Pressing time also depends on the design and functional characteristics of the press and it ranges between 40-200 seconds. Pressing time depends on the shaft speed, cake (brocken) thickness at the press exit, and physico-chemical characteristics of the meal. Pressing time is inversely proportional to shaft speed and to cake thichness. As cake thickness is greater, the pressure in pressing chamber decreases, and pressing time decreases due to the fact that the material passes easier through the pressing chamber. At high pressure presses, by reducing the thickness of sunflower brocken from 11 mm to 4 mm the pressing time increases from 93 s to 106 s.

Parameters *η*, *l* and *d* are influenced by the preparation oparations of meal thus:


In the technological diagram of pressing followed by solvent extraction, the presses can be of various types:


To obtain oil just by pressing, without solvent extraction, there are:


Depending on the press type, pressure applied on the meal gets to 250-280 daN/cm2 – for preliminary pressing, respectively 400-2000 daN/cm2 – for final or single pressing.

In Romanian oil factories, processing oleaginous seeds with high oil content is done after the diagram of pressing-extraction, so by using preliminary pressing equipments (moderate or advanced).

**Figure 4.** Volume reduction of the compressed material (Venter et al., 2007)

tional parameters on the pressing process.

the material through the slot at the end of the pressing chamber.

Thus, reduction of the total void space ocurrs, causing oil elimination (Sivalla et al., 1991). Value of the applied pressure at the point that oil leaves the interparticle voids is viewed as the oil point pressure, namely the minimum pressure that must be applied before oil expres‐ sion begins. Applied pressures below this point are regarded as effort required to mobilize

Calculus Elements for Mechanical Presses in Oil Industry

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477

The general theoretical description of expression is based on consolidation theories original‐ ly developed for soil mechanics (Terzaghi, 1954). There are several studies on the modelling of oilseed expression, resulting in the development of empirical models, Terzaghi-type mod‐ els and models mased on the nature of the cell structure of the oilseeds (Venter, 2007).

Processes and phenomena that occur during the pressing process of oleaginous materials are very complex.This paper contains a theoretical model on the power necessary to operate an oil press. The necessary components to power the press are: the power required for mate‐ rial transport along the pressing chamber, the power required to press the oleaginous mate‐ rial, the power required to overcome the friction between the screw spire and the material, the power required to push the material from the press through the exhaust cylinder head. The paper presents some diagrams showing the influence of various constructive and func‐

Theoretical elements of a functional calculus, and of the power necessary to operate the press are rather poor and based especially on simple formulas containing some correction coefficients, whose value is empirically obtained from experiments. This is due to the com‐ plexity of the processes and phenomena taking place during pressing, such as: material transport, proper pressing, overcoming the frictions between auger and material, pushing

Mechanical work for material pressing it results from the expression of the equivalent stress, which occurs in the pressing chamber after applying on the cross surface of the pressing

oil from the seed cells to the surface (Sukumaran & Singh, 1989; Mrema, 1979).

**Figure 3.** The operating principle of the continuous mechanical press 1 –front plate; 2 –back plate; 3 –clamping col‐ umns; 4 –screw; 5 –cylindrical strainer (barrel); 6 –tapered strainer (barrel); 7 –tapered exhaust end for pressure adjust‐ ment of pressing chamber; 8 –feed area of meal; 9 – evacuation area of cake from the pressing chamber.

Mechanical oil expression involves the release of oil from the seed interior into the interparti‐ cle voids on application of pressure. Filling of the interparticle voids leads to a buildup of pore pressure, thereby to the development of pressure gradient in the voids. As a result, oil flows through the porous medium and is finally expressed through the porous retaining envelope (Ajibola et al., 2002). The efficiency of expression is influenced by: the porosity of the cake, yield stress of the solid phase, the compressive force applied and viscosity of the expressed liq‐ uid (Clifford, 1973). The pressing process has been studied by several authors and they have found the following parameters influencing oil expression: applied pressure, moisture con‐ tent, heating temperature and heating time, particle size (Adeeko & Ajibola, 1990; Khan & Hanna, 1983). Thus, increasing parameters such as heating temperature, heating time and ap‐ plied pressure while reducing oilseed moisture to a certain degree will result in the increase of oil yield. A significant influence on oil yield has the postheating moisture content of some oil‐ seeds (Ajibola et al., 1993). Effects of oilseeds heat treatment are: rupture of the oil bearing cells of the seed, coagulate the protein in the meal, adjust the moisture level of the meal to optimum level for oil expression. Lower the viscosity and increase the fluidity of the oil to be expelled and destroy mould and bacteria thereby facilitating oil expression from material (Adeeko & Ajibola, 1990). Optimum heating temperature for oilseeds is found in the range of 90-110°C for an average retention time of 20 minutes (FAO, 1989).

Oil expression is accompanied by compression and consolidation process brought about by the reduction in the volume of the compressed material (figure 4).

**Figure 4.** Volume reduction of the compressed material (Venter et al., 2007)

**•** presses with a single pressing stage, mechanical presses for final pressing, which realize

**•** presses with two pressing stages, the first is used for moderate pressing, and the second

In Romanian oil factories, processing oleaginous seeds with high oil content is done after the diagram of pressing-extraction, so by using preliminary pressing equipments (moderate or

**Figure 3.** The operating principle of the continuous mechanical press 1 –front plate; 2 –back plate; 3 –clamping col‐ umns; 4 –screw; 5 –cylindrical strainer (barrel); 6 –tapered strainer (barrel); 7 –tapered exhaust end for pressure adjust‐

Mechanical oil expression involves the release of oil from the seed interior into the interparti‐ cle voids on application of pressure. Filling of the interparticle voids leads to a buildup of pore pressure, thereby to the development of pressure gradient in the voids. As a result, oil flows through the porous medium and is finally expressed through the porous retaining envelope (Ajibola et al., 2002). The efficiency of expression is influenced by: the porosity of the cake, yield stress of the solid phase, the compressive force applied and viscosity of the expressed liq‐ uid (Clifford, 1973). The pressing process has been studied by several authors and they have found the following parameters influencing oil expression: applied pressure, moisture con‐ tent, heating temperature and heating time, particle size (Adeeko & Ajibola, 1990; Khan & Hanna, 1983). Thus, increasing parameters such as heating temperature, heating time and ap‐ plied pressure while reducing oilseed moisture to a certain degree will result in the increase of oil yield. A significant influence on oil yield has the postheating moisture content of some oil‐ seeds (Ajibola et al., 1993). Effects of oilseeds heat treatment are: rupture of the oil bearing cells of the seed, coagulate the protein in the meal, adjust the moisture level of the meal to optimum level for oil expression. Lower the viscosity and increase the fluidity of the oil to be expelled and destroy mould and bacteria thereby facilitating oil expression from material (Adeeko & Ajibola, 1990). Optimum heating temperature for oilseeds is found in the range of 90-110°C for

Oil expression is accompanied by compression and consolidation process brought about by

ment of pressing chamber; 8 –feed area of meal; 9 – evacuation area of cake from the pressing chamber.

– for final or single pressing.

– for

Depending on the press type, pressure applied on the meal gets to 250-280 daN/cm2

separation with maximum 3-6 % remaining oil in brocken;

preliminary pressing, respectively 400-2000 daN/cm2

an average retention time of 20 minutes (FAO, 1989).

the reduction in the volume of the compressed material (figure 4).

stage for final pressing.

advanced).

476 Food Industry

Thus, reduction of the total void space ocurrs, causing oil elimination (Sivalla et al., 1991). Value of the applied pressure at the point that oil leaves the interparticle voids is viewed as the oil point pressure, namely the minimum pressure that must be applied before oil expres‐ sion begins. Applied pressures below this point are regarded as effort required to mobilize oil from the seed cells to the surface (Sukumaran & Singh, 1989; Mrema, 1979).

The general theoretical description of expression is based on consolidation theories original‐ ly developed for soil mechanics (Terzaghi, 1954). There are several studies on the modelling of oilseed expression, resulting in the development of empirical models, Terzaghi-type mod‐ els and models mased on the nature of the cell structure of the oilseeds (Venter, 2007).

Processes and phenomena that occur during the pressing process of oleaginous materials are very complex.This paper contains a theoretical model on the power necessary to operate an oil press. The necessary components to power the press are: the power required for mate‐ rial transport along the pressing chamber, the power required to press the oleaginous mate‐ rial, the power required to overcome the friction between the screw spire and the material, the power required to push the material from the press through the exhaust cylinder head. The paper presents some diagrams showing the influence of various constructive and func‐ tional parameters on the pressing process.

Theoretical elements of a functional calculus, and of the power necessary to operate the press are rather poor and based especially on simple formulas containing some correction coefficients, whose value is empirically obtained from experiments. This is due to the com‐ plexity of the processes and phenomena taking place during pressing, such as: material transport, proper pressing, overcoming the frictions between auger and material, pushing the material through the slot at the end of the pressing chamber.

Mechanical work for material pressing it results from the expression of the equivalent stress, which occurs in the pressing chamber after applying on the cross surface of the pressing chamber an equivalent pressing force, so that it may be produced a reduction of the volume occupied by the material, from the initial value to the final value.

The value of pressure ratio is directly proportional to the working pressure of the press, and

where: *Vte* – is the theoretical volume of the material displaced by the auger spire during a complete rotation, in the exhaust area [cu.m.]; *n* – auger rotative speed, [rpm]; *k* – coefficient taking into account the material flowing back through the spire extremities, as well as the

The theoretical volume of the material displaced by the auger spire is calculated using the

where: *s* – the auger spire pitch [m]; *δ* – thickness of the auger spire, [m]; *D* – outer diameter

d

<sup>2</sup> <sup>32</sup> ( )( <sup>4</sup> m) *V Dd s te*

of the auger spire, [m]; *d* – inner diameter of the auger spire (of the auger shaft), [m].

= - -

é ù ë û (5)

Calculus Elements for Mechanical Presses in Oil Industry

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479

é ù ë û (6)

*n*  [rpm]

é ù ë û (7)

<sup>3</sup> (1 <sup>60</sup> m) *Q V nk v te* = e

Press volume flow rate can be evaluated by using the relation [5]:

p

Qv[m3 /h] [cu.m./h]

**Figure 6.** Variation of the press flow rate depending on the auger rotative speed and the spire pitch

results the expression of the press volume flow rate under the form (see Figure 6):

<sup>2</sup> <sup>32</sup> ( ) ( ) (1 ) <sup>m</sup> /h60 <sup>4</sup> *Q D d s nk <sup>v</sup>*

= - - d

By replacing in equation (5) the expression of the theoretical volume given by equation (6), it

 e

M

p

*s*  [m]

its variation is shown in figure 5.

incomplete feed with material, (k=0,20,35).

following equation:

Based on the mathematical model developed in this study, was found the variation of the com‐ ponent parts of the power necessary for operation in the case of a press from the oil industry.

The main data taken into consideration for modelling are: the variable auger rotational speed (n=15-40 min-1), the variable pressure inside the pressing chamber (p=50 105 -200 105 Pa), the di‐ ameter of the pressing chamber (D=200 mm), the diameter of the auger shaft (d=100 mm).

The mathematical model which was created in this paper permits the high precision deter‐ mination of the functional parameters and of the necessary power for operating the presses in food industry.

From the figures presented in this paper it can be observed that the necessary power for proper pressing is the highest, being followed by the necessary power for overcoming the frictions between the auger spire and the material subdued to pressing. The values for the power necessary to push the material through the exhaust space and for the material trans‐ port through the pressing chamber are much lower than those for the presses, being possible to even be neglected.

This paper can be useful to students undertaking batchellor studies, to professors and re‐ searchers in design and development of mechanical oil presses.

## **2. Theoretical elements**

#### **2.1. Functional calculus elements**

Pressure ratio is the reduction of the material subjected to pressing and it can be calculated using equation [4], where Vi is the initial volume of the material, [m3 ] and Vf is the final volume, [m3 ].

**Figure 5.** Variation of the pressure ratio with the pressure

The value of pressure ratio is directly proportional to the working pressure of the press, and its variation is shown in figure 5.

Press volume flow rate can be evaluated by using the relation [5]:

chamber an equivalent pressing force, so that it may be produced a reduction of the volume

Based on the mathematical model developed in this study, was found the variation of the com‐ ponent parts of the power necessary for operation in the case of a press from the oil industry.

The main data taken into consideration for modelling are: the variable auger rotational speed

The mathematical model which was created in this paper permits the high precision deter‐ mination of the functional parameters and of the necessary power for operating the presses

From the figures presented in this paper it can be observed that the necessary power for proper pressing is the highest, being followed by the necessary power for overcoming the frictions between the auger spire and the material subdued to pressing. The values for the power necessary to push the material through the exhaust space and for the material trans‐ port through the pressing chamber are much lower than those for the presses, being possible

This paper can be useful to students undertaking batchellor studies, to professors and re‐

Pressure ratio is the reduction of the material subjected to pressing and it can be calculated using

*i f i*

> e*=*e(*p*)

*V V V*

] and Vf


*p* 

is the initial volume of the material, [m3

e

e

**Figure 5.** Variation of the pressure ratio with the pressure

ameter of the pressing chamber (D=200 mm), the diameter of the auger shaft (d=100 mm).


is the final volume, [m3

].

Pa), the di‐

occupied by the material, from the initial value to the final value.

searchers in design and development of mechanical oil presses.

in food industry.

478 Food Industry

to even be neglected.

equation [4], where Vi

**2. Theoretical elements**

**2.1. Functional calculus elements**

(n=15-40 min-1), the variable pressure inside the pressing chamber (p=50 105

$$Q\_v = V\_{t^c} \cdot (1 - \varepsilon) \cdot n \cdot k \cdot 60 \quad \left[\text{m}^3\right] \tag{5}$$

where: *Vte* – is the theoretical volume of the material displaced by the auger spire during a complete rotation, in the exhaust area [cu.m.]; *n* – auger rotative speed, [rpm]; *k* – coefficient taking into account the material flowing back through the spire extremities, as well as the incomplete feed with material, (k=0,20,35).

The theoretical volume of the material displaced by the auger spire is calculated using the following equation:

$$V\_{te} = \frac{\pi}{4} \cdot (D^2 - d^2) \cdot (\text{s} - \delta) \cdot \left[\text{m}^3\right] \tag{6}$$

where: *s* – the auger spire pitch [m]; *δ* – thickness of the auger spire, [m]; *D* – outer diameter of the auger spire, [m]; *d* – inner diameter of the auger spire (of the auger shaft), [m].

M **Figure 6.** Variation of the press flow rate depending on the auger rotative speed and the spire pitch

By replacing in equation (5) the expression of the theoretical volume given by equation (6), it results the expression of the press volume flow rate under the form (see Figure 6):

$$Q\_{\upsilon} = \frac{\pi}{4} \cdot \left(D^2 - d^2\right) \cdot \left(\mathbf{s} - \boldsymbol{\delta}\right) \cdot \left(\mathbf{l} - \boldsymbol{\varepsilon}\right) \cdot \boldsymbol{n} \cdot \boldsymbol{k} \cdot \boldsymbol{\delta} \, \mathbf{0} \quad \left[\text{m}^3/\text{h}\right] \tag{7}$$

#### **2.2. Calculus of the power necessary to operate the press**

**•** *The power necessary to operate the press* can be evaluated by using the equation:

$$P\_p = \frac{P\_{tr} + P\_{prs} + P\_{fr} + P\_{cap}}{\eta\_{tm}} \quad \left[\text{kW}\right] \tag{8}$$

2 2

*D d <sup>F</sup> l g*

2 2 2 2 () ()

gp

*F v D d lgv D d lgsn <sup>P</sup>*

1000 4 1000 4 1000

4 *<sup>r</sup>*

p

*tr*

*Vi*

to the final value, *Vf*

p

**•** Necessary power for pressing the material

**Figure 7.** Variation of the material volume in the pressing process

in the pressing chamber can be written as:

s

N ( )

It results the necessary power for transporting the material along the pressing chamber:

k *<sup>r</sup>*

The mechanical work for material pressing (Lpres) results from the expression of the equiva‐ lent tension (stress) σ, which appears inside the pressing chamber as a result of applying on the cross surface (*S*) of the pressing chamber an equivalent pressing force (*Fpres*), so that it may be produced a reduction of the volume occupied by the material, from the initial value,

Pa *pres pres pres pres*

*F F lL L S Sl V VV*

, as it is also shown in figure 7. Hence, the equivalent tension (stress)

D == == é ù ë û D D - (14)

*i f*



60

 g

W

http://dx.doi.org/10.5772/53167

481

Calculus Elements for Mechanical Presses in Oil Industry

y g

where: *Ptr* – represents the necessary power to transport the material from feeding chamber to exhaust head, [kW]; *Ppres* – necessary power for pressing the material, [kW]; *Pfr* – necessary power for overcoming the frictions between the auger spire and the material, [kW]; *Pcap* – necessary power for pushing the material through the exhaust space in the press, [kW]; *ηtm* – mechanical transmission yield (output).

**•** Necessary power for material transport

Taking into account the calculus equations of the slow helical conveyors, it can be written the expression of the necessary power for proper transport of the material along the auger:

$$P\_{tr} = \frac{F\_r \cdot \upsilon}{1000} \quad \left[\text{kW}\right] \tag{9}$$

where: *Fr* – represents the resistant force to the material advancing along the press auger, [N]; *v* – mean speed by which the material moves along the press auger, [m/s].

The resistant force Fr is given, on one part, by the phenomenon of outer friction between the material and the walls of the pressing chamber, and, on the other part, by the phenomenon of outer friction of the material subdued to pressing. The value of this force can be calculat‐ ed by the expression:

$$F\_r = q \cdot l \cdot g \quad \text{[N]} \tag{10}$$

where: *g* – represents the gravity acceleration, [m/sq.s]; *q* – the linear load (mass per linear meter of material) in the press, [kg/m]; *l* – length of pressing chamber, [m].

The expression of the linear load, q, can be written:

$$q = \mathbf{S} \cdot \boldsymbol{\upmu} \cdot \boldsymbol{\upgamma} = \frac{\boldsymbol{\pi} \cdot (\boldsymbol{D}^2 - \boldsymbol{d}^2)}{4} \cdot \boldsymbol{\upmu} \cdot \boldsymbol{\upgamma} \quad \left[\text{kg/m}\right] \tag{11}$$

where: *ψ* – represents the coefficient of admission for the press section; *S* – area of the cross section of the pressing chamber, [sq.m.].

By replacing into the relation (10) it results:

Calculus Elements for Mechanical Presses in Oil Industry http://dx.doi.org/10.5772/53167 481

$$F\_r = \frac{\pi \cdot \{D^2 - d^2\}}{4} \cdot \psi \cdot \gamma \cdot l \cdot \mathbf{g} \quad \text{[N]} \tag{12}$$

It results the necessary power for transporting the material along the pressing chamber:

$$P\_{tr} = \frac{F\_r \cdot \upsilon}{1000} = \frac{\pi \cdot (D^2 - d^2) \cdot \Psi \cdot \gamma \cdot l \cdot \mathbf{g} \cdot \upsilon}{4 \cdot 1000} = \frac{\pi \cdot (D^2 - d^2) \cdot \Psi \cdot \gamma \cdot l \cdot \mathbf{g} \cdot \mathbf{s} \cdot \eta}{4 \cdot 1000 \cdot 60} \tag{13}$$

**•** Necessary power for pressing the material

**2.2. Calculus of the power necessary to operate the press**

*p*

*P*

mechanical transmission yield (output).

480 Food Industry

ed by the expression:

**•** Necessary power for material transport

**•** *The power necessary to operate the press* can be evaluated by using the equation:

*PP PP*

h

1000 *tr <sup>r</sup>* kW *F v <sup>P</sup>* <sup>=</sup> é ù

[N]; *v* – mean speed by which the material moves along the press auger, [m/s].

N *<sup>r</sup> F qlg* = é ù

meter of material) in the press, [kg/m]; *l* – length of pressing chamber, [m].

p

The expression of the linear load, q, can be written:

section of the pressing chamber, [sq.m.]. By replacing into the relation (10) it results:

y g

kW *tr pres fr cap*

where: *Ptr* – represents the necessary power to transport the material from feeding chamber to exhaust head, [kW]; *Ppres* – necessary power for pressing the material, [kW]; *Pfr* – necessary power for overcoming the frictions between the auger spire and the material, [kW]; *Pcap* – necessary power for pushing the material through the exhaust space in the press, [kW]; *ηtm* –

Taking into account the calculus equations of the slow helical conveyors, it can be written the expression of the necessary power for proper transport of the material along the auger:

where: *Fr* – represents the resistant force to the material advancing along the press auger,

The resistant force Fr is given, on one part, by the phenomenon of outer friction between the material and the walls of the pressing chamber, and, on the other part, by the phenomenon of outer friction of the material subdued to pressing. The value of this force can be calculat‐

where: *g* – represents the gravity acceleration, [m/sq.s]; *q* – the linear load (mass per linear

y g

where: *ψ* – represents the coefficient of admission for the press section; *S* – area of the cross

2 2 ( ) <sup>4</sup> kg/m *D d q S*


ë û (8)

ë û (9)

ë û (10)

ë û (11)

+ ++ <sup>=</sup> é ù

*tm*

**Figure 7.** Variation of the material volume in the pressing process

The mechanical work for material pressing (Lpres) results from the expression of the equiva‐ lent tension (stress) σ, which appears inside the pressing chamber as a result of applying on the cross surface (*S*) of the pressing chamber an equivalent pressing force (*Fpres*), so that it may be produced a reduction of the volume occupied by the material, from the initial value, *Vi* to the final value, *Vf* , as it is also shown in figure 7. Hence, the equivalent tension (stress) in the pressing chamber can be written as:

$$\sigma = \frac{F\_{\rm precs}}{S} = \frac{F\_{\rm precs} \cdot \Delta l}{S \cdot \Delta l} = \frac{L\_{\rm precs}}{\Delta V} = \frac{L\_{\rm precs}}{V\_i - V\_f} \quad \left[\text{Pa}\right] \tag{14}$$

Taking into account equation (4), it results:

$$V\_f = V\_i \cdot (1 - \varepsilon) \tag{15}$$

*yz x*

(1 2 ) *xyz*

As the materials subjected to the pressing process in the food industry also contains a cer‐ tain percentage of liquid substance (oil, must, etc.), it can be considered that the hydrostatic

3 3

Thus, it results the expression of the mechanical work necessary for pressing the material:

e

1 2 <sup>J</sup> <sup>3</sup> *pres <sup>i</sup> L pV* b

1000 1000 1000 1000

*l F v <sup>F</sup> F lL <sup>t</sup> <sup>P</sup>*

D

kW *pres pres pres pres pres*

where: *Fpres* – represents the pressing force [N]; *vpres* – the pressing speed, [m/s]; *Δt* – the time interval when the reducing of the material volume is performed from the initial value *Vi* to

Taking into account equations (21), (22) and (23), the expression of the necessary power for

kW *pres <sup>i</sup>*

 e

= = é ù ë û (24)

b

(1 2 ) 1000 60 3 1000 60

*L n p Vn <sup>P</sup>* +

D <sup>D</sup> = === é ù ë û D D

respectively, the expression of the necessary power for pressing the material:

 b

(1 2 )

b

*t t*

, [s]. The value of this time interval can be calculated depending on the ro‐

+ + + = = (20)

+ = é ù ë û (21)

<sup>60</sup> *<sup>t</sup> <sup>n</sup>* D = (23)

(22)

= = (18)

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+ + = + *p* (19)

s s bs

sss

sss*xyz p*

s

where: *β* - represents the coefficient of the side pressure.

Taking into account equation (18), it results:

pressure law remains valid, respectively:

*pres*

pressing the material is obtained:

tative speed [rpm] of the press auger, respectively:

*pres*

the final value *Vf*

and from the equations (14) and (15) is obtained the expression of the mechanical work for the material pressing:

$$L\_{\rm pres} = \sigma \cdot (V\_i - V\_f) = \sigma \cdot [V\_i - V\_i \cdot (1 - \varepsilon)] = \sigma \cdot \varepsilon \cdot V\_i \quad \text{[J]} \tag{16}$$

**Figure 8.** Elementary volume of material subdued to the pressing process

To determine the value of equivalent tension (stress) *σ*, it is considered an elementary vol‐ ume of material subjected to pressing, uniformly loaded on each section, as shown in figure 8, which, during the pressing process will move only on the longitudinal direction of the press (direction *x*). Under these conditions it can by written:

$$\begin{cases} \sigma\_y = \sigma\_z\\ \sigma\_x = p \end{cases} \tag{17}$$

where: *p* [Pa] - represents the pressure performed by the auger, which is exerted on the ma‐ terial.

It is considered that the tensions (stresses) on the direction *y* and *z* occur due to the pressure oriented to the direction of material displacement, respectively:

$$
\sigma\_y = \sigma\_z = \beta \cdot \sigma\_x \tag{18}
$$

where: *β* - represents the coefficient of the side pressure.

Taking into account equation (18), it results:

Taking into account equation (4), it results:

s

**Figure 8.** Elementary volume of material subdued to the pressing process

press (direction *x*). Under these conditions it can by written:

oriented to the direction of material displacement, respectively:

the material pressing:

482 Food Industry

terial.

(1 ) *V V f i* = -

( ) [ (1 ) J] *pres i f i i <sup>i</sup> L VV VV* = - = - - =

 s e

e se

and from the equations (14) and (15) is obtained the expression of the mechanical work for

To determine the value of equivalent tension (stress) *σ*, it is considered an elementary vol‐ ume of material subjected to pressing, uniformly loaded on each section, as shown in figure 8, which, during the pressing process will move only on the longitudinal direction of the

> *y z <sup>x</sup> p*

where: *p* [Pa] - represents the pressure performed by the auger, which is exerted on the ma‐

It is considered that the tensions (stresses) on the direction *y* and *z* occur due to the pressure

s s

ìï = í ï = î

s

(15)

ë û (16)

(17)

*V* é ù

$$
\sigma\_x + \sigma\_y + \sigma\_z = p \cdot (1 + \mathcal{Z} \cdot \beta) \tag{19}
$$

As the materials subjected to the pressing process in the food industry also contains a cer‐ tain percentage of liquid substance (oil, must, etc.), it can be considered that the hydrostatic pressure law remains valid, respectively:

$$
\sigma = \frac{\sigma\_x + \sigma\_y + \sigma\_z}{3} = \frac{p \cdot (1 + 2 \cdot \beta)}{3} \tag{20}
$$

Thus, it results the expression of the mechanical work necessary for pressing the material:

$$L\_{\rm pres} = \frac{1 + 2 \cdot \beta}{3} \cdot p \cdot s \cdot V\_i \quad \text{[J]} \tag{21}$$

respectively, the expression of the necessary power for pressing the material:

$$P\_{\rm pres} = \frac{F\_{\rm pres} \cdot \upsilon\_{\rm pres}}{1000} = \frac{F\_{\rm pres} \cdot \frac{\Delta l}{\Delta t}}{1000} = \frac{F\_{\rm pres} \cdot \Delta l}{1000 \cdot \Delta t} = \frac{L\_{\rm pres}}{1000 \cdot \Delta t} \quad \left[\text{kW}\right] \tag{22}$$

where: *Fpres* – represents the pressing force [N]; *vpres* – the pressing speed, [m/s]; *Δt* – the time interval when the reducing of the material volume is performed from the initial value *Vi* to the final value *Vf* , [s]. The value of this time interval can be calculated depending on the ro‐ tative speed [rpm] of the press auger, respectively:

$$
\Delta t = \frac{60}{\pi} \tag{23}
$$

Taking into account equations (21), (22) and (23), the expression of the necessary power for pressing the material is obtained:

$$P\_{\rm pres} = \frac{L\_{\rm pres} \cdot n}{1000 \cdot 60} = \frac{(1 + 2 \cdot \beta) \cdot p \cdot \varepsilon \cdot V\_i \cdot n}{3 \cdot 1000 \cdot 60} \tag{24} \tag{24}$$

In reality, for presses in food industry, the value of the pressure, *p*, is not kept constant along the auger, having a variation which can be as that seen in figure 9.

The value of *the friction force* which occurs on the surface of the elementary ring is calculated

(26)

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(27)

3

*r R*

kW

3

1

(30)

(31)

2 *<sup>f</sup> dF dN p r dr* = =

The expression of the friction torque (moment) at the surface of the elementary ring is:

<sup>2</sup> <sup>2</sup> *f f dM r dF p r dr* = = m

22 2

 mp

3 *<sup>f</sup> R R M p* p m

9550 3 9550

**•** Power necessary for pushing the material through the exhaust space

*l*

D

*M p r dr p r dr p <sup>R</sup>* = = =

 p

2 2 2

ò ò (28)

p m


which, by integration, for the whole active cross surface of the auger spire, suitable to a

3 3 2 1 Nm2

It results the expression of the necessary power for overcoming the friction between the aug‐

2 1 2

= = é ù ë û

*M n pR R n <sup>P</sup>* p m

1000 1000 1000 1000 1000 60

D <sup>D</sup> === = = é ù ë û D D

where: *Fc* – resistant force to material pushing through, the head of the pressing chamber, [N]; *vcap* – the material speed through the head of the pressing chamber, [m/s]; *lc* – length of

*<sup>c</sup> <sup>c</sup> c cap cc c c*

*F v <sup>F</sup> F l L Ln <sup>t</sup> <sup>P</sup>*

kW *<sup>f</sup>*

For pushing the material through the exhaust space from the end of the pressing chamber

*t t*

( ) 3 3

 mp

m

2 2

*R R*

1 1

*R R*

 p

length equal to a pitch, *s*, leads to the equation:

*fr*

m

*f*

er spire and the material:

the power consumed is given by:

*cap*

exhaust canal, [m].

respectively:

by the following equation:

**Figure 9.** Pressure variation along the pressing chamber

**•** Power necessary to overcome the frictions between the auger spire and material

To calculate the necessary power for overcoming the frictions between the auger spire and the material, first it must be calculated the friction torque (moment), which occurs on the spire surface when it comes into contact with the material. For the calculus of this friction torque (moment) it is first taken into consideration an elementary ring, *dr*, situated on the auger spire on the radius *r* (Figure 10) and for the auger length suitable to a pitch, *s* it is de‐ termined *the normal force* exerted on the elementary ring:

$$dN = p \cdot dS = p \cdot \mathcal{D} \cdot \pi \cdot r \cdot dr \tag{25}$$

**Figure 10.** Elementary ring on the auger spire

The value of *the friction force* which occurs on the surface of the elementary ring is calculated by the following equation:

$$dF\_f = \mu \cdot d\mathbf{N} = \mu \cdot \mathbf{p} \cdot \mathbf{2} \cdot \boldsymbol{\pi} \cdot \mathbf{r} \cdot d\mathbf{r} \tag{26}$$

The expression of the friction torque (moment) at the surface of the elementary ring is:

$$\text{dM}\_f = r \cdot \text{dF}\_f = \mu \cdot p \cdot 2 \cdot \pi \cdot r^2 \cdot dr \tag{27}$$

which, by integration, for the whole active cross surface of the auger spire, suitable to a length equal to a pitch, *s*, leads to the equation:

$$\mathcal{M}\_f = \bigwedge\_{R\_1}^{R\_2} \mu \cdot p \cdot 2 \cdot \pi \cdot r^2 \cdot dr = 2 \cdot \mu \cdot p \cdot \pi \cdot \int\_{R\_1}^{R\_2} r^2 \cdot dr = 2 \cdot \pi \cdot \mu \cdot p \cdot \frac{r^3}{3} \Big|\_{R\_1}^{R\_2} \tag{28}$$

respectively:

In reality, for presses in food industry, the value of the pressure, *p*, is not kept constant along

*p=p*(*l*)

*p=p*(*l*)

To calculate the necessary power for overcoming the frictions between the auger spire and the material, first it must be calculated the friction torque (moment), which occurs on the spire surface when it comes into contact with the material. For the calculus of this friction torque (moment) it is first taken into consideration an elementary ring, *dr*, situated on the auger spire on the radius *r* (Figure 10) and for the auger length suitable to a pitch, *s* it is de‐

**•** Power necessary to overcome the frictions between the auger spire and material

*dN p dS p r dr* = = 2

p

*l* 

*l* 

(25)

the auger, having a variation which can be as that seen in figure 9.

*p* 

*p* 

termined *the normal force* exerted on the elementary ring:

**Figure 9.** Pressure variation along the pressing chamber

484 Food Industry

**Figure 10.** Elementary ring on the auger spire

$$\mathbf{M}\_f = 2 \cdot \boldsymbol{\pi} \cdot \boldsymbol{\mu} \cdot \boldsymbol{p} \cdot \frac{\boldsymbol{R}\_2^3 - \boldsymbol{R}\_1^3}{3} \quad \left[\text{Nm}\right] \tag{29}$$

It results the expression of the necessary power for overcoming the friction between the aug‐ er spire and the material:

$$P\_{fr} = \frac{M\_f \cdot n}{9550} = \frac{2 \cdot \pi \cdot \mu \cdot p \cdot \left(R\_2^3 - R\_1^3\right) \cdot n}{3 \cdot 9550} \quad \left[\text{kW}\right] \tag{30}$$

**•** Power necessary for pushing the material through the exhaust space

For pushing the material through the exhaust space from the end of the pressing chamber the power consumed is given by:

$$P\_{cap} = \frac{F\_c \cdot \upsilon\_{cap}}{1000} = \frac{F\_c \cdot \frac{\Delta l\_c}{\Delta t}}{1000} = \frac{F\_c \cdot \Delta l\_c}{1000 \cdot \Delta t} = \frac{L\_c}{1000 \cdot \Delta t} = \frac{L\_c \cdot n}{1000 \cdot 60} \quad \left[\text{kW}\right] \tag{31}$$

where: *Fc* – resistant force to material pushing through, the head of the pressing chamber, [N]; *vcap* – the material speed through the head of the pressing chamber, [m/s]; *lc* – length of exhaust canal, [m].

2 <sup>J</sup> <sup>4</sup> *c*

= = = é ù ë û (32)

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487

<sup>=</sup> é ù ë û (33)

200 105

Pa),

p

k

W *c c*

Using the mathematical model developed in this study, figure 12 shows the variation of the component parts of the power necessary for operation of a press from the oil industry.

The main data taken into consideration for modelling are: the variable auger rotational

the diameter of the pressing chamber (*D*=200 mm), the diameter of the auger shaft (*d*=100

The mathematical model which was created in this paper allows the high precision determi‐ nation of the functional parameters and of the necessary power for operating the presses in

In figure 12 it can by observed that the necessary power for the proper pressing *Ppres* is the highest, being followed by the necessary power to overcome the frictions between the auger spire and the material subdued to pressing *P*fr. The values for the power required to push the material through the exhaust space Pcap and for the material transport through the press‐ ing chamber Ptr are much lower than those for the presses Ppres and Pfr, so it is possible to

, Gheorghe Voicu1

, Nicoleta Ungureanu1

and

speed (*n*=1540 min-1), the variable pressure inside the pressing chamber (*p*=50 105

*c cc cc c <sup>d</sup> L F l pA l p l*

2

0 60

4 100

*p dln <sup>P</sup>* p

It results the expression for the calculus of power *Pcap*:

**3. Application**

**4. Conclusions**

food industry.

neglect them.

**Author details**

Biris Sorin-Stefan1

2 INMA Bucharest, Romania

Valentin Vladut2

, Mariana Ionescu1

1 Politehnica University of Bucharest, Romania

mm).

*cap*

**Figure 11.** End of the pressing chamber

**Figure 12.** Power variation depending on pressure and auger rotational speed

The necessary mechanical work for pushing the material through the exhaust space (Fig. 11) for the end of the pressing chamber *Lc* is calculated using the following equation:

$$L\_c = F\_c \cdot l\_c = p \cdot A\_c \cdot l\_c = p \cdot \frac{\pi \cdot d\_c^2}{4} \cdot l\_c \quad \text{[J]} \tag{32}$$

It results the expression for the calculus of power *Pcap*:

$$P\_{cap} = \frac{p \cdot \pi \cdot d\_c^2 \cdot l\_c \cdot n}{4 \cdot 1000 \cdot 60} \quad \text{[kW]} \tag{33}$$

## **3. Application**

**Figure 11.** End of the pressing chamber

486 Food Industry

**Figure 12.** Power variation depending on pressure and auger rotational speed

The necessary mechanical work for pushing the material through the exhaust space (Fig. 11)

for the end of the pressing chamber *Lc* is calculated using the following equation:

Using the mathematical model developed in this study, figure 12 shows the variation of the component parts of the power necessary for operation of a press from the oil industry.

The main data taken into consideration for modelling are: the variable auger rotational speed (*n*=1540 min-1), the variable pressure inside the pressing chamber (*p*=50 105 200 105 Pa), the diameter of the pressing chamber (*D*=200 mm), the diameter of the auger shaft (*d*=100 mm).

## **4. Conclusions**

The mathematical model which was created in this paper allows the high precision determi‐ nation of the functional parameters and of the necessary power for operating the presses in food industry.

In figure 12 it can by observed that the necessary power for the proper pressing *Ppres* is the highest, being followed by the necessary power to overcome the frictions between the auger spire and the material subdued to pressing *P*fr. The values for the power required to push the material through the exhaust space Pcap and for the material transport through the press‐ ing chamber Ptr are much lower than those for the presses Ppres and Pfr, so it is possible to neglect them.

### **Author details**

Biris Sorin-Stefan1 , Mariana Ionescu1 , Gheorghe Voicu1 , Nicoleta Ungureanu1 and Valentin Vladut2

1 Politehnica University of Bucharest, Romania

2 INMA Bucharest, Romania

## **References**

[1] Adeeko, K. A., & Ajibola, O. O. (1990). Processing factors affecting yield and quality of mechanically expressed groundnut oil, *Journal of Agricultural Engineering Research*, 45, pg. , 31-43.

[16] Mrema, G. C. (1979). Mechanisms of mechanical oil expression from rapeseed and

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[17] Ogunsina, B. S., Owolarafe, O. K., & Olatunde, G. A. (2008). Oil point pressure of cashew (Anacardium occidentale) kernels, *International Agrophysics*, 22, pg. , 53-59.

[18] Sari, P. (2006). Preliminary design and construction of a prototype canola seed oil ex‐ traction machine, Doctoral Thesis, Middle East Technical University, Ankara, Tur‐

[19] Sivalla, K., Bhole, N. C., & Mujherjee, R. K. (1991). Effects of moisture on rice bran oil

[20] Sukumaran, C. R., & Singh, B. (1989). Compression of a bed of rapeseeds- the oil

[22] Venter, M. J., Kuipers, N. J. M., & de Haan, A. B. (2007). Modelling and experimental evaluation of high-pressure expressionn of cocoa nibs, *Journal of Food Engineering*, 80,

[23] Willems, P. (2007). Gas Assisted Mechanical Expression Of Oilseeds, Doctoral Thesis,

[24] Willems, P., Kuipers, N. J. M., & De Haan, A. B. (2008). Hydraulic pressing of oil‐ seeds: Experimental determination and modelling of yield and pressing rates, *Journal*

expression, *Journal of Agricultural Engineering Research*, 50, pg. , 81-91.

point, *Journal of Agricultural Engineering Research*, 42, pg. , 77-84.

[21] Terzaghi, K. (1954). Theoretische Bodenmechanik, Berlin: Springer.

cashew, Doctoral Thesis, National University of Ireland, Dublin.

key.

pg. , 1157-1170.

University of Twente, Netherland.

*of Food Engineering*, 89, pg. , 8-16.


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45, pg. , 31-43.

Tehnică, București.

the oil presses, *Proceeding of the 37th*

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[2] Ajibola, O. O., Okunade, D. A., & Owolarafe, O. K. (2002). Oil point pressure of soy‐

[3] Ajibola, O. O., Owolarafe, O. K., Fasina, O. O., & Adeeko, K. A. (1993). Expression of

[4] Banu, C. (1999). Manualul inginerului din industria alimentară, vol. I și II, Editura

[5] Bargale, P. C. (1997). Mechanical oil expression from selected oilseeds under uniaxial

[6] Biriş, S.Şt., Manea, M., Maican, E., Ungureanu, N., & Toma, L. M. (2009). a), Studies regarding the use of Finite Elements Method for the working process modelling of

[7] Biris, S., St, , Manea, M., Paraschiv, G., Vlăduţ, V., & Bungescu, S. (2009). b), Power calculus elements for oil presses, *Proceeding of the 37th International Symposium "Ac‐ tual Tasks on Agricultural Engineering",* Croaţia, Opatija, february, pg. 475-484., 10-13.

[8] Bulley, N. R., Fattori, M., Meisen, A., & Moyls, L. (1984). Supercritical fluid extraction of vegetable oilseeds, *Journal of the American Oil Chemists' Society*, 61, pg. , 1362-1363.

[9] Clifford, W. C. (1973). Theory of filtration, *Chemical Engineers' Handbook*, 5, pg. , 19-69.

[10] Dunning, J. W. (1953). History and latest developments in expeller and screw press operation on cottonseed, *Journal of the American Oil Chemists' Society*, 30, pg., 486-492.

[11] FAO- Food and Agriculture Organization(1989). Food Composition Table for Use in Africa. US Department of Health, Education and Welfare Press, Washington D.C.

[12] Gurnham, F. C., & Masson, H. J. (1946). Expression of liquids from fibrous materials,

[13] Karaj, S., & Müller, J. (2009). Optimization of mechanical extraction of Jatropha cur‐

[14] Khan, L. M., & Hanna, M. A. (1983). Expression of oil from oilseeds- A review, *Jour‐*

[15] Masango, P. (2005). Cleaner production of essential oils by steam distillation, *Journal*

*Industrial and Engineering Chemistry*, 38, pg. , 1309-1315.

*nal of Agricultural Engineering Research*, 28, pg. , 495-503.

cas seeds, Landtechnik 64, pg. (3), 164-167.

*of Cleaner Production,* 13, pg. , 833-839.

 *International Symposium "Actual Tasks on Agricul‐*

oil from sesame seeds, *Canadian Agricultural Engineering*, 35, pg. , 83-88.

compression, Doctoral Thesis, University of Saskatchewan, Canada.

*tural Engineering",* Croaţia, Opatija, february, pg. 485-496., 10-13.

bean, *Journal of Food Process Engineering*, 25, pg. , 407-416.


**Chapter 22**

**Gastrointestinal Immunoregulation and**

Additional information is available at the end of the chapter

lian physiological and cellular environment.

**1.2. Food packaging**

MaryAnn Principato

**1. Introduction**

http://dx.doi.org/10.5772/53287

**the Challenges of Nanotechnology in Foods**

**1.1. Nanoparticles: Physicochemical characteristics and applications in foods**

Nanoparticles are elemental three dimensional structures that are typically between 1-100 nanometers (nm) in size that exhibit unique physiochemical characteristics that provide the basis for their utilization, and present unique challenges associated with the development of new applications [1, 2]. Because of their size, nanoparticles provide the opportunity to inter‐ act with human physiology at the subcellular level, affording many potential uses in nu‐ trient and drug delivery, vaccination therapies, and tissue repair. Specific physiologic applications can be achieved by chemical modification of the nanoparticle to achieve in‐ creased blood circulation parameters thus increasing their residence time in the tissues, or by the specific targeting of tissues using ligands. The uses of nanotechnology in foods are as complex and varied as the types of formulations that can be created with this technology. Current technological applications that impact foods include the manufacture of food pack‐ aging, including packaging that incorporates antimicrobial agents such as silver, [2] or de‐ tection particles (gold), flavor enhancement, and delivery of dietary supplements and nutraceuticals [2-5]. While their potential or actual application present strong advantages, it is imperative that there be a thorough understanding regarding the physiology of nanopar‐ ticle absorption, or the consequences of their containment or integration within the mamma‐

Historically, food packaging has typically consisted of conventional materials such as paper or metal-based materials. The use of polymeric formulations improved the ability to retain

and reproduction in any medium, provided the original work is properly cited.

© 2013 Principato; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Gastrointestinal Immunoregulation and the Challenges of Nanotechnology in Foods**

MaryAnn Principato

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53287

## **1. Introduction**

### **1.1. Nanoparticles: Physicochemical characteristics and applications in foods**

Nanoparticles are elemental three dimensional structures that are typically between 1-100 nanometers (nm) in size that exhibit unique physiochemical characteristics that provide the basis for their utilization, and present unique challenges associated with the development of new applications [1, 2]. Because of their size, nanoparticles provide the opportunity to inter‐ act with human physiology at the subcellular level, affording many potential uses in nu‐ trient and drug delivery, vaccination therapies, and tissue repair. Specific physiologic applications can be achieved by chemical modification of the nanoparticle to achieve in‐ creased blood circulation parameters thus increasing their residence time in the tissues, or by the specific targeting of tissues using ligands. The uses of nanotechnology in foods are as complex and varied as the types of formulations that can be created with this technology. Current technological applications that impact foods include the manufacture of food pack‐ aging, including packaging that incorporates antimicrobial agents such as silver, [2] or de‐ tection particles (gold), flavor enhancement, and delivery of dietary supplements and nutraceuticals [2-5]. While their potential or actual application present strong advantages, it is imperative that there be a thorough understanding regarding the physiology of nanopar‐ ticle absorption, or the consequences of their containment or integration within the mamma‐ lian physiological and cellular environment.

## **1.2. Food packaging**

Historically, food packaging has typically consisted of conventional materials such as paper or metal-based materials. The use of polymeric formulations improved the ability to retain

© 2013 Principato; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

moisture and provided a gas barrier, thus extending food shelf life. Typically, the Food and Drug Administration requires that the manufacturer of food contact material comply with the regulatory requirements for each individual substance that comprises the entire formu‐ lation of the food contact material [6]. These food contact materials have typically included paper, metallic-based items, and polymeric compounds such as polyethylene terephthalate (PET), polypropylene, polyethylene, polystyrene, and others. Recently the formulation of nanocomposites has improved the ability to produce food contact surfaces that are superior with respect to their heating and gas barrier resistance characteristics [2, 7, 8]. Typically, a combination of previously approved compounds and nano-material has been used for the construction of the newer nanocomposite materials that strive to enhance the storage and preservation of foods. Nanocomposites are described as a combination of inorganic nano‐ material and a continuous phase consisting of synthetic polymers [9]. Nanoclay composites consist of magnesium aluminum silicate nanoparticles (bentonite or montmorillonite), and have proven to be a superior gas barrier for the preservation of foods [7]. The production of sustainable, biodegradable polylactide (PLA)-based polymers present the potential to re‐ duce the dependence upon petrochemical based polymers by using alternative renewable sources to produce packaging materials with qualities comparable to presently used prod‐ ucts. The combination of PLA with montmorillonite (MMT) nanocomposite [10-12] has been reported to produce a short term packaging material with good O2 gas permeability, and can be converted into CO2 and H2O through decomposition by microorganisms [10].

growth of *Escherichia coli* 0157:H7, *Staphylococcus aureus*, and *Klebsiella pneumonia* on agar at

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493

The encapsulation of dietary vitamins and other nutritional supplements as a nanoparticle has gained considerable interest as a means to increase the shelf life of such materials, and to improve delivery and release within the body. The engineered particles provide potential strategies with which to overcome the impermeability of the mucosal epitheli‐ um, and offer a possible means of circumventing the degradation of the nutrient by harsh degradative gastrointestinal conditions. Several candidate materials, used successfully for the delivery of drugs and vaccines, have been examined for their ability to encapsulate nutrients. Finally, compounds such as polysaccharides and proteins that are already in use within commercial food applications are attractive candidates for the production of new nanocomposite packaging and encapsulation material, as several are generally re‐

Poly (D,L)-lactic co glycolic acid (PLGA) nanoparticles are widely used for the encapsula‐ tion and delivery of drugs due to their reported biocompatibility and lack of overt toxicity. The physicochemical properties of the PLGA particles are affected by specific formulation and processing parameters, such as drug and polymer concentration, solvent volume, poly‐ mer molecular weight, the type of emulsifier used in the processing and its concentration, and the aqueous-to-organic phase ratio [30, 31]. Thus, PLGA nanoparticles have been shown to adequately encapsulate hydrophobic and hydrophilic molecules albeit the latter present some challenges with respect to a lowered load efficiency, and many PLGA-encapsulated delivery systems have been designed for a wide variety of macromolecules including drugs,

Chitosan, an N-deacetylated derivative of chitin, has been analyzed for use in nutrient deliv‐ ery due to its wide acceptance in drug delivery, and is generally regarded as non-toxic and biocompatible. Chitosan ((1 4)-2-amino-2-deoxy-β-D-glucan) is a naturally occurring cati‐ onic polysaccharide found in the shells of shrimp, lobsters, and crab that has an intrinsic ability to bind mucin. The bioadhesive property of chitosan permits organ-specific delivery, and surface modification of the polysaccharide particle has been successfully used to alter organ delivery [34]. Chitosan has been demonstrated to induce increased permeability in Caco-2 monolayers across tight junctions as measured by changes in the measured transepi‐ thelial electrical resistance and in a 14C-mannose absorption assay [35, 36]. The improved ab‐ sorption across cell layers due to the opening of tight junctions is thought to be the result of ionic interactions between the cell membrane and chitosan polysaccharide. While these characteristics favor the polysaccharide's use as a delivery method for a variety of com‐ pounds, it is necessary to incorporate anionic alginate to prevent burst release of the encap‐ sulated material due to protonation in an acidic environment. The results obtained by encapsulation of Vitamin A within dual layered chitosan-alginate nanospheres have been reported to be successful [37, 38]. In this instance, a high encapsulation efficiency and im‐ proved storage stability was achieved using double-layered microcapsules that incorporated

levels that were 35% of the levels achieved by cefotaxime and chloramphenicol [29].

**1.3. Dietary supplements and nutraceutical delivery**

garded as safe and are biodegradable.

biologically active cytokines, and peptides [31-33].

Silver, which has long been recognized for its antimicrobial characteristics [13], has been among the inorganic constituents incorporated into nanocomposite materials. Prior to the advent of nanotechnology, silver had long been used as an ingredient within dental compo‐ site material [14], integrated into wound dressings [15-17] and other medical devices ap‐ proved by the FDA, and is recognized as a biocide by the EPA [18]. Analysis of the antimicrobial effects of silver ion on gram positive and gram negative cell walled microor‐ ganisms demonstrated similar effects [19, 20]. Exposure of microbial organisms to silver re‐ sults in the retraction of the cytoplasm from the cell wall, condensation of the DNA into electron-dense granules, and there is an accumulation of silver ions into the cytoplasm. The damage, as inferred in these studies, is due to the inability to replicate at the DNA level [19]. Additional denaturant effects attributed to the silver ion include its ability to attach to sulf‐ hydryl groups, amino groups, and the terminal phosphate and carboxyl groups of bacterial proteins [13], essentially inactivating the enzymes involved with electron transport and me‐ tabolism. Of the electron transfer functions, cytochrome reductase and cytochrome oxidase are targeted [21]. While interest in silver's use as an antimicrobial has increased due to the observed rise in hospital and community-acquired antibiotic resistances, it is important to note that a growing microbial resistance to silver has also been reported [22]. Antimicrobial properties are similarly attributed to silver (Ag) nanoparticles [20, 23-27], and this property has spurred the inclusion of this material into a wide array of products within the foods sec‐ tor including packaging and service containers, and bottles, or used as a measure to prevent or control surface contamination by *Escherichia coli* and *Staphylococcus aureus* [28]. Indeed, the incorporation of silver into MMT composite preparations was shown to inhibit the growth of *Escherichia coli* 0157:H7, *Staphylococcus aureus*, and *Klebsiella pneumonia* on agar at levels that were 35% of the levels achieved by cefotaxime and chloramphenicol [29].

#### **1.3. Dietary supplements and nutraceutical delivery**

moisture and provided a gas barrier, thus extending food shelf life. Typically, the Food and Drug Administration requires that the manufacturer of food contact material comply with the regulatory requirements for each individual substance that comprises the entire formu‐ lation of the food contact material [6]. These food contact materials have typically included paper, metallic-based items, and polymeric compounds such as polyethylene terephthalate (PET), polypropylene, polyethylene, polystyrene, and others. Recently the formulation of nanocomposites has improved the ability to produce food contact surfaces that are superior with respect to their heating and gas barrier resistance characteristics [2, 7, 8]. Typically, a combination of previously approved compounds and nano-material has been used for the construction of the newer nanocomposite materials that strive to enhance the storage and preservation of foods. Nanocomposites are described as a combination of inorganic nano‐ material and a continuous phase consisting of synthetic polymers [9]. Nanoclay composites consist of magnesium aluminum silicate nanoparticles (bentonite or montmorillonite), and have proven to be a superior gas barrier for the preservation of foods [7]. The production of sustainable, biodegradable polylactide (PLA)-based polymers present the potential to re‐ duce the dependence upon petrochemical based polymers by using alternative renewable sources to produce packaging materials with qualities comparable to presently used prod‐ ucts. The combination of PLA with montmorillonite (MMT) nanocomposite [10-12] has been reported to produce a short term packaging material with good O2 gas permeability, and

492 Food Industry

can be converted into CO2 and H2O through decomposition by microorganisms [10].

Silver, which has long been recognized for its antimicrobial characteristics [13], has been among the inorganic constituents incorporated into nanocomposite materials. Prior to the advent of nanotechnology, silver had long been used as an ingredient within dental compo‐ site material [14], integrated into wound dressings [15-17] and other medical devices ap‐ proved by the FDA, and is recognized as a biocide by the EPA [18]. Analysis of the antimicrobial effects of silver ion on gram positive and gram negative cell walled microor‐ ganisms demonstrated similar effects [19, 20]. Exposure of microbial organisms to silver re‐ sults in the retraction of the cytoplasm from the cell wall, condensation of the DNA into electron-dense granules, and there is an accumulation of silver ions into the cytoplasm. The damage, as inferred in these studies, is due to the inability to replicate at the DNA level [19]. Additional denaturant effects attributed to the silver ion include its ability to attach to sulf‐ hydryl groups, amino groups, and the terminal phosphate and carboxyl groups of bacterial proteins [13], essentially inactivating the enzymes involved with electron transport and me‐ tabolism. Of the electron transfer functions, cytochrome reductase and cytochrome oxidase are targeted [21]. While interest in silver's use as an antimicrobial has increased due to the observed rise in hospital and community-acquired antibiotic resistances, it is important to note that a growing microbial resistance to silver has also been reported [22]. Antimicrobial properties are similarly attributed to silver (Ag) nanoparticles [20, 23-27], and this property has spurred the inclusion of this material into a wide array of products within the foods sec‐ tor including packaging and service containers, and bottles, or used as a measure to prevent or control surface contamination by *Escherichia coli* and *Staphylococcus aureus* [28]. Indeed, the incorporation of silver into MMT composite preparations was shown to inhibit the

The encapsulation of dietary vitamins and other nutritional supplements as a nanoparticle has gained considerable interest as a means to increase the shelf life of such materials, and to improve delivery and release within the body. The engineered particles provide potential strategies with which to overcome the impermeability of the mucosal epitheli‐ um, and offer a possible means of circumventing the degradation of the nutrient by harsh degradative gastrointestinal conditions. Several candidate materials, used successfully for the delivery of drugs and vaccines, have been examined for their ability to encapsulate nutrients. Finally, compounds such as polysaccharides and proteins that are already in use within commercial food applications are attractive candidates for the production of new nanocomposite packaging and encapsulation material, as several are generally re‐ garded as safe and are biodegradable.

Poly (D,L)-lactic co glycolic acid (PLGA) nanoparticles are widely used for the encapsula‐ tion and delivery of drugs due to their reported biocompatibility and lack of overt toxicity. The physicochemical properties of the PLGA particles are affected by specific formulation and processing parameters, such as drug and polymer concentration, solvent volume, poly‐ mer molecular weight, the type of emulsifier used in the processing and its concentration, and the aqueous-to-organic phase ratio [30, 31]. Thus, PLGA nanoparticles have been shown to adequately encapsulate hydrophobic and hydrophilic molecules albeit the latter present some challenges with respect to a lowered load efficiency, and many PLGA-encapsulated delivery systems have been designed for a wide variety of macromolecules including drugs, biologically active cytokines, and peptides [31-33].

Chitosan, an N-deacetylated derivative of chitin, has been analyzed for use in nutrient deliv‐ ery due to its wide acceptance in drug delivery, and is generally regarded as non-toxic and biocompatible. Chitosan ((1 4)-2-amino-2-deoxy-β-D-glucan) is a naturally occurring cati‐ onic polysaccharide found in the shells of shrimp, lobsters, and crab that has an intrinsic ability to bind mucin. The bioadhesive property of chitosan permits organ-specific delivery, and surface modification of the polysaccharide particle has been successfully used to alter organ delivery [34]. Chitosan has been demonstrated to induce increased permeability in Caco-2 monolayers across tight junctions as measured by changes in the measured transepi‐ thelial electrical resistance and in a 14C-mannose absorption assay [35, 36]. The improved ab‐ sorption across cell layers due to the opening of tight junctions is thought to be the result of ionic interactions between the cell membrane and chitosan polysaccharide. While these characteristics favor the polysaccharide's use as a delivery method for a variety of com‐ pounds, it is necessary to incorporate anionic alginate to prevent burst release of the encap‐ sulated material due to protonation in an acidic environment. The results obtained by encapsulation of Vitamin A within dual layered chitosan-alginate nanospheres have been reported to be successful [37, 38]. In this instance, a high encapsulation efficiency and im‐ proved storage stability was achieved using double-layered microcapsules that incorporated chitosan, alginate, calcium chloride and Tween 20. The production of combined Chitosan/ PLGA spherical particles have also been reported for the encapsulation of Vitamin A [38].With this construct, the microspheres (averaged 283 nm) demonstrated stability within an acidic environment and a lowered release rate into the gastric environment when com‐ pared to particles composed solely of PLGA. Thus, release of the target nutrient would be mainly in the small intestine where the vitamin would be absorbed. Interestingly, the mate‐ rial was visualized in the intestinal villi, and in the endothelium of rabbit GI.

tate an understanding of the events within the mucosal immune compartment known as the Gut-Associated Lymphoid Tissue (GALT), which is critically involved in the formation and maintenance of oral tolerance to introduced nutrient-derived antigens, and the generation of mucosal immune responsiveness to ingested pathogens and their toxins. The gastrointesti‐ nal tract is responsible for the digestion and absorption of ingested nutrients. This function is aided by the intestine's mucosal lining, whose absorptive surface is greatly increased by villi which project into the lumen and are composed of a single layer of epithelial cells and a rich network of capillaries and lymphatics. While the gastrointestinal tract is responsible for the absorption of nutrients, it is also the site of ongoing immune surveillance. The intestinal lumen normally contains dietary degraded products, commensal microbial flora, and any ingested contaminants including pathogenic bacteria and their products, viruses, fungi, or parasites. The resident gastrointestinal immune system must: 1) generate immunologic tol‐ erance towards nutrients and the resident microflora, and 2) recognize and remove infec‐ tious agents and their toxins [42-44]. Oral tolerance is driven by prior administration of antigen by the oral route, generating suppressive regulatory T cells, but is also dependent upon the maintenance of an effective epithelial barrier. The role of the resident gastrointesti‐ nal CD4+ T cell population for the establishment and maintenance of the tolerant state is crit‐ ical [45, 46]. Investigators have reported the formation of exosome-like structures, designated as "tolerosomes, " which are assembled in and released from small intestinal epi‐ thelial cells, that seem to play a crucial role for the induction of tolerance [47]. Breakdown of oral tolerance is thought to lead to the development of food allergy and some autoimmune diseases, including inflammatory bowel diseases (Crohn's disease and ulcerative colitis) and

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The GALT of the gastrointestinal tract consists of Peyer's patches (PP) containing B cells, dendritics, and T cells (Figure 1), appendix, draining mesenteric lymph nodes, and lymphat‐ ic follicles distributed throughout the length of the intestinal tract. The first line of immuno‐ logic defense is the provided by antibodies of the secretory IgA type found in the mucosal secretions of the gut [48]. This is supported by the observation that individuals with IgA de‐ ficiencies demonstrate circulating immune complexes to bovine and milk proteins [49]. In this case, the lack of IgA permits the entrance of food-derived antigens into the peripheral circulation, resulting in immune complex formation. The production of IgA is now known to be induced by regulatory T cells that have been activated by CD11+ dendritic cells [50]. Additionally, lymphocytes are scattered within the columnar epithelial layer (Intraepithelial

Beneath the epithelial layer of the mammalian gastrointestinal tract lies a rich source of im‐ munocompetent cells within the submucosal lymphoid follicles known as the intraepithelial lymphocytes (IEL) that comprise a significant portion of the body's T cells. The peripheral immune system contains effector T lineage cells bearing the αβ Τ cell receptor (TCR) which

are distinguished by the predominant presence of homodimeric CD8αα<sup>+</sup> Τ cells and T line‐

T cells or class I-restricted CD8+

T cells. Intraepithelial cells

lymphocytes, or IEL) and throughout the lamina propria.

**2.1. T cells of the Gut-associated lymphoid tissue**

are either class II-restricted CD4+

celiac disease.

Whey protein, derived from dairy, is recognized for its natural ability to form films and gels [7]. Whey nanospheres containing alginate have demonstrated the controlled release of an en‐ capsulated nutrient, riboflavin, when tested in simulated gastric juices [39]. In this instance, 94 nm whey nanoparticles were constructed using an emulsification and cold gelation meth‐ od, which averts the use of toxic solvents, and modification of the alginate concentration pro‐ vides some control over degradation of the particle by pepsin in their assay. The encapsulation of viable probiotic yeast cells has been reported using whey –alginate micro‐ spheres produced by a cold gelation extrusion technique [40]. The encapsulation of a hydro‐ phobic, fat-soluble nutrient can be achieved using casein maltodextrin nanoparticles produced by the Maillard reaction. In this reaction, the ε-amine groups found on the protein's lysine residues are covalently bonded to the aldehyde of reducing sugars. Particles produced in this manner consist of an exterior composed by the bulky hydrophilic domains of casein. The result of this design is a particle with increased curvature, i.e., a smaller diameter, con‐ taining an outermost saccharide layer and a hydrophobic inner core. Once the optimal casein: maltodextrin ratios were determined for the formation of the conjugates, incorporation of oilsoluble vitamin D resulted in particles that were 30 nm in diameter and demonstrated signifi‐ cant protection of the vitamin at low pH values that simulated gastric juices [4].

Liposomes composed of polar lipids such as lecithin have been used as delivery systems for antimicrobials, colors, and antioxidants. However, best results have been reported incorpo‐ rating an additional layer of material such as the cationic polysaccharide, chitosan. Lipo‐ somes composed of soy lecithin and prepared by homogenization, and combined with chitosan with stirring and sonication, were used to encapsulate grape seed extract [3]. In this instance, the particle size increased with the addition of the grape seed extract due to surface incorporation of the grape seed extract into the liposomes' layer. This was rectified by produc‐ tion of particles containing multiple polymer layers composed of chitosan and citrus pectin; grape seed polyphenols were no longer exposed to the matrix. Finally, microspheres with a mineral composition have also been developed for the encapsulation of nutrients. In this case, the encapsulation of water soluble polyphenols extracted from green tea has been accom‐ plished using calcium carbonate salt solutions containing phosphate and carbonate[41].

## **2. The Gut-Associated Lymphoid Tissue**

The proposed and anticipated uses of orally-delivered nanoparticles, the use of nanoparti‐ cles on food-contact surfaces, and the introduction of microencapsulated nutrients, necessi‐ tate an understanding of the events within the mucosal immune compartment known as the Gut-Associated Lymphoid Tissue (GALT), which is critically involved in the formation and maintenance of oral tolerance to introduced nutrient-derived antigens, and the generation of mucosal immune responsiveness to ingested pathogens and their toxins. The gastrointesti‐ nal tract is responsible for the digestion and absorption of ingested nutrients. This function is aided by the intestine's mucosal lining, whose absorptive surface is greatly increased by villi which project into the lumen and are composed of a single layer of epithelial cells and a rich network of capillaries and lymphatics. While the gastrointestinal tract is responsible for the absorption of nutrients, it is also the site of ongoing immune surveillance. The intestinal lumen normally contains dietary degraded products, commensal microbial flora, and any ingested contaminants including pathogenic bacteria and their products, viruses, fungi, or parasites. The resident gastrointestinal immune system must: 1) generate immunologic tol‐ erance towards nutrients and the resident microflora, and 2) recognize and remove infec‐ tious agents and their toxins [42-44]. Oral tolerance is driven by prior administration of antigen by the oral route, generating suppressive regulatory T cells, but is also dependent upon the maintenance of an effective epithelial barrier. The role of the resident gastrointesti‐ nal CD4+ T cell population for the establishment and maintenance of the tolerant state is crit‐ ical [45, 46]. Investigators have reported the formation of exosome-like structures, designated as "tolerosomes, " which are assembled in and released from small intestinal epi‐ thelial cells, that seem to play a crucial role for the induction of tolerance [47]. Breakdown of oral tolerance is thought to lead to the development of food allergy and some autoimmune diseases, including inflammatory bowel diseases (Crohn's disease and ulcerative colitis) and celiac disease.

The GALT of the gastrointestinal tract consists of Peyer's patches (PP) containing B cells, dendritics, and T cells (Figure 1), appendix, draining mesenteric lymph nodes, and lymphat‐ ic follicles distributed throughout the length of the intestinal tract. The first line of immuno‐ logic defense is the provided by antibodies of the secretory IgA type found in the mucosal secretions of the gut [48]. This is supported by the observation that individuals with IgA de‐ ficiencies demonstrate circulating immune complexes to bovine and milk proteins [49]. In this case, the lack of IgA permits the entrance of food-derived antigens into the peripheral circulation, resulting in immune complex formation. The production of IgA is now known to be induced by regulatory T cells that have been activated by CD11+ dendritic cells [50]. Additionally, lymphocytes are scattered within the columnar epithelial layer (Intraepithelial lymphocytes, or IEL) and throughout the lamina propria.

#### **2.1. T cells of the Gut-associated lymphoid tissue**

chitosan, alginate, calcium chloride and Tween 20. The production of combined Chitosan/ PLGA spherical particles have also been reported for the encapsulation of Vitamin A [38].With this construct, the microspheres (averaged 283 nm) demonstrated stability within an acidic environment and a lowered release rate into the gastric environment when com‐ pared to particles composed solely of PLGA. Thus, release of the target nutrient would be mainly in the small intestine where the vitamin would be absorbed. Interestingly, the mate‐

Whey protein, derived from dairy, is recognized for its natural ability to form films and gels [7]. Whey nanospheres containing alginate have demonstrated the controlled release of an en‐ capsulated nutrient, riboflavin, when tested in simulated gastric juices [39]. In this instance, 94 nm whey nanoparticles were constructed using an emulsification and cold gelation meth‐ od, which averts the use of toxic solvents, and modification of the alginate concentration pro‐ vides some control over degradation of the particle by pepsin in their assay. The encapsulation of viable probiotic yeast cells has been reported using whey –alginate micro‐ spheres produced by a cold gelation extrusion technique [40]. The encapsulation of a hydro‐ phobic, fat-soluble nutrient can be achieved using casein maltodextrin nanoparticles produced by the Maillard reaction. In this reaction, the ε-amine groups found on the protein's lysine residues are covalently bonded to the aldehyde of reducing sugars. Particles produced in this manner consist of an exterior composed by the bulky hydrophilic domains of casein. The result of this design is a particle with increased curvature, i.e., a smaller diameter, con‐ taining an outermost saccharide layer and a hydrophobic inner core. Once the optimal casein: maltodextrin ratios were determined for the formation of the conjugates, incorporation of oilsoluble vitamin D resulted in particles that were 30 nm in diameter and demonstrated signifi‐

rial was visualized in the intestinal villi, and in the endothelium of rabbit GI.

494 Food Industry

cant protection of the vitamin at low pH values that simulated gastric juices [4].

**2. The Gut-Associated Lymphoid Tissue**

Liposomes composed of polar lipids such as lecithin have been used as delivery systems for antimicrobials, colors, and antioxidants. However, best results have been reported incorpo‐ rating an additional layer of material such as the cationic polysaccharide, chitosan. Lipo‐ somes composed of soy lecithin and prepared by homogenization, and combined with chitosan with stirring and sonication, were used to encapsulate grape seed extract [3]. In this instance, the particle size increased with the addition of the grape seed extract due to surface incorporation of the grape seed extract into the liposomes' layer. This was rectified by produc‐ tion of particles containing multiple polymer layers composed of chitosan and citrus pectin; grape seed polyphenols were no longer exposed to the matrix. Finally, microspheres with a mineral composition have also been developed for the encapsulation of nutrients. In this case, the encapsulation of water soluble polyphenols extracted from green tea has been accom‐ plished using calcium carbonate salt solutions containing phosphate and carbonate[41].

The proposed and anticipated uses of orally-delivered nanoparticles, the use of nanoparti‐ cles on food-contact surfaces, and the introduction of microencapsulated nutrients, necessi‐ Beneath the epithelial layer of the mammalian gastrointestinal tract lies a rich source of im‐ munocompetent cells within the submucosal lymphoid follicles known as the intraepithelial lymphocytes (IEL) that comprise a significant portion of the body's T cells. The peripheral immune system contains effector T lineage cells bearing the αβ Τ cell receptor (TCR) which are either class II-restricted CD4+ T cells or class I-restricted CD8+ T cells. Intraepithelial cells are distinguished by the predominant presence of homodimeric CD8αα<sup>+</sup> Τ cells and T line‐ age cells containing the γδ TCR [51]; interestingly, the TCRγδ lineage and TCRαβ<sup>+</sup> CD8αα populations do not retain immunologic memory of infection. However, the γδ-T cell en‐ riched IEL function as a surveillance system for damaged or infected epithelial cells, and may modulate local immune responses by controlling cellular traffic and limiting mucosal access of inflammatory cells [52]. The γδ Τ cells are thought to play an important role in the pathophysiologic response to infections including Staphylococcal infection. In mice, 45% of the IEL present in the small intestine are estimated to be conventional thymus-derived lym‐ phocytes that coexpress TCR-αβ and classical CD8-αβ. These cells primarily exhibit a cyto‐ lytic function and are recognized residents of the lamina propria, yet retain the ability to dessiminate to various anatomical sites including the gut epithelium following an antigen priming [53]. However, there are also TCR bearing αβ T cells in the lamina propria, the ma‐ jority of which exhibit the activated/memory phenotype; the major histocompatibility (MHC) class II-restricted CD4+ T helper (Th) cells.

overlaying the PP. The M cells can take up particulate antigen by endocytosis and transport the antigen into the interior of the PP where the dendritic cells process the antigen and present antigen to the T cell areas of the PP and MLN, initiating T cell activation and differ‐ entiation into effector cells, that will either mediate tolerance or immunologic responsive‐ ness [54, 55]. In experiments using genetically-defined mice, ingestion of the superantigenic food toxin, Staphylococcal entertoxin B (SEB), has been demonstrated to increase the TCRαβ populations in PP (Figure 2) such that the predominant response is generated as a result of the binding between the toxin, target T cell receptor-bearing populations containing the defined Vβ-8 sequence, and antigen presenting cells [56, 57].The result of this interaction is the receptor-mediated induction of cytokine-driven T cell proliferation, resulting in a prolif‐

dramatically altered following oral administration of SEB in quantities sufficient to induce illness in humans to genetically-defined C57Bl/10J mice. The B220+ populations become se‐ questered, and the interior of the node becomes predominantly B220 negative. In this case,

analysis (Principato, unpublished). γδ-T lymphocyte populations in PP, lamina propria, and

**Figure 2.** A. Distribution of B220+ B cells in normal C57Bl/10J mice Peyer's Patch. Formalin fixed tissue section was stained using a monoclonal antibody directed against murine B220 (RA3-6B2) and a horse-radish peroxidase conju‐ gated antibody [10X magnification). B200 staining (brown areas) demonstrates a diffuse presence of B220+ B cells in the unstimulated Peyer's Patch. B. Expansion of non-B220+ (i.e., T cells) within Peyer's Patches 6 days following inges‐ tion of Staphlyococcal enterotoxin B. A redistribution of the B220+ cells into aggregates forming below the PP epithe‐

The intestinal mucosa harbors all of the major T helper (Th) cell subsets (Th1, Th2, Treg (im‐ munoregulatory), Th17) that are defined by their lineage-specific transcription factor expres‐ sion, cytokine production, and immune function. The Th1 subset is critical for immune responses generated against intracellular pathogens, and provides cytokine-mediated "help" to the cytotoxic T lymphocytes. It is characterized by the production of interferon-gamma

epithelium have also been observed to increase following SE treatment [58].

T cells. As shown in Figure 2, normal PP

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T cells as determined by flow cytometric

B cells. However, the distribution of B220+ B cells becomes

Gastrointestinal Immunoregulation and the Challenges of Nanotechnology in Foods

eration and expansion of the SEB-reactive Vβ-8<sup>+</sup>

the PP lymph node becomes enriched for Vβ-8<sup>+</sup>

lial capsule is indicated by arrows. [40X magnification).

**2.3. T helper subsets of the GALT**

contain an abundance of B220+

**Figure 1.** B220+ lymphocyte localization (indicated by arrows) in Peyer's Patch derived from normal C57Bl/10J mice. Formalin fixed tissue section [10X magnification) was stained using a monoclonal directed against B220 (RA3-6B2) and a horse-radish peroxidase conjugated antibody. B220 staining demonstrates a predominance of B cells in the un‐ stimulated Peyer's Patch.

#### **2.2. T cell immune activity in the GALT**

During a gastrointestinal immune response, ingested antigens in the lumen enter the Peyer's Patches via the specialized epithelial cells known as M cells present in the epithelial layer overlaying the PP. The M cells can take up particulate antigen by endocytosis and transport the antigen into the interior of the PP where the dendritic cells process the antigen and present antigen to the T cell areas of the PP and MLN, initiating T cell activation and differ‐ entiation into effector cells, that will either mediate tolerance or immunologic responsive‐ ness [54, 55]. In experiments using genetically-defined mice, ingestion of the superantigenic food toxin, Staphylococcal entertoxin B (SEB), has been demonstrated to increase the TCRαβ populations in PP (Figure 2) such that the predominant response is generated as a result of the binding between the toxin, target T cell receptor-bearing populations containing the defined Vβ-8 sequence, and antigen presenting cells [56, 57].The result of this interaction is the receptor-mediated induction of cytokine-driven T cell proliferation, resulting in a prolif‐ eration and expansion of the SEB-reactive Vβ-8<sup>+</sup> T cells. As shown in Figure 2, normal PP contain an abundance of B220+ B cells. However, the distribution of B220+ B cells becomes dramatically altered following oral administration of SEB in quantities sufficient to induce illness in humans to genetically-defined C57Bl/10J mice. The B220+ populations become se‐ questered, and the interior of the node becomes predominantly B220 negative. In this case, the PP lymph node becomes enriched for Vβ-8<sup>+</sup> T cells as determined by flow cytometric analysis (Principato, unpublished). γδ-T lymphocyte populations in PP, lamina propria, and epithelium have also been observed to increase following SE treatment [58].

**Figure 2.** A. Distribution of B220+ B cells in normal C57Bl/10J mice Peyer's Patch. Formalin fixed tissue section was stained using a monoclonal antibody directed against murine B220 (RA3-6B2) and a horse-radish peroxidase conju‐ gated antibody [10X magnification). B200 staining (brown areas) demonstrates a diffuse presence of B220+ B cells in the unstimulated Peyer's Patch. B. Expansion of non-B220+ (i.e., T cells) within Peyer's Patches 6 days following inges‐ tion of Staphlyococcal enterotoxin B. A redistribution of the B220+ cells into aggregates forming below the PP epithe‐ lial capsule is indicated by arrows. [40X magnification).

#### **2.3. T helper subsets of the GALT**

age cells containing the γδ TCR [51]; interestingly, the TCRγδ lineage and TCRαβ<sup>+</sup>

T helper (Th) cells.

**Figure 1.** B220+ lymphocyte localization (indicated by arrows) in Peyer's Patch derived from normal C57Bl/10J mice. Formalin fixed tissue section [10X magnification) was stained using a monoclonal directed against B220 (RA3-6B2) and a horse-radish peroxidase conjugated antibody. B220 staining demonstrates a predominance of B cells in the un‐

During a gastrointestinal immune response, ingested antigens in the lumen enter the Peyer's Patches via the specialized epithelial cells known as M cells present in the epithelial layer

(MHC) class II-restricted CD4+

496 Food Industry

stimulated Peyer's Patch.

**2.2. T cell immune activity in the GALT**

populations do not retain immunologic memory of infection. However, the γδ-T cell en‐ riched IEL function as a surveillance system for damaged or infected epithelial cells, and may modulate local immune responses by controlling cellular traffic and limiting mucosal access of inflammatory cells [52]. The γδ Τ cells are thought to play an important role in the pathophysiologic response to infections including Staphylococcal infection. In mice, 45% of the IEL present in the small intestine are estimated to be conventional thymus-derived lym‐ phocytes that coexpress TCR-αβ and classical CD8-αβ. These cells primarily exhibit a cyto‐ lytic function and are recognized residents of the lamina propria, yet retain the ability to dessiminate to various anatomical sites including the gut epithelium following an antigen priming [53]. However, there are also TCR bearing αβ T cells in the lamina propria, the ma‐ jority of which exhibit the activated/memory phenotype; the major histocompatibility

CD8αα

The intestinal mucosa harbors all of the major T helper (Th) cell subsets (Th1, Th2, Treg (im‐ munoregulatory), Th17) that are defined by their lineage-specific transcription factor expres‐ sion, cytokine production, and immune function. The Th1 subset is critical for immune responses generated against intracellular pathogens, and provides cytokine-mediated "help" to the cytotoxic T lymphocytes. It is characterized by the production of interferon-gamma (IFN-γ) which is controlled by the transcription factor T-bet [59]. The Th2 subset provides help for B cells and is also implicated in allergic sensitization, including those attributable to foods [60]. The specific transcription factor for Th2 cells is GATA-3, which drives the synthe‐ sis of IL-4, IL-5, and IL-13 [61]. Tregs that have arisen from antigen-specific induction of CD4+ Foxp3+ T cells are critical for the induction of oral tolerance[62]. The Th17 cells express retinoic acid-related orphan receptors (RORγt and RORα) that are needed for the transcrip‐ tion and synthesis of IL-17 [63, 64], and provide important protection of mucosal surfaces against extracellular bacteria.

of gene sequences which must be rearranged to configure a mature, functional, TCR. This permits the specific recognition of the presented peptide sequence by the TCR of the re‐ sponding T cell, and provides for the development of the adaptive immune response, which

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Innate immunity through the Toll like receptors (TLR) is conferred with the task of recogniz‐ ing a broad range of repetitive antigenic specificities that are found on a wide array of pathogens. With this type of recognition, pathogen detection is based on the ability to recog‐ nize pathogen-associated molecular patterns using evolutionarily conserved, germline en‐ coded recognition receptors, the TLR [73-75]. Thus, while a strict sequence-dependent antigenic specificity is not required as with antigen-specific immune responsiveness, what is required is an ability to bind carbohydrate residues in a Ca++ dependent manner, and the recognition of conserved molecular patterns such as in bacterial cell wall components. Thus, LPS is the ligand for TLR 4, and targeted mutation of the TLR4 locus in mice results in LPS non-responsiveness [76]. TLR2 recognizes ligands found on yeast cell walls, bacterial lipo‐ proteins [77], and lipoteichoic acid found in the cell walls of gram positive bacteria [78]; oth‐ er TLR recognize bacterial DNA, or double stranded viral RNA. In humans and mice, there are now at least 10 such TLR identified. Unlike the sequence-specific receptors found on the antigen-binding T cells of the adaptive immune response, TLR are non-clonal and do not re‐

Upon contact with a pathogen, the cells of the innate immune system become activated, the binding of their receptors initiating signaling cascades that turn on required transcription of target genes for the production of inflammatory cytokines, and the upregulation of costimu‐ latory and MHC molecules necessary for the direct elimination of the infection or for the re‐ cruitment of adaptive immune responses. The binding of TLR with their target ligand induce costimulatory molecules that were first identified as the B7.1 and B7.2, or now refer‐ red to as CD80 and CD86. Thus, the responding T cell must recognize the modified target ligand which is expressed on the surface of the macrophage or dendritic cells in the context of both MHC and costimulator molecules with its sequence-specific TCR. It is clear that the cells of the innate immune system are critical to the establishment of an effective immune responsiveness against pathogens and for the recruitment of an efficient adaptive immune response. The extremely successful yellow fever vaccine, YF-17D, which induces both Th1/Th2 responses and generates powerful neutralizing antibodies in vaccine recipients, was shown to induce such a strong protective immunity as a result of its ability to stimulate multiple subsets of human dendritic cells and multiple TLRs [79]. Vaccine designs utilizing synthetic 300 nm PLGA nanoparticles containing antigen and ligands that bind TLR 4 and TLR 7 on the surface of dendritic cells, have successfully induced enhanced antigen-specific antibody responses against the immunizing antigen when injected into experimental mice[80]. The immunization protocol induced long-lived, high avidity antibody that was de‐ pendent upon the expression of the targeted TLR on both B cells and dendritics. The B cell

Macrophage and dendritic cells have been documented with respect to the striking speciali‐

dendritic cells of the lamina propria can sample

will also generate an immunologic memory of the peptide target.

quire gene rearrangement in order to become functionally mature.

response indicated the generation of memory-type B cells.

zations of the subsets. For instance, CD11b+

#### **2.4. Innate immunity in the GALT**

The cells of the innate immune system include macrophages, dendritic cells, and Langer‐ han's cells, and are involved in critical activities pertaining to the initiation and support of T cell-mediated, antigen-specific immunity. Significantly, the distribution of these cell types includes the skin and epithelia that line the internal organs including the gastrointestinal tract. Macrophages and dendritic cells are situated below the single layer of epithelial cells that lines the Peyer's Patches and lamina propria [65]. Macrophages have long been identi‐ fied as components of the reticuloendothelial system and are recognized for their ability to ingest extracellular matter including proteins, cellular fragments, and debris that is foreign to the body in the process known as phagocytosis. They are widely distributed within the tissues of the body, and are crucial components of immune responsiveness and inflamma‐ tion. Initial binding of the target occurs on the surface of the cell, utilizing receptors with specific capabilities. Receptors identified include surface Fc receptors that bind the Fc por‐ tion of IgG immunoglobulin, complement C3b and C3d receptors, MHC Class I and Class II, Toll like receptors (TLR), cytokine receptors, and other membrane receptors such as the Ctype lectins [66] that provide additional innate functionality which supports the binding and internalization of a wide variety of targets. Opsonization of target by plasma proteins is known to improve phagocytosis, and the endocytosing vesicles have been demonstrated to consist of clathrin structures [67, 68]. Interestingly, the endosome exhibits plasticity, and its shape has been demonstrated to change depending on the material that is engulfed [69]. In an early examination of macrophage activity, Unanue and coworkers demonstrated distinct differences in macrophage effector function based on the anatomical source of the macro‐ phage. These investigators compared the ability of alveolar and peritoneal-derived macro‐ phages to bind and present antigen, the intracellular pathogen *Listeria monocytogenes*, to previously sensitized T cells [70]. While alveolar and peritoneal macrophages both ex‐ pressed class II Ia antigen, alveolar macrophages were less efficient with respect to the up‐ take and presentation of antigen to sensitized T cells as compared to the peritoneal macrophages. However, opsonizing *Listeria* using an anti-Listeria antiserum to coat the bac‐ terium enhanced the alveolar macrophages' ability to engulf the bacterium and effectively present the antigen to sensitized T cells. Once internalized, the ingested antigen undergoes intracellular metabolic and proteolytic degradation, and modification. The resulting frag‐ ment [71, 72], is transported to the surface of the cell where it is presented in conjunction with the major histocompatibility (MHC) gene molecule. This structural relationship is criti‐ cal for the activation of the appropriate responding T cell, which contains a great variability of gene sequences which must be rearranged to configure a mature, functional, TCR. This permits the specific recognition of the presented peptide sequence by the TCR of the re‐ sponding T cell, and provides for the development of the adaptive immune response, which will also generate an immunologic memory of the peptide target.

(IFN-γ) which is controlled by the transcription factor T-bet [59]. The Th2 subset provides help for B cells and is also implicated in allergic sensitization, including those attributable to foods [60]. The specific transcription factor for Th2 cells is GATA-3, which drives the synthe‐ sis of IL-4, IL-5, and IL-13 [61]. Tregs that have arisen from antigen-specific induction of

retinoic acid-related orphan receptors (RORγt and RORα) that are needed for the transcrip‐ tion and synthesis of IL-17 [63, 64], and provide important protection of mucosal surfaces

The cells of the innate immune system include macrophages, dendritic cells, and Langer‐ han's cells, and are involved in critical activities pertaining to the initiation and support of T cell-mediated, antigen-specific immunity. Significantly, the distribution of these cell types includes the skin and epithelia that line the internal organs including the gastrointestinal tract. Macrophages and dendritic cells are situated below the single layer of epithelial cells that lines the Peyer's Patches and lamina propria [65]. Macrophages have long been identi‐ fied as components of the reticuloendothelial system and are recognized for their ability to ingest extracellular matter including proteins, cellular fragments, and debris that is foreign to the body in the process known as phagocytosis. They are widely distributed within the tissues of the body, and are crucial components of immune responsiveness and inflamma‐ tion. Initial binding of the target occurs on the surface of the cell, utilizing receptors with specific capabilities. Receptors identified include surface Fc receptors that bind the Fc por‐ tion of IgG immunoglobulin, complement C3b and C3d receptors, MHC Class I and Class II, Toll like receptors (TLR), cytokine receptors, and other membrane receptors such as the Ctype lectins [66] that provide additional innate functionality which supports the binding and internalization of a wide variety of targets. Opsonization of target by plasma proteins is known to improve phagocytosis, and the endocytosing vesicles have been demonstrated to consist of clathrin structures [67, 68]. Interestingly, the endosome exhibits plasticity, and its shape has been demonstrated to change depending on the material that is engulfed [69]. In an early examination of macrophage activity, Unanue and coworkers demonstrated distinct differences in macrophage effector function based on the anatomical source of the macro‐ phage. These investigators compared the ability of alveolar and peritoneal-derived macro‐ phages to bind and present antigen, the intracellular pathogen *Listeria monocytogenes*, to previously sensitized T cells [70]. While alveolar and peritoneal macrophages both ex‐ pressed class II Ia antigen, alveolar macrophages were less efficient with respect to the up‐ take and presentation of antigen to sensitized T cells as compared to the peritoneal macrophages. However, opsonizing *Listeria* using an anti-Listeria antiserum to coat the bac‐ terium enhanced the alveolar macrophages' ability to engulf the bacterium and effectively present the antigen to sensitized T cells. Once internalized, the ingested antigen undergoes intracellular metabolic and proteolytic degradation, and modification. The resulting frag‐ ment [71, 72], is transported to the surface of the cell where it is presented in conjunction with the major histocompatibility (MHC) gene molecule. This structural relationship is criti‐ cal for the activation of the appropriate responding T cell, which contains a great variability

T cells are critical for the induction of oral tolerance[62]. The Th17 cells express

CD4+

498 Food Industry

Foxp3+

against extracellular bacteria.

**2.4. Innate immunity in the GALT**

Innate immunity through the Toll like receptors (TLR) is conferred with the task of recogniz‐ ing a broad range of repetitive antigenic specificities that are found on a wide array of pathogens. With this type of recognition, pathogen detection is based on the ability to recog‐ nize pathogen-associated molecular patterns using evolutionarily conserved, germline en‐ coded recognition receptors, the TLR [73-75]. Thus, while a strict sequence-dependent antigenic specificity is not required as with antigen-specific immune responsiveness, what is required is an ability to bind carbohydrate residues in a Ca++ dependent manner, and the recognition of conserved molecular patterns such as in bacterial cell wall components. Thus, LPS is the ligand for TLR 4, and targeted mutation of the TLR4 locus in mice results in LPS non-responsiveness [76]. TLR2 recognizes ligands found on yeast cell walls, bacterial lipo‐ proteins [77], and lipoteichoic acid found in the cell walls of gram positive bacteria [78]; oth‐ er TLR recognize bacterial DNA, or double stranded viral RNA. In humans and mice, there are now at least 10 such TLR identified. Unlike the sequence-specific receptors found on the antigen-binding T cells of the adaptive immune response, TLR are non-clonal and do not re‐ quire gene rearrangement in order to become functionally mature.

Upon contact with a pathogen, the cells of the innate immune system become activated, the binding of their receptors initiating signaling cascades that turn on required transcription of target genes for the production of inflammatory cytokines, and the upregulation of costimu‐ latory and MHC molecules necessary for the direct elimination of the infection or for the re‐ cruitment of adaptive immune responses. The binding of TLR with their target ligand induce costimulatory molecules that were first identified as the B7.1 and B7.2, or now refer‐ red to as CD80 and CD86. Thus, the responding T cell must recognize the modified target ligand which is expressed on the surface of the macrophage or dendritic cells in the context of both MHC and costimulator molecules with its sequence-specific TCR. It is clear that the cells of the innate immune system are critical to the establishment of an effective immune responsiveness against pathogens and for the recruitment of an efficient adaptive immune response. The extremely successful yellow fever vaccine, YF-17D, which induces both Th1/Th2 responses and generates powerful neutralizing antibodies in vaccine recipients, was shown to induce such a strong protective immunity as a result of its ability to stimulate multiple subsets of human dendritic cells and multiple TLRs [79]. Vaccine designs utilizing synthetic 300 nm PLGA nanoparticles containing antigen and ligands that bind TLR 4 and TLR 7 on the surface of dendritic cells, have successfully induced enhanced antigen-specific antibody responses against the immunizing antigen when injected into experimental mice[80]. The immunization protocol induced long-lived, high avidity antibody that was de‐ pendent upon the expression of the targeted TLR on both B cells and dendritics. The B cell response indicated the generation of memory-type B cells.

Macrophage and dendritic cells have been documented with respect to the striking speciali‐ zations of the subsets. For instance, CD11b+ dendritic cells of the lamina propria can sample luminal microbes by extending their dendrites to interdigitate between neighboring intesti‐ nal epithelial cells [81], and have been reported to promote the differentiation of Th17+ regu‐ latory T cells following activation of TLR 5 due to exposure to bacterial flagellin [82]. Interestingly, as previously observed by Unanue and coworkers [70], anatomic localization can denote distinctions in the functional effector function within subsets of cells. Thus, CD11b+CD103+ dendritic cells of the lamina propia are found preferentially in the duode‐ num and rarely in the colon during the steady state, but accumulate in the lamina propria of the colon along with Th17 cells during intestinal inflammation [83]. Macrophages of the lamina propria have been demonstrated be hyporesponsive to certain inflammatory stimuli, secrete IL-10, promote the differentiation of FoxP3+ regulatory T cells [84] and are able to dampen some immune responses and intestinal inflammation. For instance, a severe dex‐ tran sulfate-associated experimental colitis can be induced in a macrophage-depleted trans‐ genic mouse or in clodronate-treated normal C57BL/6 or Babl/c mice [85]. Finally, CD103+ dendritic cells are known to assist in the antigen specific induction of FoxP3+ T regs necessa‐ ry for tolerance induction[86]. Thus, the cells of the innate immune system maintain a bal‐ ance between a normal state of tolerance, and inflammatory and autoimmune responses.

diameter of the microparticle) per square mm area of rat intestinal tissue. Infusion of the particles into gastrointestinal tissue demonstrated 100 nm particle uptake by both duodenal and ileal tissue. However, the ileum's Peyer's Patch and non-Peyer's Patch tissue demon‐ strated a higher uptake of 100 nm size particles. This observation was repeated using surro‐ gate-loaded microparticles. Histologic examination of the tissue using fluorescent microscopy confirmed a greater retention of the 100 nm nanoparticles, with a concentration

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**Figure 3.** Schematic representation of the ingestion of nanoparticles. Macrophages and dendritic cells can be found beneath the eptihelial layer of the GALT. Ingested nonionic or targetted nanoparticles distribute preferentially below the intestinal epithelium, and can meet macrophages bearing class II and TLR molecules, and are phagocytosed by the macrophage. Actively phagocytosing macrophages are represented in the foreground; engorged macrophages are

The authors' observations recollect those of an earlier study utilizing latex particles [88]. In that study, Jani and coworkers conducted a 10-day feeding study in which non-ionic latex particles ranging in size from 100 nm, 500 nm, 1 micron, and 3 microns were fed to Sprague-Dawley rats. Their histologic and radiologic examination provided unequivocal evidence of a preferential tissue distribution of 100nm particles in which the Peyer's Patches, liver, and spleen demonstrated significant uptake. Significantly, their result confirmed the potential transport of particles from the gastrointestinal tract to the periphery via the lymphatics.

A separate 5 day feeding study in rats demonstrated the effects of a hydrophilic charge upon the tissue distribution of normally hydrophobic polystyrene particles [89]. In this study, commercial non-ionized polystyrene particles with a mean diameter of 60 nm were compared to similarly-sized particles coated with poloxamer 407. Their results confirmed

below the epithelial layer.

represented containing multiple particles.

## **3. Ingestion of nanoparticles**

The ingestion of nutrients with subsequent transit throughout the lumen of the gastrointesti‐ nal tract leads to the translocation of the material across the mucosa via the M cells of the epithelial layer. M cells are specialized cells that exhibit endocytic activity, and are known to transport antigens into the interior of the PP where the dendritic cells process the antigen and present antigen to the T cell areas of the PP and MLN, initiating T cell activation and differentiation into effector cells, that will either mediate tolerance or immunologic respon‐ siveness [54, 55]. Multiple physiochemical properties, including size and surface charge, have been shown to influence nanoparticle uptake and absorption in the gut, and the extent and rate at which the particles are removed from the circulation and their ultimate biodistri‐ bution. Thus, orally-administered non-ionic nanoparticles of 100 nm or less have demon‐ strated preferential absorption in the Peyer's Patch and the small intestine. Focused, engineered targeting of particles to the GALT has reported success with respect to the in‐ duction of measurable antibody responses. However, the specific immunologic mechanisms inherent to nanoparticle intake and absorption within the gastrointestinal tract have not been adequately identified, and the effector pathways that generate the immune responses measured have not been characterized.

#### **3.1. Influence of nanoparticle size and charge**

Desai and coworkers demonstrated that 100 nm nanoparticles underwent a preferential up‐ take in the gastrointestinal tract [87] using an *in situ* rat ileal loop model. Polylactic polygly‐ colic acid (PLGA) nano- and microparticles were synthesized with averaged diameters of 100 nm, 500 nm, 1 µm, and 10 µm and infused into the tissue. Tissue uptake was quantified as weight of the nanoparticles (µg) (taking into account the density of the polymer and the diameter of the microparticle) per square mm area of rat intestinal tissue. Infusion of the particles into gastrointestinal tissue demonstrated 100 nm particle uptake by both duodenal and ileal tissue. However, the ileum's Peyer's Patch and non-Peyer's Patch tissue demon‐ strated a higher uptake of 100 nm size particles. This observation was repeated using surro‐ gate-loaded microparticles. Histologic examination of the tissue using fluorescent microscopy confirmed a greater retention of the 100 nm nanoparticles, with a concentration below the epithelial layer.

luminal microbes by extending their dendrites to interdigitate between neighboring intesti‐ nal epithelial cells [81], and have been reported to promote the differentiation of Th17+

latory T cells following activation of TLR 5 due to exposure to bacterial flagellin [82]. Interestingly, as previously observed by Unanue and coworkers [70], anatomic localization can denote distinctions in the functional effector function within subsets of cells. Thus, CD11b+CD103+ dendritic cells of the lamina propia are found preferentially in the duode‐ num and rarely in the colon during the steady state, but accumulate in the lamina propria of the colon along with Th17 cells during intestinal inflammation [83]. Macrophages of the lamina propria have been demonstrated be hyporesponsive to certain inflammatory stimuli, secrete IL-10, promote the differentiation of FoxP3+ regulatory T cells [84] and are able to dampen some immune responses and intestinal inflammation. For instance, a severe dex‐ tran sulfate-associated experimental colitis can be induced in a macrophage-depleted trans‐ genic mouse or in clodronate-treated normal C57BL/6 or Babl/c mice [85]. Finally, CD103+ dendritic cells are known to assist in the antigen specific induction of FoxP3+ T regs necessa‐ ry for tolerance induction[86]. Thus, the cells of the innate immune system maintain a bal‐ ance between a normal state of tolerance, and inflammatory and autoimmune responses.

The ingestion of nutrients with subsequent transit throughout the lumen of the gastrointesti‐ nal tract leads to the translocation of the material across the mucosa via the M cells of the epithelial layer. M cells are specialized cells that exhibit endocytic activity, and are known to transport antigens into the interior of the PP where the dendritic cells process the antigen and present antigen to the T cell areas of the PP and MLN, initiating T cell activation and differentiation into effector cells, that will either mediate tolerance or immunologic respon‐ siveness [54, 55]. Multiple physiochemical properties, including size and surface charge, have been shown to influence nanoparticle uptake and absorption in the gut, and the extent and rate at which the particles are removed from the circulation and their ultimate biodistri‐ bution. Thus, orally-administered non-ionic nanoparticles of 100 nm or less have demon‐ strated preferential absorption in the Peyer's Patch and the small intestine. Focused, engineered targeting of particles to the GALT has reported success with respect to the in‐ duction of measurable antibody responses. However, the specific immunologic mechanisms inherent to nanoparticle intake and absorption within the gastrointestinal tract have not been adequately identified, and the effector pathways that generate the immune responses

Desai and coworkers demonstrated that 100 nm nanoparticles underwent a preferential up‐ take in the gastrointestinal tract [87] using an *in situ* rat ileal loop model. Polylactic polygly‐ colic acid (PLGA) nano- and microparticles were synthesized with averaged diameters of 100 nm, 500 nm, 1 µm, and 10 µm and infused into the tissue. Tissue uptake was quantified as weight of the nanoparticles (µg) (taking into account the density of the polymer and the

**3. Ingestion of nanoparticles**

500 Food Industry

measured have not been characterized.

**3.1. Influence of nanoparticle size and charge**

regu‐

**Figure 3.** Schematic representation of the ingestion of nanoparticles. Macrophages and dendritic cells can be found beneath the eptihelial layer of the GALT. Ingested nonionic or targetted nanoparticles distribute preferentially below the intestinal epithelium, and can meet macrophages bearing class II and TLR molecules, and are phagocytosed by the macrophage. Actively phagocytosing macrophages are represented in the foreground; engorged macrophages are represented containing multiple particles.

The authors' observations recollect those of an earlier study utilizing latex particles [88]. In that study, Jani and coworkers conducted a 10-day feeding study in which non-ionic latex particles ranging in size from 100 nm, 500 nm, 1 micron, and 3 microns were fed to Sprague-Dawley rats. Their histologic and radiologic examination provided unequivocal evidence of a preferential tissue distribution of 100nm particles in which the Peyer's Patches, liver, and spleen demonstrated significant uptake. Significantly, their result confirmed the potential transport of particles from the gastrointestinal tract to the periphery via the lymphatics.

A separate 5 day feeding study in rats demonstrated the effects of a hydrophilic charge upon the tissue distribution of normally hydrophobic polystyrene particles [89]. In this study, commercial non-ionized polystyrene particles with a mean diameter of 60 nm were compared to similarly-sized particles coated with poloxamer 407. Their results confirmed the earlier observations by Jani and coworkers: a preferential uptake of uncharged polystyr‐ ene was noted in the small intestines and Peyer's Patches as measured using gel permeation chromatography to quantify polystyrene in the tissue, and by microscopy. Further, a smaller concentration of particles was observed to be in the mesenteric lymphatic tissue and liver. Collectively, these data indicate a movement of the particles from the lumen of the intestinal tract to the peripheral circulation with subsequent residence in other tissues. However, charged particles demonstrated a significant reduction in uptake, 1.5%- 2% of the total ad‐ ministered dose of particles were absorbed as opposed to 10% uptake using uncharged par‐ ticles. Interestingly, the tissue distribution was altered as a result of the poloxamer coating: the particles were particularly concentrated within the tissues of the large intestine. Taken together, these results demonstrate the importance of particle size in determining the tissue range of ingested neutrally charged particles, and the critical role of charge as a particularly strong determinant of distribution within the body.

Significant patterns in organ compartmentalization have also been described for metallic

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The fate of ingested silver salt and silver nanoparticle was examined in a feeding study in which separate groups of female Wistar rats were administered 9 mg silver acetate or 12.6 mg silver nanoparticle per kg of body weight daily for 28 days. It was estimated that 63% of the daily ingested dose was excreted in the feces. The overall accumulation of either silver ion or nanoparticle was similar, and appeared greatest in the small intestine while also de‐ tectable in liver, kidney, and stomach. Autometallographic staining (AMG) detects the pres‐ ence of either silver acetate or nanoparticle; thus, silver was localized to the lamina propia and submucosa in the ileum. Interestingly, silver was concentrated around the veins and portal circulation of the liver and was not preferentially taken up by the Kupffer cells of the liver as was reported with injected gold [90]. Transmission electron microscopy displayed similar localizations for both the silver nanoparticles and silver acetate; the material was found within the lysosomes of the macrophages within the lamina propia of the ileum [93].

**4. Immune responses and the ingested nanoparticle/microparticle**

Particulate antigens are known to induce stronger immune responsiveness to the antigen when compared to an immune response generated with the soluble form of antigen. Thus, vaccine design has recently emphasized the use of nanoparticles to maximize induction of the protective immune responsiveness. Mucosal immunizations have been viewed increas‐ ingly as an alternative to parenteral administration of vaccines, and features such as carbo‐ hydrate residue targeting by lectins has been examined by many groups [94-96]. Nevertheless, nanoparticle absorption within the GALT is still not well understood, and the effector cellular interactions involved in the generation of the induced immune response

Immunoglobulin production as a function of particle size, was measured by Gutierro and coworkers [97]. Bovine Serum Albumin (BSA) –loaded PLGA microspheres of 200 nm, 500 nm, and 1000 nm were constructed using a double emulsion technique; size was determined by laser diffractometry using a CoulterCounter® particle size analyzer. PLGA microspheres containing BSA target antigen was administered by each of three routes: subcutaneously, in‐ tranasally, or orally into 6-8 week old Balbc/J mice and the elicited immune response was measured by assaying IgG immunoglobulin production. Their results showed that IgG anti‐ bodies were elicited using each of the three sizes of microspheres when administered subcu‐ taneously; one size did not elicit greater antibody production than the others. Further, all three sizes elicited antibody responses that were greater than that elicited using either solu‐ ble antigen or conventional adjuvant approaches. The oral immunization protocol consisted of orally feeding each of the three sizes of microspheres, each containing 500 ∝g BSA, on three successive days. Interestingly, oral administration of the loaded microspheres showed that the 200 nm and 500 nm sized particles elicited fewer antibodies than an administration of antigen with either alum or Freund's adjuvant. The greatest production of serum IgG was

nanoparticles.

have not been fully defined.

#### **3.2. Biodistribution**

The macrophage is most often implicated in the uptake of nanoparticles and opsonization will influence nanoparticle uptake into the cells. Nevertheless, final biodistribution and dis‐ position is likely determined by the transport of particles by phagocytic and endocytotic cells. The intraperitoneal injection of 40 nm gold nanoparticles in mice has been demonstrat‐ ed to result in the localization of particles in the Kupffer cells of the liver. In this research, commercially-produced colloidal gold nanoparticles containing a negative surface charge, in sizes of either 2 nm or 40 nm, were injected either intraperitoneally (ip) or intravenously (iv) into C57Bl/6 mice, and detected within cryostat sections of liver and other organs by auto‐ metallography, which amplifies the detection of gold. Interestingly, a preferential uptake of the 40 nm particles by Kupffer cells of the liver was observed 24 hours after ip injection. Very little uptake was observed 1 hour after injection. Animals who received the particles by ip injection also demonstrated particle uptake within the walls of the small intestine, mesen‐ teric lymph node, and in the spleen illustrating that transit of the administered particles had occurred, most likely via the phagocytic cells [90]. Using a rabbit model, orally administered chitosan/PLGA spherical particles (averaged 283 nm) for the encapsulation of Vitamin A were found along the mucosal epithelium of the lumen, and within the intestinal epithelial cells, presumably due to endocytosis. Macrophages in the lamina propia showed evidence of particles as did the endothelial cells [38].

Variations to the particle, such as addition of a polymeric coating, will alter the biodistribu‐ tion. Thus, coating polystyrene 60 nm and 5.25 µm particles with poloxamer polymers will decrease uptake by liver and spleen macrophages. Importantly, increasing the thickness of the coating will alter uptake as well; in this case it has resulted in a reduction of uptake by the peritoneal macrophage [91]. Experiments in which hydrophilic negatively-charged algi‐ nate-coated chitosan nanoparticles were passively absorbed into gastrointestinal tissue dem‐ onstrated localization beneath the follicle associated epithelium of the Peyer's Patches and agglomeration of the particles intracellularly, although the specific nature of the cell was not described [92].

Significant patterns in organ compartmentalization have also been described for metallic nanoparticles.

the earlier observations by Jani and coworkers: a preferential uptake of uncharged polystyr‐ ene was noted in the small intestines and Peyer's Patches as measured using gel permeation chromatography to quantify polystyrene in the tissue, and by microscopy. Further, a smaller concentration of particles was observed to be in the mesenteric lymphatic tissue and liver. Collectively, these data indicate a movement of the particles from the lumen of the intestinal tract to the peripheral circulation with subsequent residence in other tissues. However, charged particles demonstrated a significant reduction in uptake, 1.5%- 2% of the total ad‐ ministered dose of particles were absorbed as opposed to 10% uptake using uncharged par‐ ticles. Interestingly, the tissue distribution was altered as a result of the poloxamer coating: the particles were particularly concentrated within the tissues of the large intestine. Taken together, these results demonstrate the importance of particle size in determining the tissue range of ingested neutrally charged particles, and the critical role of charge as a particularly

The macrophage is most often implicated in the uptake of nanoparticles and opsonization will influence nanoparticle uptake into the cells. Nevertheless, final biodistribution and dis‐ position is likely determined by the transport of particles by phagocytic and endocytotic cells. The intraperitoneal injection of 40 nm gold nanoparticles in mice has been demonstrat‐ ed to result in the localization of particles in the Kupffer cells of the liver. In this research, commercially-produced colloidal gold nanoparticles containing a negative surface charge, in sizes of either 2 nm or 40 nm, were injected either intraperitoneally (ip) or intravenously (iv) into C57Bl/6 mice, and detected within cryostat sections of liver and other organs by auto‐ metallography, which amplifies the detection of gold. Interestingly, a preferential uptake of the 40 nm particles by Kupffer cells of the liver was observed 24 hours after ip injection. Very little uptake was observed 1 hour after injection. Animals who received the particles by ip injection also demonstrated particle uptake within the walls of the small intestine, mesen‐ teric lymph node, and in the spleen illustrating that transit of the administered particles had occurred, most likely via the phagocytic cells [90]. Using a rabbit model, orally administered chitosan/PLGA spherical particles (averaged 283 nm) for the encapsulation of Vitamin A were found along the mucosal epithelium of the lumen, and within the intestinal epithelial cells, presumably due to endocytosis. Macrophages in the lamina propia showed evidence

Variations to the particle, such as addition of a polymeric coating, will alter the biodistribu‐ tion. Thus, coating polystyrene 60 nm and 5.25 µm particles with poloxamer polymers will decrease uptake by liver and spleen macrophages. Importantly, increasing the thickness of the coating will alter uptake as well; in this case it has resulted in a reduction of uptake by the peritoneal macrophage [91]. Experiments in which hydrophilic negatively-charged algi‐ nate-coated chitosan nanoparticles were passively absorbed into gastrointestinal tissue dem‐ onstrated localization beneath the follicle associated epithelium of the Peyer's Patches and agglomeration of the particles intracellularly, although the specific nature of the cell was not

strong determinant of distribution within the body.

of particles as did the endothelial cells [38].

described [92].

**3.2. Biodistribution**

502 Food Industry

The fate of ingested silver salt and silver nanoparticle was examined in a feeding study in which separate groups of female Wistar rats were administered 9 mg silver acetate or 12.6 mg silver nanoparticle per kg of body weight daily for 28 days. It was estimated that 63% of the daily ingested dose was excreted in the feces. The overall accumulation of either silver ion or nanoparticle was similar, and appeared greatest in the small intestine while also de‐ tectable in liver, kidney, and stomach. Autometallographic staining (AMG) detects the pres‐ ence of either silver acetate or nanoparticle; thus, silver was localized to the lamina propia and submucosa in the ileum. Interestingly, silver was concentrated around the veins and portal circulation of the liver and was not preferentially taken up by the Kupffer cells of the liver as was reported with injected gold [90]. Transmission electron microscopy displayed similar localizations for both the silver nanoparticles and silver acetate; the material was found within the lysosomes of the macrophages within the lamina propia of the ileum [93].

## **4. Immune responses and the ingested nanoparticle/microparticle**

Particulate antigens are known to induce stronger immune responsiveness to the antigen when compared to an immune response generated with the soluble form of antigen. Thus, vaccine design has recently emphasized the use of nanoparticles to maximize induction of the protective immune responsiveness. Mucosal immunizations have been viewed increas‐ ingly as an alternative to parenteral administration of vaccines, and features such as carbo‐ hydrate residue targeting by lectins has been examined by many groups [94-96]. Nevertheless, nanoparticle absorption within the GALT is still not well understood, and the effector cellular interactions involved in the generation of the induced immune response have not been fully defined.

Immunoglobulin production as a function of particle size, was measured by Gutierro and coworkers [97]. Bovine Serum Albumin (BSA) –loaded PLGA microspheres of 200 nm, 500 nm, and 1000 nm were constructed using a double emulsion technique; size was determined by laser diffractometry using a CoulterCounter® particle size analyzer. PLGA microspheres containing BSA target antigen was administered by each of three routes: subcutaneously, in‐ tranasally, or orally into 6-8 week old Balbc/J mice and the elicited immune response was measured by assaying IgG immunoglobulin production. Their results showed that IgG anti‐ bodies were elicited using each of the three sizes of microspheres when administered subcu‐ taneously; one size did not elicit greater antibody production than the others. Further, all three sizes elicited antibody responses that were greater than that elicited using either solu‐ ble antigen or conventional adjuvant approaches. The oral immunization protocol consisted of orally feeding each of the three sizes of microspheres, each containing 500 ∝g BSA, on three successive days. Interestingly, oral administration of the loaded microspheres showed that the 200 nm and 500 nm sized particles elicited fewer antibodies than an administration of antigen with either alum or Freund's adjuvant. The greatest production of serum IgG was demonstrated using the 1000 um size particle and this group contained the higher percent‐ age of individual responders. Analysis of serums at weeks 3 and 5 following immunization did not reveal differences in IgG2a/IgG1 isotype profiles, the latter being indicative of Th1/Th2 subset immunity and antigen-presenting differences. Ultimately, no differences were found among the various sized particles, suggesting that the method of antigen pre‐ sentation was the same for all of the sizes tested. Again, the larger particles provided the higher immunoglobulin production, regardless of the mode of immunization. These results are interesting from the perspective of what has been reported [87, 88] regarding the effect of size and nanoparticle biodistribution, and what is known about the distribution of effec‐ tor cells within the GALT. The present experiments used particles that were larger than those previously published; it is likely that the biodistribution affected the manner in which particulate antigen was presented for the induction of an immune response.

loaded with BSA test antigen, and coated with alginate. Thus, alginate was modified by using 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide to form amide linkages between the carbox‐ ylate residues on alginate and the amino group of the lectin *Ulex europaeus* agglutinin (UEA-1). The lectin *Ulex europaeus agglutinin* (UEA-1) was used to direct the microparticles towards the α-L- fucose residues found on the surface of M cells. Confocal microscopy confirmed the tar‐ geting; punctate staining was visualized using the lectin-modified microspheres. The conjuga‐ tion and loading resulted in a particle shift in size: the particle size of the original CNP particle is reported as 257+ 55.17 nm, while the lectin-modified antigen carrier CNP particle size in‐ creased to 1485 + 214.3 nm. Oral immunization of 6-8 week old Balb/c mice with each of the preparations and control antigen provided striking differences in the antibody responses against BSA antigen. The highest IgG titers were obtained using alum-absorbed BSA as the im‐ munogen's positive control, and the lowest titers were obtained using BSA loaded CNP. In contrast, antigen encapsulated in lectin-modified alginate chitosan particles (LACNP) consis‐ tently generated IgG titers that were greater than those obtained with CNP or ACNP formula‐ tions. Demonstrable levels of antigen-specific IgG2a/IgG1 were detected with all three formulations. Significantly, the highest titers of antigen-specific IgG were obtained with lectinmodified microspheres, and the results seem to indicate that there was a greater IgG2a, or Th1 response, to antigen (BSA) with that particle. The original CNP particle and the alginate chito‐

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Together, these studies demonstrate the induction of a Th1/Th2-induced immunity using en‐ gineered particles as do others [93]. However, it is not known whether α- fucose residues are found on macrophages and dendritic cells present at other body sites, possibly resulting in multiple pathways of immune responsiveness. As discussed earlier, Unanue and cowork‐ ers demonstrated distinct differences in macrophage effector function based on the anatomi‐ cal source of the macrophage [69]. Further, while TLA lectin-PLGA-HBsAg nanoparticles induced the production of sIgA in saliva and gastrointestinal fluids, it was not reported

cells. IgA has been reported to be induced by regulatory T cells that have been activated by CD11<sup>+</sup> dendritic cells [50]. Finally, the directed attachment of the particles to the endocytotic M cells of the epithelial layer presents the possibility that the particles were transcytosed by

the possibility exists for the induction of tolerance [86]. Normal exposure to ingested, digest‐ ed antigen results in the production of regulatory T cells that suppress an immune response in an antigen specific manner, resulting in tolerance and preventing food allergy. However,

Proteins used in commercial food applications include casein, whey protein, collagen, egg white, and fish myofibrillar protein, and popular plant-based proteins including soybean protein and wheat gluten [7]. Compounds such as polysaccharides and proteins that are al‐

dendritic cells, found beneath the epithelial layer. In that case,

dendritic

whether the engineered particles in these reports ultimately interacted with CD11<sup>+</sup>

the targeted microparticles document the induction of Th1and Th2 responses.

**5. Future consideration: Ingested nanoparticle and immune allergic**

san particles seemed to have induced a greater Th2 response.

the M cells towards CD103<sup>+</sup>

**dysfunction to foods**

#### **4.1. Targeting M cells in the GALT**

Directed PLGA nanoparticles, using lectins to bind onto target sugar residues, has been shown to be a means to achieve organ targeting for the induction of a systemic immune response. In one study, PLGA nanoparticles were created by the double emulsion method and loaded with hepatitis B surface antigen (HBsAg) [95]. Lectin directed to α-L- fucose residues, *Tetragonolobus purpureas,* was bound to the nanoparticle using 1-ethyl-3-(3 dimethylaminopropyl) carbodii‐ mide to produce TLA lectin-PLGA-HBsAg nanoparticles that were measured to be 270 + 23nm in size. Confocal microscopy confirmed binding of the particles to the M cells of the Peyer's Patches within immunized mice. Further, lectinized particles were stabilized by the addition of hydrophilic trehalose, which improves the release of antigen. Therefore, nanoparticles stabi‐ lized with trehalose demonstrated an increased antigen release of 43.2+2.7% after 35 days, as opposed to a release of 32.4 +2.3% by the non-stabilized equivalent. In this investigation, 10 mg of encapsulated antigen per dose was used for the oral immunization of 8 week old Balb/c mice, followed with a booster 2 weeks following the primary immunization. Thus, HBsAg -loaded PLGA nanoparticles, TLA lectin-PLGA-HBsAg nanoparticles, trehalose-stabilized HBsAg loaded PLGA nanoparticles, and trehalose-stabilized TLA lectin-PLGA-HBsAg nanoparticles were compared for the induction of antibody. Significantly, this study demonstrated the suc‐ cessful induction of antigen-specific IgG antibody by each of the engineered nanoparticles as determined by ELISA assay of the immune seras, when compared to the levels of antibody pro‐ duced by the animals immunized with an alum based antigen. Isotyping of the antibodies demonstrated induction of IgG1 antibody, indicative of a Th2 response, at levels that were twice those attained by IgG2a which is indicative of a Th1 response. While demonstrable levels of the Th1 cytokines, IL-2 and γ-IFN, were detected in the spleens of all nanoparticle-treated animals, greater levels of γ-IFN were obtained with TLA lectin-PLGA-HBsAg, with or without stabilization by trehalose. It is not known whether the engineered particle could have induced a greater γ-IFN response as PLGA nanoparticles without antigen were not used for compari‐ son in this study.

A directed approach has been extremely successful using chitosan alginate microparticles [94]. In this instance, chitosan nanoparticles (CNP) prepared by the ionic gelation method were loaded with BSA test antigen, and coated with alginate. Thus, alginate was modified by using 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide to form amide linkages between the carbox‐ ylate residues on alginate and the amino group of the lectin *Ulex europaeus* agglutinin (UEA-1). The lectin *Ulex europaeus agglutinin* (UEA-1) was used to direct the microparticles towards the α-L- fucose residues found on the surface of M cells. Confocal microscopy confirmed the tar‐ geting; punctate staining was visualized using the lectin-modified microspheres. The conjuga‐ tion and loading resulted in a particle shift in size: the particle size of the original CNP particle is reported as 257+ 55.17 nm, while the lectin-modified antigen carrier CNP particle size in‐ creased to 1485 + 214.3 nm. Oral immunization of 6-8 week old Balb/c mice with each of the preparations and control antigen provided striking differences in the antibody responses against BSA antigen. The highest IgG titers were obtained using alum-absorbed BSA as the im‐ munogen's positive control, and the lowest titers were obtained using BSA loaded CNP. In contrast, antigen encapsulated in lectin-modified alginate chitosan particles (LACNP) consis‐ tently generated IgG titers that were greater than those obtained with CNP or ACNP formula‐ tions. Demonstrable levels of antigen-specific IgG2a/IgG1 were detected with all three formulations. Significantly, the highest titers of antigen-specific IgG were obtained with lectinmodified microspheres, and the results seem to indicate that there was a greater IgG2a, or Th1 response, to antigen (BSA) with that particle. The original CNP particle and the alginate chito‐ san particles seemed to have induced a greater Th2 response.

demonstrated using the 1000 um size particle and this group contained the higher percent‐ age of individual responders. Analysis of serums at weeks 3 and 5 following immunization did not reveal differences in IgG2a/IgG1 isotype profiles, the latter being indicative of Th1/Th2 subset immunity and antigen-presenting differences. Ultimately, no differences were found among the various sized particles, suggesting that the method of antigen pre‐ sentation was the same for all of the sizes tested. Again, the larger particles provided the higher immunoglobulin production, regardless of the mode of immunization. These results are interesting from the perspective of what has been reported [87, 88] regarding the effect of size and nanoparticle biodistribution, and what is known about the distribution of effec‐ tor cells within the GALT. The present experiments used particles that were larger than those previously published; it is likely that the biodistribution affected the manner in which

Directed PLGA nanoparticles, using lectins to bind onto target sugar residues, has been shown to be a means to achieve organ targeting for the induction of a systemic immune response. In one study, PLGA nanoparticles were created by the double emulsion method and loaded with hepatitis B surface antigen (HBsAg) [95]. Lectin directed to α-L- fucose residues, *Tetragonolobus purpureas,* was bound to the nanoparticle using 1-ethyl-3-(3 dimethylaminopropyl) carbodii‐ mide to produce TLA lectin-PLGA-HBsAg nanoparticles that were measured to be 270 + 23nm in size. Confocal microscopy confirmed binding of the particles to the M cells of the Peyer's Patches within immunized mice. Further, lectinized particles were stabilized by the addition of hydrophilic trehalose, which improves the release of antigen. Therefore, nanoparticles stabi‐ lized with trehalose demonstrated an increased antigen release of 43.2+2.7% after 35 days, as opposed to a release of 32.4 +2.3% by the non-stabilized equivalent. In this investigation, 10 mg of encapsulated antigen per dose was used for the oral immunization of 8 week old Balb/c mice, followed with a booster 2 weeks following the primary immunization. Thus, HBsAg -loaded PLGA nanoparticles, TLA lectin-PLGA-HBsAg nanoparticles, trehalose-stabilized HBsAg loaded PLGA nanoparticles, and trehalose-stabilized TLA lectin-PLGA-HBsAg nanoparticles were compared for the induction of antibody. Significantly, this study demonstrated the suc‐ cessful induction of antigen-specific IgG antibody by each of the engineered nanoparticles as determined by ELISA assay of the immune seras, when compared to the levels of antibody pro‐ duced by the animals immunized with an alum based antigen. Isotyping of the antibodies demonstrated induction of IgG1 antibody, indicative of a Th2 response, at levels that were twice those attained by IgG2a which is indicative of a Th1 response. While demonstrable levels of the Th1 cytokines, IL-2 and γ-IFN, were detected in the spleens of all nanoparticle-treated animals, greater levels of γ-IFN were obtained with TLA lectin-PLGA-HBsAg, with or without stabilization by trehalose. It is not known whether the engineered particle could have induced a greater γ-IFN response as PLGA nanoparticles without antigen were not used for compari‐

A directed approach has been extremely successful using chitosan alginate microparticles [94]. In this instance, chitosan nanoparticles (CNP) prepared by the ionic gelation method were

particulate antigen was presented for the induction of an immune response.

**4.1. Targeting M cells in the GALT**

504 Food Industry

son in this study.

Together, these studies demonstrate the induction of a Th1/Th2-induced immunity using en‐ gineered particles as do others [93]. However, it is not known whether α- fucose residues are found on macrophages and dendritic cells present at other body sites, possibly resulting in multiple pathways of immune responsiveness. As discussed earlier, Unanue and cowork‐ ers demonstrated distinct differences in macrophage effector function based on the anatomi‐ cal source of the macrophage [69]. Further, while TLA lectin-PLGA-HBsAg nanoparticles induced the production of sIgA in saliva and gastrointestinal fluids, it was not reported whether the engineered particles in these reports ultimately interacted with CD11<sup>+</sup> dendritic cells. IgA has been reported to be induced by regulatory T cells that have been activated by CD11<sup>+</sup> dendritic cells [50]. Finally, the directed attachment of the particles to the endocytotic M cells of the epithelial layer presents the possibility that the particles were transcytosed by the M cells towards CD103<sup>+</sup> dendritic cells, found beneath the epithelial layer. In that case, the possibility exists for the induction of tolerance [86]. Normal exposure to ingested, digest‐ ed antigen results in the production of regulatory T cells that suppress an immune response in an antigen specific manner, resulting in tolerance and preventing food allergy. However, the targeted microparticles document the induction of Th1and Th2 responses.

## **5. Future consideration: Ingested nanoparticle and immune allergic dysfunction to foods**

Proteins used in commercial food applications include casein, whey protein, collagen, egg white, and fish myofibrillar protein, and popular plant-based proteins including soybean protein and wheat gluten [7]. Compounds such as polysaccharides and proteins that are al‐ ready in use within commercial food applications are attractive candidates for the produc‐ tion of new nanocomposite packaging and encapsulation material, as several are generally regarded as safe and are biodegradable. However, food allergy has emerged as a growing health problem throughout modern society, and current research efforts towards the identi‐ fication and characterization of clinically relevant food allergens are critical to our under‐ standing of their role in the immunopathogenic mechanisms involved in hypersensitivity reactions, and the safety of novel and proposed food-oriented nanotechnology. Thus, the characterization and identification of the proteins responsible for immune-mediated food al‐ lergies is critical.

soluble proteins, β-lactalbumin, and β-lactoglobulin, resulted in anaphylactic reactions when administered orally. Interestingly, the soluble proteins were detected in the lamina propria of the small intestine of sensitized mice indicating that these proteins were able to transcytose through the enterocytes *in vivo.* This observation was confirmed *in vitro* using Caco-2 cells. Further, the challenge with sensitizing antigen resulted in significant levels of serum IgG1, and low, but detectable levels, of serum IgE and IgG2a. Casein, normally present within micelles, demonstrated a significant difference in anaphylactic induction. Or‐ al administration did not induce anaphylaxis. Instead, casein required a systemic adminis‐ tration (i.p. injection) in order to induce anaphylaxis; and it induced significantly higher serum IgE and IgG1 (Th2) allergic responses as compared to the soluble milk allergens. Fur‐ ther, transcytosis by casein through Caco2 monolayers was poor compared to the soluble milk allergens. When the tissue was examined by fluorescence microscopy, the casein was detectable in the Peyer's patches. Thus, these data indicated that the form of the sensitizing antigen was critical to the induction of an anaphylactic response. Next, the soluble allergens, β− lactoglobulin and soluble α-lactalbumin, were next converted into particulate aggregates by pasteurization; the process reportedly abolishes the monomeric form and supports the formation of aggregates of approximately 670 kDa. Pasteurization does not alter casein, and it exists in two predominant types as it would in its natural state: 180 kDa and 670 kDa. The conversion of soluble β− lactoglobulin and soluble α-lactalbumin into particulate aggregates by pasteurization altered the immunogenicity of the proteins such that they now required a systemic administration to induce anaphylaxis. Oral administration of either protein aggre‐ gate in sensitized mice did not induce anaphylaxis. The magnitude of the elicited serum IgG1 and IgE immunoglobulin production was much greater than that induced by their soluble forms. Further, the proteins were now detectable in association with the Peyer's Patches. Casein's induction was not altered by the process. Taken together, these results present the critical role of antigenic structure and its uptake across the epithelium as critical

Gastrointestinal Immunoregulation and the Challenges of Nanotechnology in Foods

http://dx.doi.org/10.5772/53287

507

The allergic state presents serious challenges to the incorporation of nanoparticles in food and food-associated products, particularly when considering the composition and ultimate biodistribution of the particles. The engineering of nanoparticle containing materials impli‐ cated, related, or identified as allergens raises concern for the initiation of alternate allergyinducing pathways in the host. For instance, while casein is incorporated in a variety of foods and is generally regarded as safe, it is also known to elicit strong allergic responses in afflicted individuals with dairy intolerance. Disruption of the epithelial barrier is known to result in gastrointestinal illness [100]. Infection and inflammation are conditions associated with a disruption of the epithelial layer leading to the increased paracellular transport of lu‐ minal antigen. Cytokines such as IFN-γ and TNF-α directly affect barrier function of the epi‐ thelium, the latter being implicated in milk allergy [101-103]. Thus, the transit of nanoparticle through a sensitized gastrointestinal system might result in a more complicat‐ ed scenario, depending upon the sensitizing antigen and the composition of the nanoparticle itself. Thus, casein nanoparticle constructs, with or without targeting lectins, might not be advisable for individuals with casein sensitivity. In this instance, the transit of the nanoparti‐ cle might be hastened across the layer, due to the pre-existing sensitivity, resulting in in‐

factors contributing to the allergic state.

In view of reported differences with respect to nanoparticle size and organ biodistribution, it is interesting to note that particles are often detected below the epithelium of the gastroin‐ testinal tract. Since the Intraepithelial lymphocytes (IEL) reside below the epithelial layer of the mammalian gastrointestinal tract, an understanding of the interactions between particle and resident IEL is crucial. Following the ingestion of food, digested protein fragments, or antigens, cross the epithelium to be processed and presented on the surface of class II mole‐ cule- bearing antigen presenting cells for recognition by specific TCR-bearing T cells. Aller‐ gic sensitization in the presence of IL-4 results in the generation of Th2 cells that will assist the development of IgE+ B cells. A repeat encounter with the antigen will result in a food allergic response. This event generates a skewed Th2 response, and will occur when luminal antigen is introduced to IgE bound onto IgE Fc receptor on the surface of mast cells. Thus, crossing the epithelial barrier to reach the mast cells is a critical step. The binding of antigen to the receptor-bound complex will result in the release of histamine, serotonin and prosta‐ glandins in anaphylactic reactions including those generated by food.

Recent studies suggest that intestinal epithelial cells play a central regulatory role in deter‐ mining the rate and pattern of uptake of ingested antigens. This is particularly critical in food allergy within the antigen-sensitized gastrointestinal tract. Studies using rats sensitized to horseradish peroxidase (HRP) showed that intestinal antigen transport is keenly affected by antigen-specific sensitization and is composed of 2 phases. The first phase consists of the rapid transepithelial transport of specific antigen from the lumen, via endocytosis, into the lamina propia. This phase is antigen specific, implying the existence of an antigen -specific receptor on the surface of the epithelial cells, and occurs within 2 minutes in sensitized rats as compared to a transit time of 20 minutes in non-sensitized, normal control animals. This is followed by a flow of the antigen in tight junctions resulting in an increase of antigen across the tissue. The second phase of antigen transport is not antigen specific, but is mark‐ edly increased by antigen challenge in sensitized rats compared with non-sensitized controls [98], indicative of the paracellular penetration through the epithelium by antigen. These studies clearly demonstrate that the kinetics of transport of antigen during IgE-mediated re‐ actions in the gastrointestinal tract is markedly increased across the epithelium. The result of this transport is the generation of a Th2 response.

Finally, a feeding study using mice orally sensitized to the known milk allergens, casein, βlactalbumin, and β-lactoglobulin, provided compelling evidence regarding the importance of the form of the antigen (soluble vs. particulate) for the induction of anaphylaxis [99]. The soluble proteins, β-lactalbumin, and β-lactoglobulin, resulted in anaphylactic reactions when administered orally. Interestingly, the soluble proteins were detected in the lamina propria of the small intestine of sensitized mice indicating that these proteins were able to transcytose through the enterocytes *in vivo.* This observation was confirmed *in vitro* using Caco-2 cells. Further, the challenge with sensitizing antigen resulted in significant levels of serum IgG1, and low, but detectable levels, of serum IgE and IgG2a. Casein, normally present within micelles, demonstrated a significant difference in anaphylactic induction. Or‐ al administration did not induce anaphylaxis. Instead, casein required a systemic adminis‐ tration (i.p. injection) in order to induce anaphylaxis; and it induced significantly higher serum IgE and IgG1 (Th2) allergic responses as compared to the soluble milk allergens. Fur‐ ther, transcytosis by casein through Caco2 monolayers was poor compared to the soluble milk allergens. When the tissue was examined by fluorescence microscopy, the casein was detectable in the Peyer's patches. Thus, these data indicated that the form of the sensitizing antigen was critical to the induction of an anaphylactic response. Next, the soluble allergens, β− lactoglobulin and soluble α-lactalbumin, were next converted into particulate aggregates by pasteurization; the process reportedly abolishes the monomeric form and supports the formation of aggregates of approximately 670 kDa. Pasteurization does not alter casein, and it exists in two predominant types as it would in its natural state: 180 kDa and 670 kDa. The conversion of soluble β− lactoglobulin and soluble α-lactalbumin into particulate aggregates by pasteurization altered the immunogenicity of the proteins such that they now required a systemic administration to induce anaphylaxis. Oral administration of either protein aggre‐ gate in sensitized mice did not induce anaphylaxis. The magnitude of the elicited serum IgG1 and IgE immunoglobulin production was much greater than that induced by their soluble forms. Further, the proteins were now detectable in association with the Peyer's Patches. Casein's induction was not altered by the process. Taken together, these results present the critical role of antigenic structure and its uptake across the epithelium as critical factors contributing to the allergic state.

ready in use within commercial food applications are attractive candidates for the produc‐ tion of new nanocomposite packaging and encapsulation material, as several are generally regarded as safe and are biodegradable. However, food allergy has emerged as a growing health problem throughout modern society, and current research efforts towards the identi‐ fication and characterization of clinically relevant food allergens are critical to our under‐ standing of their role in the immunopathogenic mechanisms involved in hypersensitivity reactions, and the safety of novel and proposed food-oriented nanotechnology. Thus, the characterization and identification of the proteins responsible for immune-mediated food al‐

In view of reported differences with respect to nanoparticle size and organ biodistribution, it is interesting to note that particles are often detected below the epithelium of the gastroin‐ testinal tract. Since the Intraepithelial lymphocytes (IEL) reside below the epithelial layer of the mammalian gastrointestinal tract, an understanding of the interactions between particle and resident IEL is crucial. Following the ingestion of food, digested protein fragments, or antigens, cross the epithelium to be processed and presented on the surface of class II mole‐ cule- bearing antigen presenting cells for recognition by specific TCR-bearing T cells. Aller‐ gic sensitization in the presence of IL-4 results in the generation of Th2 cells that will assist

allergic response. This event generates a skewed Th2 response, and will occur when luminal antigen is introduced to IgE bound onto IgE Fc receptor on the surface of mast cells. Thus, crossing the epithelial barrier to reach the mast cells is a critical step. The binding of antigen to the receptor-bound complex will result in the release of histamine, serotonin and prosta‐

Recent studies suggest that intestinal epithelial cells play a central regulatory role in deter‐ mining the rate and pattern of uptake of ingested antigens. This is particularly critical in food allergy within the antigen-sensitized gastrointestinal tract. Studies using rats sensitized to horseradish peroxidase (HRP) showed that intestinal antigen transport is keenly affected by antigen-specific sensitization and is composed of 2 phases. The first phase consists of the rapid transepithelial transport of specific antigen from the lumen, via endocytosis, into the lamina propia. This phase is antigen specific, implying the existence of an antigen -specific receptor on the surface of the epithelial cells, and occurs within 2 minutes in sensitized rats as compared to a transit time of 20 minutes in non-sensitized, normal control animals. This is followed by a flow of the antigen in tight junctions resulting in an increase of antigen across the tissue. The second phase of antigen transport is not antigen specific, but is mark‐ edly increased by antigen challenge in sensitized rats compared with non-sensitized controls [98], indicative of the paracellular penetration through the epithelium by antigen. These studies clearly demonstrate that the kinetics of transport of antigen during IgE-mediated re‐ actions in the gastrointestinal tract is markedly increased across the epithelium. The result of

Finally, a feeding study using mice orally sensitized to the known milk allergens, casein, βlactalbumin, and β-lactoglobulin, provided compelling evidence regarding the importance of the form of the antigen (soluble vs. particulate) for the induction of anaphylaxis [99]. The

glandins in anaphylactic reactions including those generated by food.

this transport is the generation of a Th2 response.

B cells. A repeat encounter with the antigen will result in a food

lergies is critical.

506 Food Industry

the development of IgE+

The allergic state presents serious challenges to the incorporation of nanoparticles in food and food-associated products, particularly when considering the composition and ultimate biodistribution of the particles. The engineering of nanoparticle containing materials impli‐ cated, related, or identified as allergens raises concern for the initiation of alternate allergyinducing pathways in the host. For instance, while casein is incorporated in a variety of foods and is generally regarded as safe, it is also known to elicit strong allergic responses in afflicted individuals with dairy intolerance. Disruption of the epithelial barrier is known to result in gastrointestinal illness [100]. Infection and inflammation are conditions associated with a disruption of the epithelial layer leading to the increased paracellular transport of lu‐ minal antigen. Cytokines such as IFN-γ and TNF-α directly affect barrier function of the epi‐ thelium, the latter being implicated in milk allergy [101-103]. Thus, the transit of nanoparticle through a sensitized gastrointestinal system might result in a more complicat‐ ed scenario, depending upon the sensitizing antigen and the composition of the nanoparticle itself. Thus, casein nanoparticle constructs, with or without targeting lectins, might not be advisable for individuals with casein sensitivity. In this instance, the transit of the nanoparti‐ cle might be hastened across the layer, due to the pre-existing sensitivity, resulting in in‐ creased transit through the layer, perhaps overwhelming the resident macrophage phagocytic activity (Figure 4), leading to exacerbation of the allergic state or the generation of alternative immunologic reactions.

**Abbreviations**

**Acknowledgement**

**Author details**

MaryAnn Principato

**References**

3/22/12)

2012;3:263-70.

and Technology. 2012;24:30-46.

gram/default.htm (accessed 3/22/12)

ence. 2010;75(1):R43-9. Epub 2010/05/25.

GALT, gut-associated lymphoid tissue; Ig, immunoglobulin; M cell, microfold/membranous cell; CD, Cluster designation; sIgA, surface IgA; MHC, major histocompatibility complex;

Gastrointestinal Immunoregulation and the Challenges of Nanotechnology in Foods

http://dx.doi.org/10.5772/53287

509

[1] Anonymous. Nanotechnology 101. Nano.gov/nanotech-101/special. 2009. (accessed

[2] Cushen M, Kerry J, Morris M, Cruz-Romero M, Cummins E. Nanotechnologies in the food industry-Recent developments, risks and regulation. . Trends in Food Science

[3] Gibis M, Vogt E, Weiss J. Encapsulation of polyphenolic grape seed extract in poly‐ mer-coated liposomes. Food & function. 2012;3(3):246-54. Epub 2011/11/26.

[4] Markman G and Yoav L. Maillard-conjugate based core shell co-assemblies for nano‐ encapsulation of hydrophobic nutraceuticals in clear beverges. Food & function.

[5] Huang Q, Yu H, Ru Q. Bioavailability and delivery of nutraceuticals using nanotech‐

[6] Anonymous FaDA. About the FCS Review Program 2010. http://www.fda.gov/Food/ FoodIngredientsPackaging/FoodContactSubstancesFCS/AbouttheFCSReviewPro‐

[7] Arora A, Padua GW. Review: nanocomposites in food packaging. Journal of food sci‐

nology. Journal of food science. 2010;75(1):R50-7. Epub 2010/05/25.

BSA, bovine serum albumin; PP, Peyer's patch: MLN, mesenteric lymph node

The author thanks Dr. Jeffrey Yourick for careful reading of the manuscript.

Address all correspondence to: maryann.principato@fda.hhs.gov

Food and Drug Administration, CFSAN/OARSA, Laurel, MD, USA

## **6. Conclusions**

The choice of material used in the formulation of nanoparticles and spheres during the for‐ mulation of encapsulated nutrients or supplements intended for ingestion can be critical to the possible outcomes in mucosal immunity. A crucial consideration is whether the material will influence the induction of either tolerance or active immunity to the introduced nutrient as a result of its deposition within the gastrointestinal tract and possible interaction with res‐ ident effector cells.

The specific targeting of the nanoparticles and spheres using specific ligand interactions pro‐ vides an advantage in this respect. While polymers containing natural biodegradable mate‐ rials such as chitosan, PLGA, whey, casein, and others offer great advantages within this technology, they also present further challenges towards an understanding of the mecha‐ nism involved in the maintenance of gastrointestinal immune homeostasis, and preventing the induction or potentiation of immune dysfunction.

## **Abbreviations**

creased transit through the layer, perhaps overwhelming the resident macrophage phagocytic activity (Figure 4), leading to exacerbation of the allergic state or the generation

**Figure 4.** Schematic representation of nanoparticle transit through the epithelial layer and the allergic state. An in‐ creased rate of transit by the nanoparticles is theorized as a result of the induction or presence of an allergic state.

The choice of material used in the formulation of nanoparticles and spheres during the for‐ mulation of encapsulated nutrients or supplements intended for ingestion can be critical to the possible outcomes in mucosal immunity. A crucial consideration is whether the material will influence the induction of either tolerance or active immunity to the introduced nutrient as a result of its deposition within the gastrointestinal tract and possible interaction with res‐

The specific targeting of the nanoparticles and spheres using specific ligand interactions pro‐ vides an advantage in this respect. While polymers containing natural biodegradable mate‐ rials such as chitosan, PLGA, whey, casein, and others offer great advantages within this technology, they also present further challenges towards an understanding of the mecha‐ nism involved in the maintenance of gastrointestinal immune homeostasis, and preventing

of alternative immunologic reactions.

508 Food Industry

**6. Conclusions**

ident effector cells.

the induction or potentiation of immune dysfunction.

GALT, gut-associated lymphoid tissue; Ig, immunoglobulin; M cell, microfold/membranous cell; CD, Cluster designation; sIgA, surface IgA; MHC, major histocompatibility complex; BSA, bovine serum albumin; PP, Peyer's patch: MLN, mesenteric lymph node

## **Acknowledgement**

The author thanks Dr. Jeffrey Yourick for careful reading of the manuscript.

## **Author details**

MaryAnn Principato

Address all correspondence to: maryann.principato@fda.hhs.gov

Food and Drug Administration, CFSAN/OARSA, Laurel, MD, USA

## **References**


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**Chapter 23**

**Yeast: World's Finest** *Chef*

http://dx.doi.org/10.5772/53156

scribed approximately 1500 species [1].

**1. Introduction**

Fábio Faria-Oliveira, Sónia Puga and Célia Ferreira

Yeast is the simplest eukaryotic organism of our days. They are unicellular microorganisms classified in the kingdom Fungi. Nevertheless, yeasts were probably the first microorganism to be domesticated and since early in human history have been used on a daily basis in bread making and in alcoholic beverages. Nowadays, yeast has become a key microorgan‐ ism for many types of industrial and food processing manufactures, including the produc‐ tion of beer, wine, cheese and bread. In particular, its use in baking industry is quite relevant due to the central role of bread as a dietary product all over the world. Moreover, yeasts are regarded with reasonably interest as nutrients and health provider sources both for humans

as well as for animals. We dare to appoint yeast as the one of the world finest chefs.

Yeasts are found in diverse natural environments; colonizing from terrestrial, to aerial and aquatic environments. They can be found on decomposing fruit, on soils, as opportunistic pathogens in human beings, in the gut of the fish and free living in the sea. In general they contribute to the decay of organic material, but their successful colonization is intimately re‐ lated to their capacity of physiologically adapt at diverse milieus. Hitherto, it has been de‐

This chapter aims at contribute to a comprehensible analysis of the role of yeasts on the ac‐ tual feed lifestyle, mainly in what regards the yeast *Saccharomyces cerevisiae*. This yeast is known with by many appellations: "Baker's yeast" in baking and confectionery fields, "Brewer's Yeast" by all beer industrial and artisanal producers, and perhaps less familiar "Wine's Yeast" by wine-like alcoholic beverages producers. We will first go over several physiologic aspects of this yeast metabolism, specifically associated with glucose catabolism, under anaerobic environments (fermentation) as well as aerobic conditions. Most of our at‐ tention is given to glycolysis pathway and to alcoholic fermentation in order to prepare the reader for the issues discussed later. Considerable notice will be paid to the intervention of

and reproduction in any medium, provided the original work is properly cited.

© 2013 Faria-Oliveira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

## **Chapter 23**

## **Yeast: World's Finest** *Chef*

Fábio Faria-Oliveira, Sónia Puga and Célia Ferreira

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53156

## **1. Introduction**

Yeast is the simplest eukaryotic organism of our days. They are unicellular microorganisms classified in the kingdom Fungi. Nevertheless, yeasts were probably the first microorganism to be domesticated and since early in human history have been used on a daily basis in bread making and in alcoholic beverages. Nowadays, yeast has become a key microorgan‐ ism for many types of industrial and food processing manufactures, including the produc‐ tion of beer, wine, cheese and bread. In particular, its use in baking industry is quite relevant due to the central role of bread as a dietary product all over the world. Moreover, yeasts are regarded with reasonably interest as nutrients and health provider sources both for humans as well as for animals. We dare to appoint yeast as the one of the world finest chefs.

Yeasts are found in diverse natural environments; colonizing from terrestrial, to aerial and aquatic environments. They can be found on decomposing fruit, on soils, as opportunistic pathogens in human beings, in the gut of the fish and free living in the sea. In general they contribute to the decay of organic material, but their successful colonization is intimately re‐ lated to their capacity of physiologically adapt at diverse milieus. Hitherto, it has been de‐ scribed approximately 1500 species [1].

This chapter aims at contribute to a comprehensible analysis of the role of yeasts on the ac‐ tual feed lifestyle, mainly in what regards the yeast *Saccharomyces cerevisiae*. This yeast is known with by many appellations: "Baker's yeast" in baking and confectionery fields, "Brewer's Yeast" by all beer industrial and artisanal producers, and perhaps less familiar "Wine's Yeast" by wine-like alcoholic beverages producers. We will first go over several physiologic aspects of this yeast metabolism, specifically associated with glucose catabolism, under anaerobic environments (fermentation) as well as aerobic conditions. Most of our at‐ tention is given to glycolysis pathway and to alcoholic fermentation in order to prepare the reader for the issues discussed later. Considerable notice will be paid to the intervention of

© 2013 Faria-Oliveira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

yeast in alcoholic beverages, in particular beer and wine, important economical industries of our times. The particular role of *S. cerevisiae* in baking industry, interactions with lactic acid bacteria (LAB) in sourdoughs, and also the scientific approaches/advances for sustainability were exhaustively reviewed, recently by us in [2] but also by [3], therefore we will focus preferentially on the production of commercial yeast for baking. In the end, we will briefly review the other applications of *S. cerevisiae* in less familiar products, including animals and fish feeding rations and biotic supplements.

The gene family of hexose transporters in *S. cerevisiae* consists of more than 20 members: i) 18 genes encoding transporters (*HXT*1-*HXT*17, *GAL*2), being the most relevant Hxt1p and Hxt3p, with a low affinity for glucose and high transport capacity, and Hxt2p, Hxt4p and Hxt7p, with a high affinity and low transport capacity and; ii) at least two genes encoding sensors (*SNF*3, *RGT*2), although several points of evidence suggest that *GPR*1 and *HXK*2 also sense and signal glucose levels [6, 8]. All these, sensors and transporters are therefore the primary interveners on sugar metabolism. After glucose uptake, it enters in the glycolytic pathway (Figure 1 – Steps from glucose to pyruvate) in order to be metabolized to pyruvate, whereby production of ener‐ gy in form of ATP is coupled to the generation of intermediates and reducing power in form of

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 521

The first step of the glycolytic pathway consists on the phosphorylation of glucose to glu‐ cose 6-phosphate by the action of the hexokinases (Hxkp) and the glucokinase (Glkp); which are linked to high-affinity glucose uptake. Then glucose-6-phosphate is isomerized by the phosphoglucose isomerase, encoded by *PGI* gene, to fructose-6-phosphate. The next step, done by the phosphofructokinase (Pfkp) requires energy, in the form of ATP, to convert

NADH for biosynthetic pathways (for reviews see [5, 6, 9]) [10].

**Figure 1.** Alcoholic fermentation - enzymatic steps on *S. cerevisiae* (adapted from [11]).

fructose-6-phosphate into fructose 1,6-biphosphate.

#### **1.1. Yeast metabolism**

Yeasts, resembling other heterotrophic organisms, have the energy and carbon metabolism operating in concert, *i.e.*, anabolism is coupled with catabolism. Their chemical energy, in the form of ATP, results from the oxidation of organic molecules and is used as energy re‐ source by the cell. On the other hand, those organic molecules can also operate as carbon sources for biosynthesis. Yeast environmental diversity leads to a vast metabolic complexity, due to the multiplicity of carbon and the energy sources available in nature. This includes polyols, alcohols, organic acids and amino acids yet, yeasts preferentially metabolize sugars.

#### *1.1.1. Greedy yeasts – Sugar metabolism*

The yeast metabolize diverse sugars, hexoses such as glucose, fructose, galactose or man‐ nose, some can use pentoses like xylose or arabinose, disaccharides as maltose or sucrose; yet, glucose and fructose are the preferred substrates. The metabolic routes for the dissimu‐ lation of hexoses and disaccharides share the same pathways, with the great majority of the metabolic elements arising from intermediaries of glycolysis, the tricarboxylic acid cycle (TCA) and the pentose phosphate pathway, and differ only in the initial basic steps of me‐ tabolism.

The sugar dissimilation may occur in anaerobic or in aerobic environment. In the first case is called **fermentation** and in the presence of oxygen is named **respiration**. The most common process is the glucose dissimilation, generally known as **alcoholic fermentation**, which oc‐ curs anaerobically and yields as final products: ethanol and CO2.

For the sugar utilization, yeast has primarily to sense the presence of glucose in the environ‐ ment and then to transport it across the plasma membrane [4, 5]. The presence and levels of glucose sensed by the yeast can influence the enzyme levels through several processes, alter‐ ation of mRNA translation rates; mRNA stability or protein degradation, but also the con‐ centration of intracellular metabolites (for a review see [6]). Yet, the major outcome is the extensive transcriptional regulation of a large number of genes leading to the adaptation to fermentative metabolism (alcoholic fermentation). These encompasses the induction of genes required for the utilization of glucose, such as genes encoding glycolytic pathway en‐ zymes (discussed below), whereas genes required for the metabolism of alternative sub‐ strates, and those encoding proteins in the gluconeogenic and respiratory pathways are repressed by glucose (for reviews see [6] and [7]).

The gene family of hexose transporters in *S. cerevisiae* consists of more than 20 members: i) 18 genes encoding transporters (*HXT*1-*HXT*17, *GAL*2), being the most relevant Hxt1p and Hxt3p, with a low affinity for glucose and high transport capacity, and Hxt2p, Hxt4p and Hxt7p, with a high affinity and low transport capacity and; ii) at least two genes encoding sensors (*SNF*3, *RGT*2), although several points of evidence suggest that *GPR*1 and *HXK*2 also sense and signal glucose levels [6, 8]. All these, sensors and transporters are therefore the primary interveners on sugar metabolism. After glucose uptake, it enters in the glycolytic pathway (Figure 1 – Steps from glucose to pyruvate) in order to be metabolized to pyruvate, whereby production of ener‐ gy in form of ATP is coupled to the generation of intermediates and reducing power in form of NADH for biosynthetic pathways (for reviews see [5, 6, 9]) [10].

yeast in alcoholic beverages, in particular beer and wine, important economical industries of our times. The particular role of *S. cerevisiae* in baking industry, interactions with lactic acid bacteria (LAB) in sourdoughs, and also the scientific approaches/advances for sustainability were exhaustively reviewed, recently by us in [2] but also by [3], therefore we will focus preferentially on the production of commercial yeast for baking. In the end, we will briefly review the other applications of *S. cerevisiae* in less familiar products, including animals and

Yeasts, resembling other heterotrophic organisms, have the energy and carbon metabolism operating in concert, *i.e.*, anabolism is coupled with catabolism. Their chemical energy, in the form of ATP, results from the oxidation of organic molecules and is used as energy re‐ source by the cell. On the other hand, those organic molecules can also operate as carbon sources for biosynthesis. Yeast environmental diversity leads to a vast metabolic complexity, due to the multiplicity of carbon and the energy sources available in nature. This includes polyols, alcohols, organic acids and amino acids yet, yeasts preferentially metabolize sugars.

The yeast metabolize diverse sugars, hexoses such as glucose, fructose, galactose or man‐ nose, some can use pentoses like xylose or arabinose, disaccharides as maltose or sucrose; yet, glucose and fructose are the preferred substrates. The metabolic routes for the dissimu‐ lation of hexoses and disaccharides share the same pathways, with the great majority of the metabolic elements arising from intermediaries of glycolysis, the tricarboxylic acid cycle (TCA) and the pentose phosphate pathway, and differ only in the initial basic steps of me‐

The sugar dissimilation may occur in anaerobic or in aerobic environment. In the first case is called **fermentation** and in the presence of oxygen is named **respiration**. The most common process is the glucose dissimilation, generally known as **alcoholic fermentation**, which oc‐

For the sugar utilization, yeast has primarily to sense the presence of glucose in the environ‐ ment and then to transport it across the plasma membrane [4, 5]. The presence and levels of glucose sensed by the yeast can influence the enzyme levels through several processes, alter‐ ation of mRNA translation rates; mRNA stability or protein degradation, but also the con‐ centration of intracellular metabolites (for a review see [6]). Yet, the major outcome is the extensive transcriptional regulation of a large number of genes leading to the adaptation to fermentative metabolism (alcoholic fermentation). These encompasses the induction of genes required for the utilization of glucose, such as genes encoding glycolytic pathway en‐ zymes (discussed below), whereas genes required for the metabolism of alternative sub‐ strates, and those encoding proteins in the gluconeogenic and respiratory pathways are

curs anaerobically and yields as final products: ethanol and CO2.

repressed by glucose (for reviews see [6] and [7]).

fish feeding rations and biotic supplements.

*1.1.1. Greedy yeasts – Sugar metabolism*

**1.1. Yeast metabolism**

520 Food Industry

tabolism.

The first step of the glycolytic pathway consists on the phosphorylation of glucose to glu‐ cose 6-phosphate by the action of the hexokinases (Hxkp) and the glucokinase (Glkp); which are linked to high-affinity glucose uptake. Then glucose-6-phosphate is isomerized by the phosphoglucose isomerase, encoded by *PGI* gene, to fructose-6-phosphate. The next step, done by the phosphofructokinase (Pfkp) requires energy, in the form of ATP, to convert fructose-6-phosphate into fructose 1,6-biphosphate.

**Figure 1.** Alcoholic fermentation - enzymatic steps on *S. cerevisiae* (adapted from [11]).

Yeast phosphofructokinase, Pfkp, is a heterooctameric enzyme subject to a complex alloster‐ ic regulation. Aldolase (fructose 1,6-bisphosphate aldolase- Fbap) in turn, catalyses the re‐ versible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. These two compounds can be converted one into another, again in a reversible way, by the triosephosphate isomerase (Tpip). Subsequently, glyceral‐ dehyde 3-phosphate yields the pyruvate by the action of a series of acting enzymes, whereas some of the dihydroxyacetone phosphate follows gluconeogenesis. Glyceraldehyde 3-phos‐ phate is firstly oxidised by NAD+ (with the production of a reducing equivalent, which will take part in the latter steps of glycolysis when acetaldehyde gives ethanol (Figure 1)) and then phosphorylated, under the catalysis of the 3-phosphate dehydrogenase (Tdhp). The re‐ sulting 1,3-diphosphoglycerate, by the action of phosphoglycerate kinase (Pgkp), donates a phosphate group to an ADP molecule originating the 3 phosphoglycerate and releasing 1 molecule of energy (ATP). The next step is just a relocation of the phosphate group on posi‐ tion 2, done by the phosphoglycerate mutase (Pgmp); preparing this way the following reac‐ tion, the dehydration by the enolase (Enop) and from which results the phosphoenol pyruvate, a high energetic molecule. This is then phosphorylated by the pyruvate kinase (Pykp) giving the pyruvate and also releasing another molecule of ATP.

In alcoholic fermentation, pyruvate is decarboxylated to give acetaldehyde and CO2, by the pyruvate decarboxylase (Pdc1p). In the final reaction, catalysed by the alcohol dehydrogen‐ ase (Adhp), acetaldehyde is reduced yielding the ethanol and promoting the re-oxidation of

formed per molecule of glucose, the sugar is incorporated into other by-products such as yeast biomass, acids (pyruvic, acetaldehyde, ketoglutaric, lactic) and also importantly glyc‐ erol. This is generated from dihydroxyacetone-phosphate and is, to a certain extent, very de‐ sired by the wine producers in order to get fuller bodied wines (discussed below). Furthermore, alcoholic fermentation is a redox-neutral process; given that the NADH pro‐ duced during the oxidation of glyceraldehyde 3-phosphate is afterwards reoxidized in the reduction of acetaldehyde to ethanol [13]. Yet, one must keep in mind that with fermenta‐ tion is associated culture growth and, biomass composition is more oxidized than glucose, consequently an excess of reducing equivalents may be attained. The way yeast circumvent this problem, under anaerobic conditions, consists on the production of glycerol by reduc‐ tion of the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate cat‐

isogenes *GPD*1 and *GPD*2), and its subsequent dephosphorylation due to the action of glyc‐

Although fermentation usually happens in the absence of oxygen, this is not a strict rule. Even in the presence of high levels of oxygen, if the sugars are fully accessible to be metabo‐ lized, yeasts choose to ferment instead of respire. This phenomenon is called the Crabtree effect [17], defined as the inhibition of aerobic metabolism when glucose is available, which occurs both in the presence or absence of oxygen. For instance, *S. cerevisiae* is known as Crabtree negative yeast, since is able to produce ethanol aerobically in the presence of high external glucose concentrations. These high concentrations promote the acceleration of gly‐ colysis, producing appreciable amounts of ATP through substrate-level phosphorylation. Si‐ multaneously, it reduces the need of oxidative phosphorylation done by the TCA cycle via the electron transport chain, inhibits respiration and ATP synthesis, and therefore decreases oxygen consumption. Conversely, Crabtree negative yeasts produce biomass via TCA cycle, but these should not be mystified with obligate aerobes: Crabtree-negative yeasts are able to ferment, yet usually only ferment under anaerobic conditions, since there is no inhibition of aerobic respiration in the presence of glucose and this is a more efficient form of energy me‐ tabolism. Obligate aerobe yeasts, on the other hand, cannot ferment and only respire aerobi‐ cally, providing another category of metabolic diversity. Moreover, Crabtree effect is not specific to yeasts: many mammalian tumour cells display a Crabtree effect as well [18-20].

In **aerobic respiration** (Figure 3), the pyruvate is converted to Acetyl-CoA due to an oxida‐ tive decarboxylation, catalysed by the pyruvate dehydrogenase multi enzyme complex. In this way starts TCA cycle, which major issue is to supply the respiratory chain with reduc‐ ing equivalents (in form of NADH and FADH2) obtained from the oxidative decarboxylation of Acetyl-CoA, which is then used to generate energy through the highly conserved electron transport chain. Moreover TCA cycle also has anabolic functions, almost all intermediates

erol 3-phosphatase (encoded by *GPP*1 and *GPP*2) [14-16].

. At the same time, and in addition to the 2 molecules of CO2 and of ethanol,

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 523


NADH to NAD+

alysed by NAD+

At this point, pyruvate can follow distinguished metabolic routes (Figure 2) depending on the environmental conditions, which in turn regulate the enzymes involved as well as their kinet‐ ics properties, but also of the yeast species [12]. Conversely, the carbon flux gets to a branch‐ ing point in which may be divided among the respiratory and the fermentative pathways.

**Figure 2.** Pyruvate formed in glycolysis alternative metabolic routes. Pyruvate can be converted into 2 intermediates of TCA cycle: acetyl–CoA by the pyruvate dehydrogenase complex (Pdhp) and transported to the mitochondria by mi‐ tochondrial oxaloacetate carrier (Oacp); and/or oxaloacetate by pyruvate carboxylase (Pyc1p/2p) whose mitochondri‐ al carrier is (Mpc1p/2p). Pyruvate can also be decarboxylated to give acetaldehyde by the pyruvate decarboxylase (Pdc1p). Adh1p - alcohol dehydrogenase; Ald5p - acetaldehyde dehydrogenase; Acs1p/2p - acetyl–CoA synthase; Yat1p/2p - carnitine acetyltransferase (adapted from [9]).

In alcoholic fermentation, pyruvate is decarboxylated to give acetaldehyde and CO2, by the pyruvate decarboxylase (Pdc1p). In the final reaction, catalysed by the alcohol dehydrogen‐ ase (Adhp), acetaldehyde is reduced yielding the ethanol and promoting the re-oxidation of NADH to NAD+ . At the same time, and in addition to the 2 molecules of CO2 and of ethanol, formed per molecule of glucose, the sugar is incorporated into other by-products such as yeast biomass, acids (pyruvic, acetaldehyde, ketoglutaric, lactic) and also importantly glyc‐ erol. This is generated from dihydroxyacetone-phosphate and is, to a certain extent, very de‐ sired by the wine producers in order to get fuller bodied wines (discussed below). Furthermore, alcoholic fermentation is a redox-neutral process; given that the NADH pro‐ duced during the oxidation of glyceraldehyde 3-phosphate is afterwards reoxidized in the reduction of acetaldehyde to ethanol [13]. Yet, one must keep in mind that with fermenta‐ tion is associated culture growth and, biomass composition is more oxidized than glucose, consequently an excess of reducing equivalents may be attained. The way yeast circumvent this problem, under anaerobic conditions, consists on the production of glycerol by reduc‐ tion of the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate cat‐ alysed by NAD+ -dependent glycerol 3-phosphate dehydrogenase (encoded by the two isogenes *GPD*1 and *GPD*2), and its subsequent dephosphorylation due to the action of glyc‐ erol 3-phosphatase (encoded by *GPP*1 and *GPP*2) [14-16].

Yeast phosphofructokinase, Pfkp, is a heterooctameric enzyme subject to a complex alloster‐ ic regulation. Aldolase (fructose 1,6-bisphosphate aldolase- Fbap) in turn, catalyses the re‐ versible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. These two compounds can be converted one into another, again in a reversible way, by the triosephosphate isomerase (Tpip). Subsequently, glyceral‐ dehyde 3-phosphate yields the pyruvate by the action of a series of acting enzymes, whereas some of the dihydroxyacetone phosphate follows gluconeogenesis. Glyceraldehyde 3-phos‐

take part in the latter steps of glycolysis when acetaldehyde gives ethanol (Figure 1)) and then phosphorylated, under the catalysis of the 3-phosphate dehydrogenase (Tdhp). The re‐ sulting 1,3-diphosphoglycerate, by the action of phosphoglycerate kinase (Pgkp), donates a phosphate group to an ADP molecule originating the 3 phosphoglycerate and releasing 1 molecule of energy (ATP). The next step is just a relocation of the phosphate group on posi‐ tion 2, done by the phosphoglycerate mutase (Pgmp); preparing this way the following reac‐ tion, the dehydration by the enolase (Enop) and from which results the phosphoenol pyruvate, a high energetic molecule. This is then phosphorylated by the pyruvate kinase

At this point, pyruvate can follow distinguished metabolic routes (Figure 2) depending on the environmental conditions, which in turn regulate the enzymes involved as well as their kinet‐ ics properties, but also of the yeast species [12]. Conversely, the carbon flux gets to a branch‐ ing point in which may be divided among the respiratory and the fermentative pathways.

**Figure 2.** Pyruvate formed in glycolysis alternative metabolic routes. Pyruvate can be converted into 2 intermediates of TCA cycle: acetyl–CoA by the pyruvate dehydrogenase complex (Pdhp) and transported to the mitochondria by mi‐ tochondrial oxaloacetate carrier (Oacp); and/or oxaloacetate by pyruvate carboxylase (Pyc1p/2p) whose mitochondri‐ al carrier is (Mpc1p/2p). Pyruvate can also be decarboxylated to give acetaldehyde by the pyruvate decarboxylase (Pdc1p). Adh1p - alcohol dehydrogenase; Ald5p - acetaldehyde dehydrogenase; Acs1p/2p - acetyl–CoA synthase;

Yat1p/2p - carnitine acetyltransferase (adapted from [9]).

(Pykp) giving the pyruvate and also releasing another molecule of ATP.

(with the production of a reducing equivalent, which will

phate is firstly oxidised by NAD+

522 Food Industry

Although fermentation usually happens in the absence of oxygen, this is not a strict rule. Even in the presence of high levels of oxygen, if the sugars are fully accessible to be metabo‐ lized, yeasts choose to ferment instead of respire. This phenomenon is called the Crabtree effect [17], defined as the inhibition of aerobic metabolism when glucose is available, which occurs both in the presence or absence of oxygen. For instance, *S. cerevisiae* is known as Crabtree negative yeast, since is able to produce ethanol aerobically in the presence of high external glucose concentrations. These high concentrations promote the acceleration of gly‐ colysis, producing appreciable amounts of ATP through substrate-level phosphorylation. Si‐ multaneously, it reduces the need of oxidative phosphorylation done by the TCA cycle via the electron transport chain, inhibits respiration and ATP synthesis, and therefore decreases oxygen consumption. Conversely, Crabtree negative yeasts produce biomass via TCA cycle, but these should not be mystified with obligate aerobes: Crabtree-negative yeasts are able to ferment, yet usually only ferment under anaerobic conditions, since there is no inhibition of aerobic respiration in the presence of glucose and this is a more efficient form of energy me‐ tabolism. Obligate aerobe yeasts, on the other hand, cannot ferment and only respire aerobi‐ cally, providing another category of metabolic diversity. Moreover, Crabtree effect is not specific to yeasts: many mammalian tumour cells display a Crabtree effect as well [18-20].

In **aerobic respiration** (Figure 3), the pyruvate is converted to Acetyl-CoA due to an oxida‐ tive decarboxylation, catalysed by the pyruvate dehydrogenase multi enzyme complex. In this way starts TCA cycle, which major issue is to supply the respiratory chain with reduc‐ ing equivalents (in form of NADH and FADH2) obtained from the oxidative decarboxylation of Acetyl-CoA, which is then used to generate energy through the highly conserved electron transport chain. Moreover TCA cycle also has anabolic functions, almost all intermediates are utilized in other metabolic reactions; exception is made to isocitrate, including the syn‐ thesis of amino acids and nucleotides (for reviews see [9, 21]).

pecially relevant role in religious ceremonies to Ninkasi, the Sumerian Goddess of beer [26]. Babylonians succeeded the Sumerians and kept producing the fermented beverage. They be‐ came so skilful at this art that at least 20 different brews were produced and exported, to as far as Egypt, at the zenith of Babylonian empire. Egyptians were so adept of this "imported beer" that they started producing their own from unbaked dough and even created a special hieroglyph for this new craft. Furthermore, records show that pyramid workers were paid in beer, a readily storable merchandise, and Pharaohs were entombed with model breweries to ensure an afterlife beer supply [26, 27]. Beer popularity grew and this beverage spread for

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 525

Wine was also very popular in the ancient cultures, with references to this beverage in reli‐ gious ceremonies of Egypt and Phoenicia. Pharaohs tombs were frequently adorned with vin‐ tage scenes and jars filled with wine accompanied the Kings afterlife [11, 23]. Wine consumption spread with the rise of the Greek and Roman Empires. Under the Greek and Ro‐ man influence, wine earned the status of "Civilized" drink, becoming very popular with the Empire upper classes, and beer was labelled as a "Barbarian" drink. Wine production and vine cultivation spread across Europe and replaced beer as the main drink in many countries. Some of these are still nowadays associated with wine production like Portugal, Spain and France. The production and consumption of beer continued mainly in northern borders of the Roman

In Middle Ages, wine and beer production gained a new impetus with the shift from the fa‐ miliar production to a more centralized production in monasteries [28]. Such happened be‐ cause, at the time, water was frequently polluted, so alcoholic beverages were safer than water for the monks' consumption. Additionally, during the long fasting periods that the monks subjected themselves, the drinking of these highly nutritious beverages became com‐ mon to satisfy hunger. This happened because wine and beer were considered similar to water and didn't constitute a breach of fast. In fact, in some monasteries monks were al‐ lowed to drink up to 5 litres of beer per day [28]. Southern monasteries produced mainly wine, as the weather was warmer and suitable for vines, but in the north the colder weather was more fitting barley and wheat growth and therefore Northern monasteries were more devoted to beer production. Each monastery developed its own methodologies for wine and beer making, leading to new wines and new brews and to a great technical improvement.

From the sixteenth century, with the discovery of the New World by Portuguese and the Spanish explorers, wine and beer spread to new territories. Vines were introduced in Brazil by the Portuguese around 1500 [29], and in Africa by the Dutch around 1650. In the Austral‐

In the nineteenth century, wine and beer making suffered probably the major scientific ad‐ vances. Around 1860 Louis Pasteur, a name forever associated with wine and beer produc‐ tion, developed studies on the conservation of wine through a heating-cooling process later known as "pasteurization", showing that wine could be stored for longer periods after such treatment. Moreover, in 1870 Pasteur made known to the world the role of *S. cerevisiae* in the fermentation process. Later, in 1876 Pasteur conducted similar studies in beer in his work

Empire, where Germanic tribes ruled and Roman influence was weaker [26, 28].

Later these products become a source of income for the monasteries.

ian continent and North America this happened later, around 1800.

the entire Europe, especially in the Mediterranean region [28].

**Figure 3.** Aerobic respiration in *S. cerevisiae* (adapted from [22]).

## **2.** *S. cerevisiae***, the party starter**

Beverages with an alcoholic content are largely consumed by mankind since ancient times. Such beverages made from fermentation of sugar-rich goods, namely cereals and fruits, are present in oldest records [23]. Beer, made from germinated barley, and wine, produced from grapes, are among the most popular and their worldwide consumption is second only to non-alcoholic drinks as water, tea and coffee [24].

Wine and beer history is hand to hand with human civilization history, as most likely only the agriculture advent and the establishment of permanent settlements provided the condi‐ tions for its production. Nevertheless, wine and beer are most probably result of an "acci‐ dent", as some harvested grapes were not consumed rapidly enough or some cereal wet pulp was left aside, and *S. cerevisiae* took advantage of the sugary free meal. The result should have been pleasant enough, especially the mild psychotropic effect, therefore the ear‐ ly farmers must have tried to repeat such "accident". The high ethyl alcohol content of this beverage and its analgesic, disinfectant and conservative properties contributed to a wide‐ spread utilization as a drug. Hence, our successful partnership with this yeast began.

In fact, our relationship with *S. cerevisiae* can be traced back as far as 7,000 years ago in Chi‐ na, with the first fermented beverages similar to beer, and Mesopotamia, with the first wines as well as domesticated vines [11, 25]. Conversely, the oldest known written account on the production of beer was found in Sumeria, modern day Iraq, in a stone tablet dating from 2,000 BC. This tablet, called "Hymn to Ninkasi", describes the production of beer and its es‐ pecially relevant role in religious ceremonies to Ninkasi, the Sumerian Goddess of beer [26]. Babylonians succeeded the Sumerians and kept producing the fermented beverage. They be‐ came so skilful at this art that at least 20 different brews were produced and exported, to as far as Egypt, at the zenith of Babylonian empire. Egyptians were so adept of this "imported beer" that they started producing their own from unbaked dough and even created a special hieroglyph for this new craft. Furthermore, records show that pyramid workers were paid in beer, a readily storable merchandise, and Pharaohs were entombed with model breweries to ensure an afterlife beer supply [26, 27]. Beer popularity grew and this beverage spread for the entire Europe, especially in the Mediterranean region [28].

are utilized in other metabolic reactions; exception is made to isocitrate, including the syn‐

Beverages with an alcoholic content are largely consumed by mankind since ancient times. Such beverages made from fermentation of sugar-rich goods, namely cereals and fruits, are present in oldest records [23]. Beer, made from germinated barley, and wine, produced from grapes, are among the most popular and their worldwide consumption is second only to

Wine and beer history is hand to hand with human civilization history, as most likely only the agriculture advent and the establishment of permanent settlements provided the condi‐ tions for its production. Nevertheless, wine and beer are most probably result of an "acci‐ dent", as some harvested grapes were not consumed rapidly enough or some cereal wet pulp was left aside, and *S. cerevisiae* took advantage of the sugary free meal. The result should have been pleasant enough, especially the mild psychotropic effect, therefore the ear‐ ly farmers must have tried to repeat such "accident". The high ethyl alcohol content of this beverage and its analgesic, disinfectant and conservative properties contributed to a wide‐

spread utilization as a drug. Hence, our successful partnership with this yeast began.

In fact, our relationship with *S. cerevisiae* can be traced back as far as 7,000 years ago in Chi‐ na, with the first fermented beverages similar to beer, and Mesopotamia, with the first wines as well as domesticated vines [11, 25]. Conversely, the oldest known written account on the production of beer was found in Sumeria, modern day Iraq, in a stone tablet dating from 2,000 BC. This tablet, called "Hymn to Ninkasi", describes the production of beer and its es‐

thesis of amino acids and nucleotides (for reviews see [9, 21]).

524 Food Industry

**Figure 3.** Aerobic respiration in *S. cerevisiae* (adapted from [22]).

non-alcoholic drinks as water, tea and coffee [24].

**2.** *S. cerevisiae***, the party starter**

Wine was also very popular in the ancient cultures, with references to this beverage in reli‐ gious ceremonies of Egypt and Phoenicia. Pharaohs tombs were frequently adorned with vin‐ tage scenes and jars filled with wine accompanied the Kings afterlife [11, 23]. Wine consumption spread with the rise of the Greek and Roman Empires. Under the Greek and Ro‐ man influence, wine earned the status of "Civilized" drink, becoming very popular with the Empire upper classes, and beer was labelled as a "Barbarian" drink. Wine production and vine cultivation spread across Europe and replaced beer as the main drink in many countries. Some of these are still nowadays associated with wine production like Portugal, Spain and France. The production and consumption of beer continued mainly in northern borders of the Roman Empire, where Germanic tribes ruled and Roman influence was weaker [26, 28].

In Middle Ages, wine and beer production gained a new impetus with the shift from the fa‐ miliar production to a more centralized production in monasteries [28]. Such happened be‐ cause, at the time, water was frequently polluted, so alcoholic beverages were safer than water for the monks' consumption. Additionally, during the long fasting periods that the monks subjected themselves, the drinking of these highly nutritious beverages became com‐ mon to satisfy hunger. This happened because wine and beer were considered similar to water and didn't constitute a breach of fast. In fact, in some monasteries monks were al‐ lowed to drink up to 5 litres of beer per day [28]. Southern monasteries produced mainly wine, as the weather was warmer and suitable for vines, but in the north the colder weather was more fitting barley and wheat growth and therefore Northern monasteries were more devoted to beer production. Each monastery developed its own methodologies for wine and beer making, leading to new wines and new brews and to a great technical improvement. Later these products become a source of income for the monasteries.

From the sixteenth century, with the discovery of the New World by Portuguese and the Spanish explorers, wine and beer spread to new territories. Vines were introduced in Brazil by the Portuguese around 1500 [29], and in Africa by the Dutch around 1650. In the Austral‐ ian continent and North America this happened later, around 1800.

In the nineteenth century, wine and beer making suffered probably the major scientific ad‐ vances. Around 1860 Louis Pasteur, a name forever associated with wine and beer produc‐ tion, developed studies on the conservation of wine through a heating-cooling process later known as "pasteurization", showing that wine could be stored for longer periods after such treatment. Moreover, in 1870 Pasteur made known to the world the role of *S. cerevisiae* in the fermentation process. Later, in 1876 Pasteur conducted similar studies in beer in his work "Études sur la bière". Such works were based in the first observations of yeast and bacteria by Antonie Van Leeuwenhoek in late seventeenth century [11, 26, 27]. Only almost a decade after it was isolated a pure yeast culture resulting from a single cell by Emil Christian Han‐ sen at the Carlsberg brewery, Denmark and to that followed the name of several yeast spe‐ cies [30]. A few years later, based on Hansen's work, Hermann Müller-Thurgau introduced the notion of inoculating wine fermentations with pure yeast starter cultures [11].

colour and flavour development. Conversely, another important aspect is the intervention of hop, the female flower cluster of *Humulus lupulus*, which acts as bacteriostatic agent against Gram-positive bacteria, helping to control unwanted microorganism during brewing. It also functions as bittering agent, disguising beer natural sweet taste [31, 32]. In winemaking, the maceration of grapes is the starting point to wine production. The variety of grapevines, as well as the weather and cultivation/soil conditions, greatly influences the wine final proper‐ ties. In fact, the environment has such influence that some type of wines can only be pro‐ duced in certain regions, like the Porto wine in Douro region, Portugal, and Champagne

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 527

Brewer's yeast can be distinguished in top fermenting and bottom fermenting yeasts, based in the position at which the fermentation occurs. This division accounts with the yeast floc‐ culation behaviour, and it is such an important element of brewing that defines the two main classes: **ale beers** (top fermenting) and **lager beers** (bottom fermenting). Such catego‐ ries were devised as soon as the first pure yeast culture was isolated. Hansen was able to purify two different species, a top fermenting appropriate for ale brewing, *S. cerevisiae*, and a bottom fermenting, *S. carlsbergensis*, suitable for lager beer [26]. Such taxonomic classifica‐ tion was reviewed several times [1], and the top fermenting yeasts are now included in the *S. cerevisiae* and *S. bayanus* species and the bottom fermenting yeasts fit to the *S. pastorianus*

These brewer's yeasts present several differences in their genomes. **Lager yeast** strains present complex polyploidy genomes, with evidences of contribution from distinct *Saccharo‐ myces* species [33]. Usually these complex genomes are tetraploid, which may result from the fusion of diploid parental strains or from duplication of the genetic information after the original cell fusion. Analysis on lager yeast genomes revealed other changes such as chro‐ mosome loss and/or duplications, likely due to human selection of relevant phenotypes [33, 34]. In reference [34] lager yeast genomes were analysed and classified in two groups. In group I, cells present one *S. cerevisiae* genome equivalent and, in group II, cells present two *S. cerevisiae* genome equivalents. Both groups exhibit one *S. bayanus* genome equivalent and the remaining genome was mostly hybrid chromosomes from both species. These different yeast must be related with the conditions they are exposed, meaning for instance to lager beer fermentation, yeast has to react to conditions inherent to beer production, as cropping and pitching, and to bottom fermentation specificities, *e.g.*, temperature of reaction [35].

As for **ale yeasts**, studies revealed that these strains are closely to *S. cerevisiae* [34, 36]. In fact, a genotype analysis of 651 *S. cerevisiae* strains revealed that ale strains were more closely re‐ lated to wine and bread strains (above referred as Baker's yeast), than to lager brewer's yeast strains [36]. Reports of hybrids in ale yeasts showed that strains traditionally classified as *S. cerevisiae* may indeed be the result of hybridization events [37]. Ale beer has less repre‐ sentation in worldwide markets, and as a consequence less studies and information are available on the corresponding yeasts. As such, the considerations on beer yeast physiology

In winemaking, most wine yeasts belong to *S. cerevisiae* species, but *S. bayanus* has also been detected. Yet, wine fermentations also present yeast, derived from vineyard environment,

species, all belonging to the *Saccharomyces sensu stricto* genus [27, 33].

wine in Champagne region, France.

will be focused on lager strains.

The intensive study of this amazing microorganism and its role in fermentation showed the specificities of each yeast species and strains. The necessity of consistent properties and quality in different fermentations, both in brewing and winemaking, paved the way for the selection of the right yeast for the job. The quest for stable and improved yeast began.

## **2.1. The "Right" yeast for the job**

*S. cerevisiae*, known as "Wine's Yeast" or "Brewer's Yeast" (and also as "Baker Yeast", see below), is the main responsible for some of the world's most important fermented beverag‐ es. However, brewing and winemaking have inherent differences: i) the culture medium, ii) the bioreactor and iii) the yeast starter culture.

The final product, either wine or beer, is greatly influenced by the sugar-rich fermentable broth, grape juice or malted cereals, with different composition in fermentable sugars and nitrogen sources. The progression of the fermentation is another very important aspect of winemaking and brewing, *e.g.,* the oxygen available*,* the temperature and pH variations during substrate consumption and ethanol and CO2 production. But the most significant dif‐ ference is the yeast starter culture, its physiological state, whether it is dried yeast or a fresh inoculum, how well it ferments the available sugars and resists to fermentation by-products and its ability to flocculate at the right moment.

All *S. cerevisiae* strains described so far are capable of fermenting sugars to ethanol, but cen‐ turies of partnership with mankind directed the yeast evolution. Such evolutionary pressure resulted in a selection of distinct yeast strains for different applications, to produce wine and beer you need "Wine's Yeast" and "Brewer's Yeast", respectively.

#### *2.1.1. Yeast physiology*

Beer is the denomination commonly attributed to a carbonated alcoholic beverage produced by fermentation of malted barley, while wine is made of the fermented juice of any of sever‐ al types of grapes. However, there are as many different wines and beers as there are differ‐ ent producers, all with their unique character and flavour influenced by the selected ingredients, kind of fermentation and yeast selected.

As said, the choice of the ingredients greatly impacts the fermentation final product; usually beer is the product of malt, hops, water and yeast. Malt is the result of germinating and dry‐ ing (kilning) barley, yet other cereals besides barley can be used to produce beer, as wheat and rye. Malt extract will provide the entirety of the carbohydrates and nitrogen to the fer‐ mentation process and as such, it will influence the final ethanol concentration as well as colour and flavour development. Conversely, another important aspect is the intervention of hop, the female flower cluster of *Humulus lupulus*, which acts as bacteriostatic agent against Gram-positive bacteria, helping to control unwanted microorganism during brewing. It also functions as bittering agent, disguising beer natural sweet taste [31, 32]. In winemaking, the maceration of grapes is the starting point to wine production. The variety of grapevines, as well as the weather and cultivation/soil conditions, greatly influences the wine final proper‐ ties. In fact, the environment has such influence that some type of wines can only be pro‐ duced in certain regions, like the Porto wine in Douro region, Portugal, and Champagne wine in Champagne region, France.

"Études sur la bière". Such works were based in the first observations of yeast and bacteria by Antonie Van Leeuwenhoek in late seventeenth century [11, 26, 27]. Only almost a decade after it was isolated a pure yeast culture resulting from a single cell by Emil Christian Han‐ sen at the Carlsberg brewery, Denmark and to that followed the name of several yeast spe‐ cies [30]. A few years later, based on Hansen's work, Hermann Müller-Thurgau introduced

The intensive study of this amazing microorganism and its role in fermentation showed the specificities of each yeast species and strains. The necessity of consistent properties and quality in different fermentations, both in brewing and winemaking, paved the way for the selection of the right yeast for the job. The quest for stable and improved yeast began.

*S. cerevisiae*, known as "Wine's Yeast" or "Brewer's Yeast" (and also as "Baker Yeast", see below), is the main responsible for some of the world's most important fermented beverag‐ es. However, brewing and winemaking have inherent differences: i) the culture medium, ii)

The final product, either wine or beer, is greatly influenced by the sugar-rich fermentable broth, grape juice or malted cereals, with different composition in fermentable sugars and nitrogen sources. The progression of the fermentation is another very important aspect of winemaking and brewing, *e.g.,* the oxygen available*,* the temperature and pH variations during substrate consumption and ethanol and CO2 production. But the most significant dif‐ ference is the yeast starter culture, its physiological state, whether it is dried yeast or a fresh inoculum, how well it ferments the available sugars and resists to fermentation by-products

All *S. cerevisiae* strains described so far are capable of fermenting sugars to ethanol, but cen‐ turies of partnership with mankind directed the yeast evolution. Such evolutionary pressure resulted in a selection of distinct yeast strains for different applications, to produce wine and

Beer is the denomination commonly attributed to a carbonated alcoholic beverage produced by fermentation of malted barley, while wine is made of the fermented juice of any of sever‐ al types of grapes. However, there are as many different wines and beers as there are differ‐ ent producers, all with their unique character and flavour influenced by the selected

As said, the choice of the ingredients greatly impacts the fermentation final product; usually beer is the product of malt, hops, water and yeast. Malt is the result of germinating and dry‐ ing (kilning) barley, yet other cereals besides barley can be used to produce beer, as wheat and rye. Malt extract will provide the entirety of the carbohydrates and nitrogen to the fer‐ mentation process and as such, it will influence the final ethanol concentration as well as

the notion of inoculating wine fermentations with pure yeast starter cultures [11].

**2.1. The "Right" yeast for the job**

526 Food Industry

the bioreactor and iii) the yeast starter culture.

and its ability to flocculate at the right moment.

ingredients, kind of fermentation and yeast selected.

*2.1.1. Yeast physiology*

beer you need "Wine's Yeast" and "Brewer's Yeast", respectively.

Brewer's yeast can be distinguished in top fermenting and bottom fermenting yeasts, based in the position at which the fermentation occurs. This division accounts with the yeast floc‐ culation behaviour, and it is such an important element of brewing that defines the two main classes: **ale beers** (top fermenting) and **lager beers** (bottom fermenting). Such catego‐ ries were devised as soon as the first pure yeast culture was isolated. Hansen was able to purify two different species, a top fermenting appropriate for ale brewing, *S. cerevisiae*, and a bottom fermenting, *S. carlsbergensis*, suitable for lager beer [26]. Such taxonomic classifica‐ tion was reviewed several times [1], and the top fermenting yeasts are now included in the *S. cerevisiae* and *S. bayanus* species and the bottom fermenting yeasts fit to the *S. pastorianus* species, all belonging to the *Saccharomyces sensu stricto* genus [27, 33].

These brewer's yeasts present several differences in their genomes. **Lager yeast** strains present complex polyploidy genomes, with evidences of contribution from distinct *Saccharo‐ myces* species [33]. Usually these complex genomes are tetraploid, which may result from the fusion of diploid parental strains or from duplication of the genetic information after the original cell fusion. Analysis on lager yeast genomes revealed other changes such as chro‐ mosome loss and/or duplications, likely due to human selection of relevant phenotypes [33, 34]. In reference [34] lager yeast genomes were analysed and classified in two groups. In group I, cells present one *S. cerevisiae* genome equivalent and, in group II, cells present two *S. cerevisiae* genome equivalents. Both groups exhibit one *S. bayanus* genome equivalent and the remaining genome was mostly hybrid chromosomes from both species. These different yeast must be related with the conditions they are exposed, meaning for instance to lager beer fermentation, yeast has to react to conditions inherent to beer production, as cropping and pitching, and to bottom fermentation specificities, *e.g.*, temperature of reaction [35].

As for **ale yeasts**, studies revealed that these strains are closely to *S. cerevisiae* [34, 36]. In fact, a genotype analysis of 651 *S. cerevisiae* strains revealed that ale strains were more closely re‐ lated to wine and bread strains (above referred as Baker's yeast), than to lager brewer's yeast strains [36]. Reports of hybrids in ale yeasts showed that strains traditionally classified as *S. cerevisiae* may indeed be the result of hybridization events [37]. Ale beer has less repre‐ sentation in worldwide markets, and as a consequence less studies and information are available on the corresponding yeasts. As such, the considerations on beer yeast physiology will be focused on lager strains.

In winemaking, most wine yeasts belong to *S. cerevisiae* species, but *S. bayanus* has also been detected. Yet, wine fermentations also present yeast, derived from vineyard environment, belonging to the genera *Candida*, *Debaryomyces* and *Brettanomyces*. But, yeast is mainly select‐ ed for its resistance to ethanol, favouring *S. cerevisiae*. There is also selection for capacity to float or to flocculate, important for some specific wines. While in most wines the ability to flocculate is important to improve the filtration, in some wines such as sherry wine, the for‐ mation of a floating film is vital. This *vellum* is formed at the surface of the wine and pro‐ motes oxidative metabolism. Sherry wine is characterized by high ethanol content and low aldehyde. Its featured nutty flavour can be ascribed to partial oxidation of ethanol to acetal‐ dehyde [11].

cells are adapting its physiology. In wine the use of active dried yeast (ADY) is common and

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 529

Nitrogen assimilation is especially important in flavour development. The main sources of nitrogen are free amino acids and ammonium ions, which are used by the cell for protein formation [35, 40]. Such amino acids are also relevant for the production of alcohols and es‐ ters, important in these beverages flavour. During fermentation, amino acids are always used following a certain order, independent from the fermentation conditions. Group A, in‐ cluding arginine, asparagine, aspartate, glutamate, glutamine, lysine, serine and threonine, are used first. Group B amino acids are utilized slowly and include histidine, isoleucine, leu‐ cine, methionine and valine. Group C is composed by alanine, glycine, phenylalanine, tyro‐ sine, tryptophan, and are only absorbed after the complete exhaustion of group A. Group D is composed of proline, which require an aerobic metabolism for its uptake and it is poorly

In winemaking, fermentations are usually developed under anaerobic conditions, but it is common in brewing to oxygenate the *wort*. So another important stressor is the dissolved oxygen, which may lead to the formation of reactive oxygen species (ROS). ROS, as hydro‐ gen peroxide or superoxide radical, can promote damages in cell main constituents, DNA, proteins and lipids. However, oxygen is very important for the synthesis of sterols and un‐ saturated fatty acids ensuring the physiological fitness for cell replication. The control of dis‐ solved oxygen is vital to ensure a healthy population. An important problem is the excessive growth of yeast cells when exposed to high amounts of dissolved oxygen, at the expenses of

The inorganic ions are necessary, but at nanomolar concentrations. These trace elements, as calcium, zinc or copper, are mainly required as cofactors of enzymes or in the flocculation process. For instance, the response to oxidative stress is dependent on enzymes such as the different superoxide dismutase isoforms that require manganese, zinc or copper [32]. On the other hand, calcium is vital for the flocculation advance [42]. Conversely, insufficient amounts of such elements can lead to cellular damage and stress, and consequent stuck fer‐

The use of antimicrobials in vineyards is common to control fungi that spoil grapes. But, when grapes are macerated these compounds are incorporated into the juice. Even though they may help to prevent the wine oxidation and microbial spoilage, a concentration to high may lead to off-flavours and in worst case, yeast death. So antimicrobials, especially sulphur dioxide, are an important stress to yeast during fermentation. Commercially available yeast also has to deal with toxins produced by wild yeasts derived from the vineyards. These tox‐ ins are produced to give those wild yeast advantages over others species in accessing to the nutrients. Isolation of strains resistant to both antimicrobials and natural toxins is an impor‐

Certain compounds are extremely important for brewing and wine making not as substrates but as by-products. Such metabolites greatly influence the final product's colour and fla‐ vour, as well as its stability. In fact, the importance of these compounds is such that lager

no effect on fermentation time was reported [39].

used during fermentations [40].

ethanol production [41].

tant research field [11, 43].

mentations.

The utilization of dried yeast as a starter culture is very common in the wine industry. Cells are dehydrated through a cycle of filtrations and centrifugation to remove external water and then submitted to streams of dehumidified hot air. Such procedure can reduce yeast cells' content in water to as low as 6%. However, even though yeast cells can survive such treatment, it causes cellular damage. Damages to cell wall and plasma membranes caused by the changes on cell size and shape, as well as damages to proteins produced by free radi‐ cals were reported [35].

One of beer brewing specificities is the utilization of a freshly grown starter culture. In one hand, it ensures a healthy population fully adapted to growth medium. Cells are usually collected at the late exponential phase, preventing a large percentage of aged cells, and at the same time ensuring metabolic fitness. On the other hand, it meets the requirement for flavour consistency of the final product, even though it is more expensive than the alterna‐ tives. The pattern of metabolic products of yeast is highly dependent on its growth condi‐ tions, and cells fully adapted to *wort* produce a more consistent flavour. The first batches inoculated with dried yeast are often of inferior quality, with by-products of fermentation conferring off-flavours, which compromises the regularity of a brand product [30]. An alter‐ native that meets the requirements of fresh grown cells and flavour consistency, but at the same time reduces the process duration and costs, is the fed-batch technology. Cells are kept in late exponential phase by leaving a certain amount of yeast in the reactor and adding fresh *wort*, shortening the fermentation time and maintaining the beers properties [38].

During fermentation, yeast is constantly facing new pressures. The high osmotic stress due to the sugar high content of *wort* and *must* is just the beginning. *Wort* is a rich and complex medium, composed of carbohydrates (90%), nitrogen sources (5%) and small amounts of in‐ organic ions, lipids and polyphenols. *Wort* composition, being highly dependable of the quality of the cereal and the process used to malt it, is usually enriched in fermentable su‐ crose (5%), monosaccharides (10%), maltotriose (15%) and maltose (50%). About 20-30% of total carbohydrates are non-fermentable dextrins, polysaccharides result from starch degra‐ dation [35]. On the other hand, grape juice is rich in fructose and glucose, presenting small amounts of sucrose. Grape variety influence the ratio glucose/fructose, Chardonnay is a high fructose variety, whereas Zinfandel is regarded as high glucose variety. Such high-gravity *wort*s, 12-18 g of extract per 100 mL, subject cells to high osmotic pressure. The production of compatible solutes, as glycerol and trehalose, and a "robust" plasma membrane composition seem to be the main adaptations to withstand such stress. The cells fully adapted to *wort* used to inoculate (pitch) fermentations are important to avoid extended lag phases where cells are adapting its physiology. In wine the use of active dried yeast (ADY) is common and no effect on fermentation time was reported [39].

belonging to the genera *Candida*, *Debaryomyces* and *Brettanomyces*. But, yeast is mainly select‐ ed for its resistance to ethanol, favouring *S. cerevisiae*. There is also selection for capacity to float or to flocculate, important for some specific wines. While in most wines the ability to flocculate is important to improve the filtration, in some wines such as sherry wine, the for‐ mation of a floating film is vital. This *vellum* is formed at the surface of the wine and pro‐ motes oxidative metabolism. Sherry wine is characterized by high ethanol content and low aldehyde. Its featured nutty flavour can be ascribed to partial oxidation of ethanol to acetal‐

The utilization of dried yeast as a starter culture is very common in the wine industry. Cells are dehydrated through a cycle of filtrations and centrifugation to remove external water and then submitted to streams of dehumidified hot air. Such procedure can reduce yeast cells' content in water to as low as 6%. However, even though yeast cells can survive such treatment, it causes cellular damage. Damages to cell wall and plasma membranes caused by the changes on cell size and shape, as well as damages to proteins produced by free radi‐

One of beer brewing specificities is the utilization of a freshly grown starter culture. In one hand, it ensures a healthy population fully adapted to growth medium. Cells are usually collected at the late exponential phase, preventing a large percentage of aged cells, and at the same time ensuring metabolic fitness. On the other hand, it meets the requirement for flavour consistency of the final product, even though it is more expensive than the alterna‐ tives. The pattern of metabolic products of yeast is highly dependent on its growth condi‐ tions, and cells fully adapted to *wort* produce a more consistent flavour. The first batches inoculated with dried yeast are often of inferior quality, with by-products of fermentation conferring off-flavours, which compromises the regularity of a brand product [30]. An alter‐ native that meets the requirements of fresh grown cells and flavour consistency, but at the same time reduces the process duration and costs, is the fed-batch technology. Cells are kept in late exponential phase by leaving a certain amount of yeast in the reactor and adding fresh *wort*, shortening the fermentation time and maintaining the beers properties [38].

During fermentation, yeast is constantly facing new pressures. The high osmotic stress due to the sugar high content of *wort* and *must* is just the beginning. *Wort* is a rich and complex medium, composed of carbohydrates (90%), nitrogen sources (5%) and small amounts of in‐ organic ions, lipids and polyphenols. *Wort* composition, being highly dependable of the quality of the cereal and the process used to malt it, is usually enriched in fermentable su‐ crose (5%), monosaccharides (10%), maltotriose (15%) and maltose (50%). About 20-30% of total carbohydrates are non-fermentable dextrins, polysaccharides result from starch degra‐ dation [35]. On the other hand, grape juice is rich in fructose and glucose, presenting small amounts of sucrose. Grape variety influence the ratio glucose/fructose, Chardonnay is a high fructose variety, whereas Zinfandel is regarded as high glucose variety. Such high-gravity *wort*s, 12-18 g of extract per 100 mL, subject cells to high osmotic pressure. The production of compatible solutes, as glycerol and trehalose, and a "robust" plasma membrane composition seem to be the main adaptations to withstand such stress. The cells fully adapted to *wort* used to inoculate (pitch) fermentations are important to avoid extended lag phases where

dehyde [11].

528 Food Industry

cals were reported [35].

Nitrogen assimilation is especially important in flavour development. The main sources of nitrogen are free amino acids and ammonium ions, which are used by the cell for protein formation [35, 40]. Such amino acids are also relevant for the production of alcohols and es‐ ters, important in these beverages flavour. During fermentation, amino acids are always used following a certain order, independent from the fermentation conditions. Group A, in‐ cluding arginine, asparagine, aspartate, glutamate, glutamine, lysine, serine and threonine, are used first. Group B amino acids are utilized slowly and include histidine, isoleucine, leu‐ cine, methionine and valine. Group C is composed by alanine, glycine, phenylalanine, tyro‐ sine, tryptophan, and are only absorbed after the complete exhaustion of group A. Group D is composed of proline, which require an aerobic metabolism for its uptake and it is poorly used during fermentations [40].

In winemaking, fermentations are usually developed under anaerobic conditions, but it is common in brewing to oxygenate the *wort*. So another important stressor is the dissolved oxygen, which may lead to the formation of reactive oxygen species (ROS). ROS, as hydro‐ gen peroxide or superoxide radical, can promote damages in cell main constituents, DNA, proteins and lipids. However, oxygen is very important for the synthesis of sterols and un‐ saturated fatty acids ensuring the physiological fitness for cell replication. The control of dis‐ solved oxygen is vital to ensure a healthy population. An important problem is the excessive growth of yeast cells when exposed to high amounts of dissolved oxygen, at the expenses of ethanol production [41].

The inorganic ions are necessary, but at nanomolar concentrations. These trace elements, as calcium, zinc or copper, are mainly required as cofactors of enzymes or in the flocculation process. For instance, the response to oxidative stress is dependent on enzymes such as the different superoxide dismutase isoforms that require manganese, zinc or copper [32]. On the other hand, calcium is vital for the flocculation advance [42]. Conversely, insufficient amounts of such elements can lead to cellular damage and stress, and consequent stuck fer‐ mentations.

The use of antimicrobials in vineyards is common to control fungi that spoil grapes. But, when grapes are macerated these compounds are incorporated into the juice. Even though they may help to prevent the wine oxidation and microbial spoilage, a concentration to high may lead to off-flavours and in worst case, yeast death. So antimicrobials, especially sulphur dioxide, are an important stress to yeast during fermentation. Commercially available yeast also has to deal with toxins produced by wild yeasts derived from the vineyards. These tox‐ ins are produced to give those wild yeast advantages over others species in accessing to the nutrients. Isolation of strains resistant to both antimicrobials and natural toxins is an impor‐ tant research field [11, 43].

Certain compounds are extremely important for brewing and wine making not as substrates but as by-products. Such metabolites greatly influence the final product's colour and fla‐ vour, as well as its stability. In fact, the importance of these compounds is such that lager beers are usually stored from several days to weeks, lagering, solely to remove diacetyl, an off-flavour causing metabolite. This time consuming maturation phase consists in a second fermentation at low temperature to eliminate the butter-like flavour caused by this vicinal diketone. Studies are being conducted in order to minimize this metabolite formation and reduce the maturation time [30]. Sulphur containing compounds are other family of byproducts receiving great attention. Such group comprises sulphite, sulphide and dimethyl sulphide, and while sulphite is a beneficial and flavour stabilizing metabolite, the remaining compounds are responsible for off-flavours. The equilibrium of such compounds formation could lead to better wine and beer and shorter fermentations [44].

Large collections of yeast were assembled, as the Centraalbureau voor Schimmelcultures (CBS) collection, in The Netherlands, and the Carlsberg collection, in Denmark. Manipula‐ tion of these strains to improve wine and beer properties has been performed in several ways, from spores manoeuvring and natural mutants' survey to genetic engineering (GE). A rather recent and extensive review in strategies for the improvement of *S. cerevisiae* industri‐ al strains can be found in [2]. As referred above, an efficient utilization of substrates by yeast during fermentation is extremely important for wine and beer industries. Such efficiency will yield higher amounts of by-products, as ethanol, and reduce the fermentation time. Fur‐ thermore, glucose repression on other sugars and consumption of unusual nitrogen sources

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 531

The presence of glucose, even in small amounts, represses the simultaneous uptake and con‐ sumption of several sugars, namely maltose and galactose. Maltose (50%), maltotriose (15%) and sucrose (5%) are the main sugars in *wort* under glucose repression. Such repression leads to late fermentation of these sugars and slower fermentations, as their utilization is de‐ pendent on glucose depletion. This process is controlled at the transcriptional level, through the action of the proteins Mig1p, Ssn6p and Tup1p. Mig1p, a zinc finger protein, binds to specific sequences in the promoter region of the glucose-repressed genes and recruits the SSN6-TUP1 complex, the responsible for the actual repression [49]. Mig1p binding sites were found in genes associated with the utilization of sucrose (*SUC*2), maltose (*MALR*, *MALS* and *MALT*) and galactose (*GAL*1-5) [50]. Therefore, *MIG1* presents itself as a potential

Conversely, in sucrose metabolism, glucose repression addresses the sucrose conversion in fructose and glucose under the action of Suc2p. Studies showed that the disruption of *MIG*1 lessen the glucose repression on the transcription of this excreted invertase in both lab and industrial strains. Therefore, the lag in sucrose utilization was greatly diminished. Besides Mig1p, another zinc finger protein, Mig2p, was associated with glucose repression of *SUC*2 [51]. The interruption of both *MIG*1 and *MIG*2, in *S. cerevisiae* strains led a high sucrose me‐

Maltose metabolism is more complex than sucrose, as it responds to both glucose repression and maltose induction. Maltose induction is under the influence of the locus *MAL*, a closely integrated group of genes. This sugar presence induces *MALR*, a transcription factor, which in turn will induce *MALT*, coding for a maltose permease, and *MALS*, coding for a maltase [30]. Up to 5 different *loci* have been detected in *S. cerevisiae* industrial strains; still haploid lab strains present a single *locus*. In both situations these *loci* are under repression of Mig1p [53]. However, disruption of *MIG*1 in industrial strains only alleviated the sucrose metabo‐ lism [50], but didn't cause any effect regarding maltose metabolism. Even though, in haploid lab strains *MIG*1 disruption lifted the glucose repression; the same has not happen in the polyploid strains, presenting multiple *loci*. Complex regulation between the different genes

Finally, maltotriose, a glucose tri-saccharide, is the second most abundant sugar in *wort* (15%). This is under similar regulation by the presence of glucose and maltose, therefore most studies have focused in an efficient uptake of this carbohydrate [54]. Those works

are vital research areas [30].

target to improve yeast sugar consumption.

tabolism in the presence of high glucose concentrations [52].

must be under way and most probably not solely controlled by Mig1p [53].

Ethanol is one of the most important by-products of beer fermentation. Nevertheless, it rep‐ resents an important stressor for yeast cells due to its high toxicity. Ethanol concentration can reach 10% in higher gravity fermentations, and acts especially upon biological mem‐ branes [35]. Reports showed ethanol effects in growth inhibition [45], lipid modification and loss of proton motive force across the membrane and increased membrane permeability/ fluidity [46]. Yet, cells exposed to oxygen, with high levels of sterols in membranes, and ade‐ quate levels of nutrients, amino acids and trace elements in the fermentation broth are able to respond efficiently to such effects [35].

Nutritional stress occurs at the end of fermentation and cells enter stationary phase. This oc‐ curs because fermentable carbon sources tend to be depleted, and cells have to change their metabolism from fermentative to respiratory (explained in section 1), entering in a quiescent state [30]. Such phenomenon induces flocculation, a cell-cell interaction process dependent of lectins and calcium that promotes sedimentation. Flocculation in turn is influenced by several other factors besides nutrient depletion. Reports showed the influence of ethanol content, calcium concentration, pH changes, oxygen concentration and temperature [47]. The onset of flocculation is an important area of interest in brewing. If flocculation occurs to soon, stuck fermentation may occur, which results in a high sugar and low ethanol content. If, on the other hand, happens in a later stage, it has a high impact in beer filtration as most cells tend to be kept in suspension.

Fermentation is the most yeast-dependent phase of these alcoholic beverages production, but yeast also interferes with others proceedings. The metabolic fitness of the starter culture, the storage and maintenance of both dried and fresh yeasts, and the storage of the products submit yeast to different conditions to which they have to respond/adapt. To obtain detailed information on such processes please see reviews [35] and [43].

#### **2.2. Old beverages, new solutions**

Wine and beer industrial production led to a demand for better and more efficient yeast. Yeasts with improved utilization of substrates, carbohydrates and nitrogen, and consistent flocculent behaviour, as well as high fermentative capacity and high ethanol production are the industry goal. Enhancing beer and wine flavour through modification of by-products formation is another field of intensive research [30, 39]. As it is the improvement of the fer‐ mentation process, through encapsulation/immobilization of yeast [48].

Large collections of yeast were assembled, as the Centraalbureau voor Schimmelcultures (CBS) collection, in The Netherlands, and the Carlsberg collection, in Denmark. Manipula‐ tion of these strains to improve wine and beer properties has been performed in several ways, from spores manoeuvring and natural mutants' survey to genetic engineering (GE). A rather recent and extensive review in strategies for the improvement of *S. cerevisiae* industri‐ al strains can be found in [2]. As referred above, an efficient utilization of substrates by yeast during fermentation is extremely important for wine and beer industries. Such efficiency will yield higher amounts of by-products, as ethanol, and reduce the fermentation time. Fur‐ thermore, glucose repression on other sugars and consumption of unusual nitrogen sources are vital research areas [30].

beers are usually stored from several days to weeks, lagering, solely to remove diacetyl, an off-flavour causing metabolite. This time consuming maturation phase consists in a second fermentation at low temperature to eliminate the butter-like flavour caused by this vicinal diketone. Studies are being conducted in order to minimize this metabolite formation and reduce the maturation time [30]. Sulphur containing compounds are other family of byproducts receiving great attention. Such group comprises sulphite, sulphide and dimethyl sulphide, and while sulphite is a beneficial and flavour stabilizing metabolite, the remaining compounds are responsible for off-flavours. The equilibrium of such compounds formation

Ethanol is one of the most important by-products of beer fermentation. Nevertheless, it rep‐ resents an important stressor for yeast cells due to its high toxicity. Ethanol concentration can reach 10% in higher gravity fermentations, and acts especially upon biological mem‐ branes [35]. Reports showed ethanol effects in growth inhibition [45], lipid modification and loss of proton motive force across the membrane and increased membrane permeability/ fluidity [46]. Yet, cells exposed to oxygen, with high levels of sterols in membranes, and ade‐ quate levels of nutrients, amino acids and trace elements in the fermentation broth are able

Nutritional stress occurs at the end of fermentation and cells enter stationary phase. This oc‐ curs because fermentable carbon sources tend to be depleted, and cells have to change their metabolism from fermentative to respiratory (explained in section 1), entering in a quiescent state [30]. Such phenomenon induces flocculation, a cell-cell interaction process dependent of lectins and calcium that promotes sedimentation. Flocculation in turn is influenced by several other factors besides nutrient depletion. Reports showed the influence of ethanol content, calcium concentration, pH changes, oxygen concentration and temperature [47]. The onset of flocculation is an important area of interest in brewing. If flocculation occurs to soon, stuck fermentation may occur, which results in a high sugar and low ethanol content. If, on the other hand, happens in a later stage, it has a high impact in beer filtration as most

Fermentation is the most yeast-dependent phase of these alcoholic beverages production, but yeast also interferes with others proceedings. The metabolic fitness of the starter culture, the storage and maintenance of both dried and fresh yeasts, and the storage of the products submit yeast to different conditions to which they have to respond/adapt. To obtain detailed

Wine and beer industrial production led to a demand for better and more efficient yeast. Yeasts with improved utilization of substrates, carbohydrates and nitrogen, and consistent flocculent behaviour, as well as high fermentative capacity and high ethanol production are the industry goal. Enhancing beer and wine flavour through modification of by-products formation is another field of intensive research [30, 39]. As it is the improvement of the fer‐

could lead to better wine and beer and shorter fermentations [44].

information on such processes please see reviews [35] and [43].

mentation process, through encapsulation/immobilization of yeast [48].

to respond efficiently to such effects [35].

530 Food Industry

cells tend to be kept in suspension.

**2.2. Old beverages, new solutions**

The presence of glucose, even in small amounts, represses the simultaneous uptake and con‐ sumption of several sugars, namely maltose and galactose. Maltose (50%), maltotriose (15%) and sucrose (5%) are the main sugars in *wort* under glucose repression. Such repression leads to late fermentation of these sugars and slower fermentations, as their utilization is de‐ pendent on glucose depletion. This process is controlled at the transcriptional level, through the action of the proteins Mig1p, Ssn6p and Tup1p. Mig1p, a zinc finger protein, binds to specific sequences in the promoter region of the glucose-repressed genes and recruits the SSN6-TUP1 complex, the responsible for the actual repression [49]. Mig1p binding sites were found in genes associated with the utilization of sucrose (*SUC*2), maltose (*MALR*, *MALS* and *MALT*) and galactose (*GAL*1-5) [50]. Therefore, *MIG1* presents itself as a potential target to improve yeast sugar consumption.

Conversely, in sucrose metabolism, glucose repression addresses the sucrose conversion in fructose and glucose under the action of Suc2p. Studies showed that the disruption of *MIG*1 lessen the glucose repression on the transcription of this excreted invertase in both lab and industrial strains. Therefore, the lag in sucrose utilization was greatly diminished. Besides Mig1p, another zinc finger protein, Mig2p, was associated with glucose repression of *SUC*2 [51]. The interruption of both *MIG*1 and *MIG*2, in *S. cerevisiae* strains led a high sucrose me‐ tabolism in the presence of high glucose concentrations [52].

Maltose metabolism is more complex than sucrose, as it responds to both glucose repression and maltose induction. Maltose induction is under the influence of the locus *MAL*, a closely integrated group of genes. This sugar presence induces *MALR*, a transcription factor, which in turn will induce *MALT*, coding for a maltose permease, and *MALS*, coding for a maltase [30]. Up to 5 different *loci* have been detected in *S. cerevisiae* industrial strains; still haploid lab strains present a single *locus*. In both situations these *loci* are under repression of Mig1p [53]. However, disruption of *MIG*1 in industrial strains only alleviated the sucrose metabo‐ lism [50], but didn't cause any effect regarding maltose metabolism. Even though, in haploid lab strains *MIG*1 disruption lifted the glucose repression; the same has not happen in the polyploid strains, presenting multiple *loci*. Complex regulation between the different genes must be under way and most probably not solely controlled by Mig1p [53].

Finally, maltotriose, a glucose tri-saccharide, is the second most abundant sugar in *wort* (15%). This is under similar regulation by the presence of glucose and maltose, therefore most studies have focused in an efficient uptake of this carbohydrate [54]. Those works showed that overexpression of maltotriose transporters lead to positive effects on its metab‐ olism [54, 55].

tolactate decarboxylase (ALDC) in yeast is the main approach to reduce the amounts of this compound. ALDC catalyses the reaction of α-acetolactate to acetoin, preventing the forma‐ tion of diacetyl. However, after heterologous expression of ALDC, the yeast became auxo‐ trophic for some amino acids and the growth rate was very low in *wort*. An alternative approach was the interruption of *ILV*2, encoding acetolactate synthase. Such strategy also resulted in an auxotrophic strain with slow growth. The search for natural mutants in *ILV*2

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 533

Improvement of yeast to render fermentations faster and cheaper is an industry goal, but the enhancing of the fermentation process itself is another alternative. The fed-batch technology has already proved its benefits [38], and improvements of such process with yeast immobili‐ zation/encapsulation are now under the spotlight. This results in much faster fermentation rates as compared to the existing free cell fermentations. However, it has some disadvantag‐ es, such as: i) complexity of production process including the choice of the suitable carrier materials, ii) bioreactors design, iii) fine-tuning of the flavour formation during fermentation

The process of bread making relies on the fermentation carried out by a mixture of yeast and bacteria. Even when all this was unknown and the flour leavening seen as "magic", bread was already produced and extensively consumed. On those ancient times, the leavening re‐ sulted presumably due to the action (fermentation) of the natural microbial contaminants of flour or dough ingredients. This was obviously not a controlled process, yet with the prac‐ tice of maintaining a fresh inoculum from one preparation to the next, promoted the selec‐ tion of yeast and bacteria biodiversity. Nowadays, some types of bread are still prepared in this fashion, sourdoughs are one example (for a review see [2]), but the baking industry moved for the use of commercially baker's yeast, typically the strain *S. cerevisiae*, for the bread production. And, while the flour types, geographical origin and mixtures introduce organoleptic differences in bread, the globalization of commercial baker's yeast market de‐

Commercial baker's yeast is produced in several forms in order to meet specific require‐ ments of climate, technology, methodology, transportation, storage and final product. As with all biotechnology processes, this is in constant development/undergoing research not only to optimize the process technology and its components, but as to produce faster grow‐

Molasses (beet and cane molasses), the common carbon and energy source used in the production of baker's yeast, is a by-product of sugar refining industries, therefore cheap‐ er than the formerly used cereals grain. Furthermore the sugars present on those molass‐ es (around 50%), consisting on a mixture of sucrose, fructose and glucose, are ready to be

with an appropriated growth rate is now the major strategy [30].

processes, and iv) cost constraints [59].

**3. Baker's yeast – Magic on bread making**

creased worldwide bread diversity (for a review see [2]).

**3.1. Commercial baker's yeast production – The break of spell**

ing strains with the characteristics to deliver better quality end products.

In winemaking and brewing, where flavour has such importance, amino acids metabolism has a notorious place. As said, amino acids are involved in formation of higher alcohols and esters that significantly contribute to beer and wine flavour. Since yeast cannot hydrolyse *must* and *wort* proteins, it depends on the available ammonium and amino acids in solution [11, 30]. However, the predominant amino acid in both *must* and *wort*, proline, is the less as‐ similated [40]. As such, improvements in yeast ability to uptake this amino acid has been attempted. Efficient proline uptake was reported in lager beer yeast expressing a mutagen‐ ized proline permease, Put4p. The site-directed mutagenesis stabilized the permease and en‐ hanced amino acid utilization without affecting the beer quality [56]. The study of the same problematic in winemaking led to the disruption of *URE*1, a repressor of permease encoding *PUT*1 and pyrroline-5-carboxylate dehydrogenase *PUT*2, with significant improvements in fermentation rate and vigour described [57].

Flocculation is a phenotype of industrial interest. It facilitates the filtration process in the end of fermentation, saving both time and money. In the case of brewing, it also serves the cropping (recover of part of the yeast population of the fermentation to pitch the next). Floc‐ culation is a reversible aggregation of cells, where lectins recognize sugar residues in neigh‐ bour cells. Two industrially relevant flocculation phenotypes are well-known, Flo1 and NewFlo. Both are under the control of *FLO* genes, Flo1 phenotype is repressed by mannose and NewFlo by mannose, glucose and sucrose. Almost every industrial strain is NewFlo, as‐ sociated with *FLO*10 [30]. The main approach to improve these phenotypes is to put *FLO* genes under a promoter active only in stationary phase. Promoters of *HSP26* and *HSP30* were proven as the most suitable for induction at this late growth phase in lab strains [30].

The control of by-products production in order to improve wine and beer organoleptic properties is an expanding research area. The production of glycerol, to improve wine and beer's fullness, as well as sulphite, to improve stability, and the reduction in diacetyl and sulphide content are the main targets. Glycerol, as the second fermentation metabolite, is rather important to wine and beer; the increase of its concentration to improve these bever‐ ages sensory character is an active field. Overexpression of *GPD*1, encoding glycerol-3-pho‐ phate dehydrogenase, is the main approach. However, this change resulted in a redox imbalance with increased production of unwanted metabolites [39]. This point has been fully discussed in [2].

The presence of sulphite, an antioxidant and flavour stabilizer, and reduction of off-flavour producing sulphide is another important problem addressed by the industry. Both these goals can be achieved at the same time with the directed mutagenesis of NADPH-dependent sulphite reductase, an important enzyme in sulphur–containing amino acids synthesis. This strategy lowered this enzyme activity and increased the amount of sulphite in wine while reducing the sulphide presence in wine [58].

The reduction of diacetyl has special importance, as the maturation time (lagering) is direct‐ ly dependent on this compound concentration. The expression of the bacterial enzyme ace‐ tolactate decarboxylase (ALDC) in yeast is the main approach to reduce the amounts of this compound. ALDC catalyses the reaction of α-acetolactate to acetoin, preventing the forma‐ tion of diacetyl. However, after heterologous expression of ALDC, the yeast became auxo‐ trophic for some amino acids and the growth rate was very low in *wort*. An alternative approach was the interruption of *ILV*2, encoding acetolactate synthase. Such strategy also resulted in an auxotrophic strain with slow growth. The search for natural mutants in *ILV*2 with an appropriated growth rate is now the major strategy [30].

Improvement of yeast to render fermentations faster and cheaper is an industry goal, but the enhancing of the fermentation process itself is another alternative. The fed-batch technology has already proved its benefits [38], and improvements of such process with yeast immobili‐ zation/encapsulation are now under the spotlight. This results in much faster fermentation rates as compared to the existing free cell fermentations. However, it has some disadvantag‐ es, such as: i) complexity of production process including the choice of the suitable carrier materials, ii) bioreactors design, iii) fine-tuning of the flavour formation during fermentation processes, and iv) cost constraints [59].

## **3. Baker's yeast – Magic on bread making**

showed that overexpression of maltotriose transporters lead to positive effects on its metab‐

In winemaking and brewing, where flavour has such importance, amino acids metabolism has a notorious place. As said, amino acids are involved in formation of higher alcohols and esters that significantly contribute to beer and wine flavour. Since yeast cannot hydrolyse *must* and *wort* proteins, it depends on the available ammonium and amino acids in solution [11, 30]. However, the predominant amino acid in both *must* and *wort*, proline, is the less as‐ similated [40]. As such, improvements in yeast ability to uptake this amino acid has been attempted. Efficient proline uptake was reported in lager beer yeast expressing a mutagen‐ ized proline permease, Put4p. The site-directed mutagenesis stabilized the permease and en‐ hanced amino acid utilization without affecting the beer quality [56]. The study of the same problematic in winemaking led to the disruption of *URE*1, a repressor of permease encoding *PUT*1 and pyrroline-5-carboxylate dehydrogenase *PUT*2, with significant improvements in

Flocculation is a phenotype of industrial interest. It facilitates the filtration process in the end of fermentation, saving both time and money. In the case of brewing, it also serves the cropping (recover of part of the yeast population of the fermentation to pitch the next). Floc‐ culation is a reversible aggregation of cells, where lectins recognize sugar residues in neigh‐ bour cells. Two industrially relevant flocculation phenotypes are well-known, Flo1 and NewFlo. Both are under the control of *FLO* genes, Flo1 phenotype is repressed by mannose and NewFlo by mannose, glucose and sucrose. Almost every industrial strain is NewFlo, as‐ sociated with *FLO*10 [30]. The main approach to improve these phenotypes is to put *FLO* genes under a promoter active only in stationary phase. Promoters of *HSP26* and *HSP30* were proven as the most suitable for induction at this late growth phase in lab strains [30].

The control of by-products production in order to improve wine and beer organoleptic properties is an expanding research area. The production of glycerol, to improve wine and beer's fullness, as well as sulphite, to improve stability, and the reduction in diacetyl and sulphide content are the main targets. Glycerol, as the second fermentation metabolite, is rather important to wine and beer; the increase of its concentration to improve these bever‐ ages sensory character is an active field. Overexpression of *GPD*1, encoding glycerol-3-pho‐ phate dehydrogenase, is the main approach. However, this change resulted in a redox imbalance with increased production of unwanted metabolites [39]. This point has been

The presence of sulphite, an antioxidant and flavour stabilizer, and reduction of off-flavour producing sulphide is another important problem addressed by the industry. Both these goals can be achieved at the same time with the directed mutagenesis of NADPH-dependent sulphite reductase, an important enzyme in sulphur–containing amino acids synthesis. This strategy lowered this enzyme activity and increased the amount of sulphite in wine while

The reduction of diacetyl has special importance, as the maturation time (lagering) is direct‐ ly dependent on this compound concentration. The expression of the bacterial enzyme ace‐

olism [54, 55].

532 Food Industry

fermentation rate and vigour described [57].

fully discussed in [2].

reducing the sulphide presence in wine [58].

The process of bread making relies on the fermentation carried out by a mixture of yeast and bacteria. Even when all this was unknown and the flour leavening seen as "magic", bread was already produced and extensively consumed. On those ancient times, the leavening re‐ sulted presumably due to the action (fermentation) of the natural microbial contaminants of flour or dough ingredients. This was obviously not a controlled process, yet with the prac‐ tice of maintaining a fresh inoculum from one preparation to the next, promoted the selec‐ tion of yeast and bacteria biodiversity. Nowadays, some types of bread are still prepared in this fashion, sourdoughs are one example (for a review see [2]), but the baking industry moved for the use of commercially baker's yeast, typically the strain *S. cerevisiae*, for the bread production. And, while the flour types, geographical origin and mixtures introduce organoleptic differences in bread, the globalization of commercial baker's yeast market de‐ creased worldwide bread diversity (for a review see [2]).

#### **3.1. Commercial baker's yeast production – The break of spell**

Commercial baker's yeast is produced in several forms in order to meet specific require‐ ments of climate, technology, methodology, transportation, storage and final product. As with all biotechnology processes, this is in constant development/undergoing research not only to optimize the process technology and its components, but as to produce faster grow‐ ing strains with the characteristics to deliver better quality end products.

Molasses (beet and cane molasses), the common carbon and energy source used in the production of baker's yeast, is a by-product of sugar refining industries, therefore cheap‐ er than the formerly used cereals grain. Furthermore the sugars present on those molass‐ es (around 50%), consisting on a mixture of sucrose, fructose and glucose, are ready to be fermented by the yeast. In order to obtain the proper broth for the optimum yeast bio‐ mass yield; the mixture of molasses has to be supplemented with nitrogen sources, miner‐ als, salts and vitamins [60, 61].

ed, with incubation at optimal temperatures, the CO2 starts to saturate the liquid phase of the dough and starts to accumulate in the bubble *nuclei* [66]. This leads to dough rising pro‐ vided that a mature gluten network, capable of ensure the dough foam-like structure, is formed. The amount of time such process occurs will influence dough gumminess and rheology, as well as crust colour, crumb texture, and firmness of the bread (reviewed by [2, 3]). The amount of sugars present in the dough that are actually fermented, as well as the efficient secretion of enzymes as invertase, responsible for the conversion of sucrose to glu‐ cose and fructose, has a great impact in the flavour characteristics of the bread. The action of several enzymes, namely proteases, lecithinases, lipases α-glucosidase and β-fructosidase, leads to different utilization of the dough substrates. In bread making, the flavour is also greatly influenced by the metabolic by-products of the yeast fermentation, and while the most important by-product of yeast metabolism is certainly the CO2, the production of me‐ tabolites such as alcohols, esters, and carbonyl compounds have also a deep impact. More than 300 volatile compounds associated with bread's flavour and aroma, are produced by yeast. Although, some are dependent more on the substrates, the vast majority of such com‐

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 535

pounds are yeast dependent, introducing an important variability to bread [67].

year [2] as well as on [3, 68].

**4. Yeast à la Carte**

metals [70, 71].

**4.1. Yeast treats for animals**

As mentioned, the common procedure of bread making today, at least in developed coun‐ tries, consists of using this commercial baker's yeast. Its quality/individuality depends on storage stability, osmotolerance and freeze-thaw resistance. Considerable efforts have been made to obtain the Idol Yeast, including evolutionary engineering, genetic engineering (mainly to provide yeast with high capacity to tolerate freeze-thaw treatments). Yet, there is still considerable space for improvement. Those several strategies to achieve the Idol Yeast has been thoroughly revised and discussed in a previous work from the beginning of this

Fresh *S. cerevisiae* consists of approximately 30–33% of dry materials, 6.5–9.3% of nitrogen, 40.6–58.0% of proteins, 35.0–45.0% of carbohydrates, 4.0–6.0% of lipids, 5.0–7.5% of minerals and various amounts of vitamins, depending on its growth conditions [69]. So, today yeasts are acquiring increasingly more attention for other uses, besides the production of alcoholic beverages and the baling industry. The products of modern yeast biotechnology form the backbone of many commercially important sectors, including functional foods (for animals, fish and humans), health food supplements, including additives, conditioners and flavour‐ ing agents, as pharmaceutical products, for the production of microbiology media and ex‐ tracts, as well as livestock feed or even as agents of detoxifying effluents containing heavy

The pioneering research conducted almost a century ago by Max Delbrück and his collea‐ gues was the first to highlight the value of surplus brewer's yeast as a feeding supplement

After the preparation and sterilization of the broth, the production of baker yeast can take place. It begins by the inoculation of a small closed test flask containing the prepared steri‐ lized broth with a pure yeast culture. The growth is allowed and careful screened, and when the culture reaches an elevated density, it is transferred to larger vessels and supplied with more broth, fed-batch reactors. This scale-up process continues until a desirable biomass quantity is attained, the so-called commercial starter, able to inoculate industrial fermenters/ reactors, which production ranges from 40,000 to 200,000L [62].

The entire fermentation process of baker's yeast has to be directed towards maximum bio‐ mass production; by-products such as ethanol are not desired. As we saw in section 1.1.1, in anaerobic dissimilation of sugars (alcoholic fermentation) the ATP yield is quite low com‐ paring with respiratory dissimilation, affecting drastically the biomass yield. The way to avoid anaerobic ethanol production is the use of the mentioned fed-batch reactors, in which is possible to control the specific growth rate and sugar concentration by controlling the fed of reactors with fresh broth [63].

Nowadays, during the industrial large reactors the addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. In this way, the tones of baker's yeast obtained in the end of the fermentation have the same quality/characteristics/properties as the original pure yeast culture that started the process. These tones of yeast are suspended in a large amount of water, resulting in a creamy suspension of active yeast, being necessary the so-called **downstream processes** to obtain the concentrated yeast [64, 65]. At the end of the fermenta‐ tion, the yeast culture is concentrated using a series of combined centrifugation and washing steps, into a yeast cream with concentration of approximately 20%. The yeast is then cooled to approximately 4°C, and stored. It can be sold in this form – **Cream Yeast**- however is quite expensive due to the manipulations required because of high content in water. Cream yeast can be further processed, compressed or dried originating the **Granular Yeast** or **In‐ stant Dried Yeast**, if then converted in small granules, or **Cake Yeast** or **Active Dry Yeast**, if as an alternative the dried yeast is extruded or cut into blocks/cakes. All these yeast types are then packaged, typically vacuum packed to reduce the risk of contamination, and dis‐ tributed to wholesalers or traders. The shelf life of Active Dry/Cake Yeast and Instant Dry/ Granular Yeast at ambient temperature is 1 to 2 years.

#### **3.2. Idol baker's yeast**

Yeast has a significant role on bread making, greatly influencing the final product proper‐ ties. The most important contribution is in the leavening phase; after the dough has been kneaded and the gluten network start to develop, yeast starts to consume the available fer‐ mentable sugars and to produce ethanol and CO2, as mentioned in section 1. As fermenta‐ tion occurs, the dough is gradually depleted of O2 present in the air bubbles trapped in the dough, leaving small bubble *nuclei* full of N2. As the metabolism is more and more stimulat‐ ed, with incubation at optimal temperatures, the CO2 starts to saturate the liquid phase of the dough and starts to accumulate in the bubble *nuclei* [66]. This leads to dough rising pro‐ vided that a mature gluten network, capable of ensure the dough foam-like structure, is formed. The amount of time such process occurs will influence dough gumminess and rheology, as well as crust colour, crumb texture, and firmness of the bread (reviewed by [2, 3]). The amount of sugars present in the dough that are actually fermented, as well as the efficient secretion of enzymes as invertase, responsible for the conversion of sucrose to glu‐ cose and fructose, has a great impact in the flavour characteristics of the bread. The action of several enzymes, namely proteases, lecithinases, lipases α-glucosidase and β-fructosidase, leads to different utilization of the dough substrates. In bread making, the flavour is also greatly influenced by the metabolic by-products of the yeast fermentation, and while the most important by-product of yeast metabolism is certainly the CO2, the production of me‐ tabolites such as alcohols, esters, and carbonyl compounds have also a deep impact. More than 300 volatile compounds associated with bread's flavour and aroma, are produced by yeast. Although, some are dependent more on the substrates, the vast majority of such com‐ pounds are yeast dependent, introducing an important variability to bread [67].

As mentioned, the common procedure of bread making today, at least in developed coun‐ tries, consists of using this commercial baker's yeast. Its quality/individuality depends on storage stability, osmotolerance and freeze-thaw resistance. Considerable efforts have been made to obtain the Idol Yeast, including evolutionary engineering, genetic engineering (mainly to provide yeast with high capacity to tolerate freeze-thaw treatments). Yet, there is still considerable space for improvement. Those several strategies to achieve the Idol Yeast has been thoroughly revised and discussed in a previous work from the beginning of this year [2] as well as on [3, 68].

## **4. Yeast à la Carte**

fermented by the yeast. In order to obtain the proper broth for the optimum yeast bio‐ mass yield; the mixture of molasses has to be supplemented with nitrogen sources, miner‐

After the preparation and sterilization of the broth, the production of baker yeast can take place. It begins by the inoculation of a small closed test flask containing the prepared steri‐ lized broth with a pure yeast culture. The growth is allowed and careful screened, and when the culture reaches an elevated density, it is transferred to larger vessels and supplied with more broth, fed-batch reactors. This scale-up process continues until a desirable biomass quantity is attained, the so-called commercial starter, able to inoculate industrial fermenters/

The entire fermentation process of baker's yeast has to be directed towards maximum bio‐ mass production; by-products such as ethanol are not desired. As we saw in section 1.1.1, in anaerobic dissimilation of sugars (alcoholic fermentation) the ATP yield is quite low com‐ paring with respiratory dissimilation, affecting drastically the biomass yield. The way to avoid anaerobic ethanol production is the use of the mentioned fed-batch reactors, in which is possible to control the specific growth rate and sugar concentration by controlling the fed

Nowadays, during the industrial large reactors the addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. In this way, the tones of baker's yeast obtained in the end of the fermentation have the same quality/characteristics/properties as the original pure yeast culture that started the process. These tones of yeast are suspended in a large amount of water, resulting in a creamy suspension of active yeast, being necessary the so-called **downstream processes** to obtain the concentrated yeast [64, 65]. At the end of the fermenta‐ tion, the yeast culture is concentrated using a series of combined centrifugation and washing steps, into a yeast cream with concentration of approximately 20%. The yeast is then cooled to approximately 4°C, and stored. It can be sold in this form – **Cream Yeast**- however is quite expensive due to the manipulations required because of high content in water. Cream yeast can be further processed, compressed or dried originating the **Granular Yeast** or **In‐ stant Dried Yeast**, if then converted in small granules, or **Cake Yeast** or **Active Dry Yeast**, if as an alternative the dried yeast is extruded or cut into blocks/cakes. All these yeast types are then packaged, typically vacuum packed to reduce the risk of contamination, and dis‐ tributed to wholesalers or traders. The shelf life of Active Dry/Cake Yeast and Instant Dry/

Yeast has a significant role on bread making, greatly influencing the final product proper‐ ties. The most important contribution is in the leavening phase; after the dough has been kneaded and the gluten network start to develop, yeast starts to consume the available fer‐ mentable sugars and to produce ethanol and CO2, as mentioned in section 1. As fermenta‐ tion occurs, the dough is gradually depleted of O2 present in the air bubbles trapped in the dough, leaving small bubble *nuclei* full of N2. As the metabolism is more and more stimulat‐

reactors, which production ranges from 40,000 to 200,000L [62].

Granular Yeast at ambient temperature is 1 to 2 years.

als, salts and vitamins [60, 61].

534 Food Industry

of reactors with fresh broth [63].

**3.2. Idol baker's yeast**

Fresh *S. cerevisiae* consists of approximately 30–33% of dry materials, 6.5–9.3% of nitrogen, 40.6–58.0% of proteins, 35.0–45.0% of carbohydrates, 4.0–6.0% of lipids, 5.0–7.5% of minerals and various amounts of vitamins, depending on its growth conditions [69]. So, today yeasts are acquiring increasingly more attention for other uses, besides the production of alcoholic beverages and the baling industry. The products of modern yeast biotechnology form the backbone of many commercially important sectors, including functional foods (for animals, fish and humans), health food supplements, including additives, conditioners and flavour‐ ing agents, as pharmaceutical products, for the production of microbiology media and ex‐ tracts, as well as livestock feed or even as agents of detoxifying effluents containing heavy metals [70, 71].

#### **4.1. Yeast treats for animals**

The pioneering research conducted almost a century ago by Max Delbrück and his collea‐ gues was the first to highlight the value of surplus brewer's yeast as a feeding supplement for animals [72]. Yeasts have been fed to animals for more than a hundred years, either in the form of yeast fermented mash produced on the farm, yeast by-products from breweries or distilleries, or commercial yeast products specifically produced for animal feeding. In ani‐ mals, including pets, this practice is used to compensate for the amino acid and vitamin de‐ ficiencies of cereals [73, 74], and in fish as a substitute for other ingredients [71].

digestion of the fibrous and cellulolytic portion of the diet, which leads to a greater intake of

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 537

Dried yeast was used traditionally in poultry diets in the past as a source of aminoacids and micronutrients, and though the broiler growth was improving this practice was largely dis‐ continued for economic reasons [77]. On the other hand, brewer's yeast appears to be espe‐ cially beneficial for breeder turkeys and laying hens [85]. Reproductive improvements are attributed to the high level of dietary biotin and selenium in yeast, which is more beneficial than inorganic selenium added to poultry diets [86] and it contributes for the prevention of biotin deficiency in poultry diets, which may result in reduced feed conversion, low egg production, and poor hatchability [87]. Brewer's yeast is also very rich in folic acid, an im‐

Brewer's yeast has been recognized to have potential as well as a substitute for live food in the production of certain fish or as a potential replacement for fishmeal [88-90]. In addition, it has low content in phosphorous, meaning less water and environmental contamination than common fish meal and other plant-based alternate protein sources [91]. Multiple stud‐ ies have demonstrated the immunostimulant properties of yeasts, such as their ability to en‐ hance non-specific immune activity [92]. That reaction can be related to β-glucans, nucleic acids as well as mannan oligosaccharides [93]. Brewer's yeast may serve as an excellent health promoter for fish culture as even when administered for relatively long periods is able to enhance immune responses as well as growth of various fish species, without caus‐ ing immunosuppression [94, 95]. Furthermore, the relative high levels of nucleic acid nitro‐ gen present (mostly in the form of RNA) that in humans and most monogastric animals can became toxic if taken in excess, as the capacity of excretion of the uric acid formed is limited

In a world of rapidly increasing population and low agricultural production, yeasts are rela‐ tively cheap and easily produced on an industrial scale representing a sustainable alternate protein source to cover the population nutritional demands. The first time that yeast was cultivated in large scale for human nutritional use was in Germany during both World Wars [72]. The yeasts *S. cerevisiae* as baker yeast, *Candida utilis* as torula yeast, and *Kluyveromyces fragilis* as whey yeast, when produced on suitable, food grade substrates (*e.g.*, sugars, etha‐ nol, and lactose), are permitted in many foods around the world [70]. Besides the alcoholic beverages and baking products referred above, yeast are used in the health food industry; as additives, conditioners, and flavouring agents; as sources of high nutritional value proteins, enzymes, nucleic acids, nucleotides, and cell wall polysaccharides [98] as well as for the pro‐ duction of food-grade yeast extracts and autolysates [69, 99]. Yeast extract from dried brew‐ er's yeast cells can be used by enzymatic treatment in a wide variety of foods (*e.g.*, meat products, sauces and gravies, soups, chips and crackers) as flavours enhancers or potentia‐ tors [73, 100]. β-Glucan obtained from brewer's yeast can be used in food products as a thickening, water-holding, or oil-binding agent and emulsifying stabilizer [101]. The probi‐ otic activity is an additional role of some yeast that is attracting increasing interest [102].

[96], in fish does not happen due to their very active liver uricase [97].

food and better performance [83, 84].

portant vitamin for turkeys [73].

**4.2. Human little treats, big benefits**

Brewer's yeast biomass, as described above, which results from the cultivation of *S. cerevisiae* on malted barley, separated after the *wort* fermentation, debittered and then dried, is the second major by-product from brewing industry, just after the brewer spent grain [73]. This biomass is an excellent source of proteins, peptides and amino acids, vitamins (especially of B-group: B1, B2, PP, B5, B6, B8, B12), minerals and trace elements (calcium, phosphorus, po‐ tassium, magnesium, copper, iron, zinc, manganese, chromium, selenium), carbohydrates (glucans and mannans), as well as phospholipids [75]. The winemaking industry also gener‐ ates a huge amount of microbial biomass - **leeds**. Yet, this incorporates the yeasts (that die due to nutrient depletion) but also other microorganisms, suspended solids, colloids, and organic matter, and have been shown to display quite low nutritional value to be considered for use as a supplement in animal feed [76].

Those yeast used for monogastrics food or feeding rations is generally inactivated because feeding of live yeast might cause avitaminosis due to the depletion of B-vitamins in the in‐ testine [77]. They can also cause adverse fermentation in the digestive tract of swine leading to diarrhoea and bloating [75]. Yeasts can be killed through application of heat or using chemicals. High temperature destroys the yeast membrane, but does not necessarily inacti‐ vate all yeast enzymes, unless quite elevated temperatures are applied. Alternatively, there are the chemical treatments with propionic acid or formic acid, which also act as a preserva‐ tives for yeast, and contribute to the feed value of the yeast [73].

Yeasts have been used in diets of numerous species with varying levels of success. Yeast for swines is sold for feed applications as wet slurry, as dried brewer's yeast, or in mixtures with other brewery by-products [73]. It is ideal for their feed as a good protein source, it contains most of the essential amino acids in adequate quantities, and numerous vitamins, selenium, copper, and phosphorus. Selenium concentrations are much higher in yeast than in soybean meal, and deficiency of this compound in the swine's alimentation has been the cause of high‐ er swine mortality [78, 79]. Additionally, dried brewer's yeast contains mannan oligosacchar‐ ides, which have been reported to increase the growth performance and intestinal health of pigs [80]. Benefits have been described as well for nursing and weanling pigs [81].

Either live or inactivated brewer's yeast have been used as well in ruminants diets, conse‐ quently it was observed an increase in productivity of animal meat or milk [73, 82], but also live yeast cultures have been used. These are prepared by inoculating wet cereal grains or grain by-products with live yeast, partially fermenting the mash, and then drying the entire medium without killing yeast or destroying vitamins and enzymes [73]. Live yeast is report‐ ed to stimulate fermentation in the rumen through its ability to stimulate the development of anaerobic, cellulolytic and acid lactic bacteria fermentations. In addition, the ingestion of yeast offers continuous supply of vitamins, dicarboxylic acids, removal of oxygen, buffering effect, and reduction in the number of protozoa. As a result, there is an improvement of the digestion of the fibrous and cellulolytic portion of the diet, which leads to a greater intake of food and better performance [83, 84].

Dried yeast was used traditionally in poultry diets in the past as a source of aminoacids and micronutrients, and though the broiler growth was improving this practice was largely dis‐ continued for economic reasons [77]. On the other hand, brewer's yeast appears to be espe‐ cially beneficial for breeder turkeys and laying hens [85]. Reproductive improvements are attributed to the high level of dietary biotin and selenium in yeast, which is more beneficial than inorganic selenium added to poultry diets [86] and it contributes for the prevention of biotin deficiency in poultry diets, which may result in reduced feed conversion, low egg production, and poor hatchability [87]. Brewer's yeast is also very rich in folic acid, an im‐ portant vitamin for turkeys [73].

Brewer's yeast has been recognized to have potential as well as a substitute for live food in the production of certain fish or as a potential replacement for fishmeal [88-90]. In addition, it has low content in phosphorous, meaning less water and environmental contamination than common fish meal and other plant-based alternate protein sources [91]. Multiple stud‐ ies have demonstrated the immunostimulant properties of yeasts, such as their ability to en‐ hance non-specific immune activity [92]. That reaction can be related to β-glucans, nucleic acids as well as mannan oligosaccharides [93]. Brewer's yeast may serve as an excellent health promoter for fish culture as even when administered for relatively long periods is able to enhance immune responses as well as growth of various fish species, without caus‐ ing immunosuppression [94, 95]. Furthermore, the relative high levels of nucleic acid nitro‐ gen present (mostly in the form of RNA) that in humans and most monogastric animals can became toxic if taken in excess, as the capacity of excretion of the uric acid formed is limited [96], in fish does not happen due to their very active liver uricase [97].

#### **4.2. Human little treats, big benefits**

for animals [72]. Yeasts have been fed to animals for more than a hundred years, either in the form of yeast fermented mash produced on the farm, yeast by-products from breweries or distilleries, or commercial yeast products specifically produced for animal feeding. In ani‐ mals, including pets, this practice is used to compensate for the amino acid and vitamin de‐

Brewer's yeast biomass, as described above, which results from the cultivation of *S. cerevisiae* on malted barley, separated after the *wort* fermentation, debittered and then dried, is the second major by-product from brewing industry, just after the brewer spent grain [73]. This biomass is an excellent source of proteins, peptides and amino acids, vitamins (especially of B-group: B1, B2, PP, B5, B6, B8, B12), minerals and trace elements (calcium, phosphorus, po‐ tassium, magnesium, copper, iron, zinc, manganese, chromium, selenium), carbohydrates (glucans and mannans), as well as phospholipids [75]. The winemaking industry also gener‐ ates a huge amount of microbial biomass - **leeds**. Yet, this incorporates the yeasts (that die due to nutrient depletion) but also other microorganisms, suspended solids, colloids, and organic matter, and have been shown to display quite low nutritional value to be considered

Those yeast used for monogastrics food or feeding rations is generally inactivated because feeding of live yeast might cause avitaminosis due to the depletion of B-vitamins in the in‐ testine [77]. They can also cause adverse fermentation in the digestive tract of swine leading to diarrhoea and bloating [75]. Yeasts can be killed through application of heat or using chemicals. High temperature destroys the yeast membrane, but does not necessarily inacti‐ vate all yeast enzymes, unless quite elevated temperatures are applied. Alternatively, there are the chemical treatments with propionic acid or formic acid, which also act as a preserva‐

Yeasts have been used in diets of numerous species with varying levels of success. Yeast for swines is sold for feed applications as wet slurry, as dried brewer's yeast, or in mixtures with other brewery by-products [73]. It is ideal for their feed as a good protein source, it contains most of the essential amino acids in adequate quantities, and numerous vitamins, selenium, copper, and phosphorus. Selenium concentrations are much higher in yeast than in soybean meal, and deficiency of this compound in the swine's alimentation has been the cause of high‐ er swine mortality [78, 79]. Additionally, dried brewer's yeast contains mannan oligosacchar‐ ides, which have been reported to increase the growth performance and intestinal health of

Either live or inactivated brewer's yeast have been used as well in ruminants diets, conse‐ quently it was observed an increase in productivity of animal meat or milk [73, 82], but also live yeast cultures have been used. These are prepared by inoculating wet cereal grains or grain by-products with live yeast, partially fermenting the mash, and then drying the entire medium without killing yeast or destroying vitamins and enzymes [73]. Live yeast is report‐ ed to stimulate fermentation in the rumen through its ability to stimulate the development of anaerobic, cellulolytic and acid lactic bacteria fermentations. In addition, the ingestion of yeast offers continuous supply of vitamins, dicarboxylic acids, removal of oxygen, buffering effect, and reduction in the number of protozoa. As a result, there is an improvement of the

pigs [80]. Benefits have been described as well for nursing and weanling pigs [81].

ficiencies of cereals [73, 74], and in fish as a substitute for other ingredients [71].

for use as a supplement in animal feed [76].

536 Food Industry

tives for yeast, and contribute to the feed value of the yeast [73].

In a world of rapidly increasing population and low agricultural production, yeasts are rela‐ tively cheap and easily produced on an industrial scale representing a sustainable alternate protein source to cover the population nutritional demands. The first time that yeast was cultivated in large scale for human nutritional use was in Germany during both World Wars [72]. The yeasts *S. cerevisiae* as baker yeast, *Candida utilis* as torula yeast, and *Kluyveromyces fragilis* as whey yeast, when produced on suitable, food grade substrates (*e.g.*, sugars, etha‐ nol, and lactose), are permitted in many foods around the world [70]. Besides the alcoholic beverages and baking products referred above, yeast are used in the health food industry; as additives, conditioners, and flavouring agents; as sources of high nutritional value proteins, enzymes, nucleic acids, nucleotides, and cell wall polysaccharides [98] as well as for the pro‐ duction of food-grade yeast extracts and autolysates [69, 99]. Yeast extract from dried brew‐ er's yeast cells can be used by enzymatic treatment in a wide variety of foods (*e.g.*, meat products, sauces and gravies, soups, chips and crackers) as flavours enhancers or potentia‐ tors [73, 100]. β-Glucan obtained from brewer's yeast can be used in food products as a thickening, water-holding, or oil-binding agent and emulsifying stabilizer [101]. The probi‐ otic activity is an additional role of some yeast that is attracting increasing interest [102].

*S. cerevisiae* has been studied extensively for its medicinal properties and several beneficial/ probiotic effects on human health and well-being have been reported, including prevention and treatment of intestinal diseases and immunomodulatory actions, are the most wellknown. Probiotics are viable microorganisms that are beneficial to the host when consumed in appropriate quantities [103]. The probiotic properties of yeasts reported refer the ability to sur‐ vive through the gastrointestinal tract and interact antagonistically with gastrointestinal pathogens. As described above, *S. cerevisiae* have been used as supplements to animal and fish feeds with reported improvements on the growth and health of the hosts [104]. Regarding hu‐ mans, *S. cerevisiae* var. *boulardii* has been successfully used as an oral biotherapeutic agent to treat patients with severe cases of diarrhoea (*e.g.,* antibiotic-associated diarrhoea and travel‐ ler's diarrhoea) and other gastrointestinal disorders (*e.g.*, irritable bowel syndrome and Crohn's disease) [105]. Several studies have shown that *S. cerevisiae* var. *boulardii* confer benefi‐ cial effects against various enteric pathogens, involving different mechanisms as: i) preven‐ tion of bacterial adherence and translocation in the intestinal epithelial cells, ii) production of factors that neutralize bacterial toxins and iii) modulation of the host cell signalling pathway associated with pro-inflammatory response during bacterial infection [106]. As reviewed in detail in [106] prevention of bacterial adherence and translocation in the intestinal epithelial cells is due to the fact that the cell wall of *S. cerevisiae* var. *boulardii* has the ability to bind enter‐ opathogens, which results in a decrease of their adherence to host epithelial cells. This yeast al‐ so produces proteins that are responsible for degradation, neutralisation or dephosphorylation of bacterial toxins. Moreover, the mechanism by which *S. cerevisiae* var. *boulardii* modifies host cell signalling pathways associated with pro-inflammatory response is based on blocking activation of nuclear factor-kappa B (NF-κB) and mitogen activated pro‐ tein kinase (MAPK) which decreases the expression of inflammation-associated cytokines such as interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-α) and interferon gamma (IFNγ). Conversely, *S. cerevisiae* var. *boulardii* also stimulates the peroxisome proliferator-activated receptor gamma (PPAR-γ) expression in human colonocytes and reduces the response of hu‐ man colon cells to pro-inflammatory cytokines. There are several studies indicating the stimu‐ lation of the host cell immunity, both innate and adaptive immunity, by yeast in response to pathogen infections. Furthermore, it has been shown that *S. cerevisiae* var. *boulardii* also has a role in the maintenance of epithelial barrier integrity; during bacterial infection the tight junc‐ tions are disrupted and this yeast enhances the ability of intestinal epithelial cells to restore the tight-junction structure and the barrier permeability [106].

**4.3. What the future holds**

**Acknowledgements**

**Author details**

**References**

Elsevier; 2011.

Since *S. cerevisiae* var. *boulardii* is recognised as a member of the species *S. cerevisiae*, it is most likely that also other strains within *S. cerevisiae* might show probiotics properties. So far, great efforts have been placed on utilising the probiotic effects of especially LAB, where‐ as rather limited emphasis has been placed on the beneficial effects offered by yeast. How‐ ever, yeasts offer several advantages compared to LAB. They have a more diverse enzymatic profile and appear to have a more versatile effect on the immune system. They also provide protection against pathogenic bacteria and toxic compounds by surface binding and appear to be better suited for nutritional enrichment and delivery of bio-active molecules. Besides, yeast is much more robust than LAB and therefore easier to produce and to distribute, espe‐ cially in less developed areas [106]. Furthermore and though there is still much room for im‐ provement, also the encapsulation technology applied to probiotics has shown benefits, *e.g.*, *S. cerevisiae* var. *boulardii* in microspheres protect the yeast from destruction in the gastroin‐

Yeast: World's Finest *Chef* http://dx.doi.org/10.5772/53156 539

testinal tract and therefore increase intestinal delivery of the viable probiotic [114].

FCT (Fundação para a Ciência e a Tecnologia) project PEst-C/BIA/UI4050/2011.

University of Minho/ CBMA (Centre of Molecular and Environmental Biology), Portugal

[1] Kurtzman CP, Fell JW, Boekhout T. The Yeasts: A Taxonomic Study. Amsterdam:

[2] Tulha J, Carvalho J, Armada R, Faria-Oliveira F, Lucas C, Pais C, Almeida J, Ferreira C. Yeast, the Man's Best Friend. In: Valdez B. (ed.) Scientific, Health and Social As‐

pects of the Food Industry. Rijeka: InTech; 2012. p255-278.

Fábio Faria-Oliveira, Sónia Puga and Célia Ferreira

Authors would like to acknowledge Hugh S. Johnson for the several critical readings of the manuscript regarding proper English usage and Maria Manuel Azevedo for reviewing the manuscript and for valuable suggestions. Fábio Faria-Oliveira is supported by a PhD grant from FCT-SFRH/BD/45368/2008. This work was financed by FEDER through COMPETE Programme (Programa Operacional Factores de Competitividade) and national funds from

The benefits from ingesting yeasts do not stop here, many other have been reported as: se‐ lectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon [107], *e.g.,* fructooligosaccharides [108]; decreasing the serum cholesterol levels [109], in the treatment of diabetes (regulation of insulin levels) and chronic acne, reducing the ap‐ petite, for healthy hair and nails [70, 110, 111] and also for promoting the bioavailability of minerals through the hydrolysis of phytate, folate biofortification and detoxification of my‐ cotoxins due to surface binding to the yeast cell wall [106]. Yet, still are some concerns about public health safety as cause of crescent reports associating the intake of yeast with cases of fungaemia [112, 113].

### **4.3. What the future holds**

*S. cerevisiae* has been studied extensively for its medicinal properties and several beneficial/ probiotic effects on human health and well-being have been reported, including prevention and treatment of intestinal diseases and immunomodulatory actions, are the most wellknown. Probiotics are viable microorganisms that are beneficial to the host when consumed in appropriate quantities [103]. The probiotic properties of yeasts reported refer the ability to sur‐ vive through the gastrointestinal tract and interact antagonistically with gastrointestinal pathogens. As described above, *S. cerevisiae* have been used as supplements to animal and fish feeds with reported improvements on the growth and health of the hosts [104]. Regarding hu‐ mans, *S. cerevisiae* var. *boulardii* has been successfully used as an oral biotherapeutic agent to treat patients with severe cases of diarrhoea (*e.g.,* antibiotic-associated diarrhoea and travel‐ ler's diarrhoea) and other gastrointestinal disorders (*e.g.*, irritable bowel syndrome and Crohn's disease) [105]. Several studies have shown that *S. cerevisiae* var. *boulardii* confer benefi‐ cial effects against various enteric pathogens, involving different mechanisms as: i) preven‐ tion of bacterial adherence and translocation in the intestinal epithelial cells, ii) production of factors that neutralize bacterial toxins and iii) modulation of the host cell signalling pathway associated with pro-inflammatory response during bacterial infection [106]. As reviewed in detail in [106] prevention of bacterial adherence and translocation in the intestinal epithelial cells is due to the fact that the cell wall of *S. cerevisiae* var. *boulardii* has the ability to bind enter‐ opathogens, which results in a decrease of their adherence to host epithelial cells. This yeast al‐ so produces proteins that are responsible for degradation, neutralisation or dephosphorylation of bacterial toxins. Moreover, the mechanism by which *S. cerevisiae* var. *boulardii* modifies host cell signalling pathways associated with pro-inflammatory response is based on blocking activation of nuclear factor-kappa B (NF-κB) and mitogen activated pro‐ tein kinase (MAPK) which decreases the expression of inflammation-associated cytokines such as interleukin 8 (IL-8), tumor necrosis factor alpha (TNF-α) and interferon gamma (IFNγ). Conversely, *S. cerevisiae* var. *boulardii* also stimulates the peroxisome proliferator-activated receptor gamma (PPAR-γ) expression in human colonocytes and reduces the response of hu‐ man colon cells to pro-inflammatory cytokines. There are several studies indicating the stimu‐ lation of the host cell immunity, both innate and adaptive immunity, by yeast in response to pathogen infections. Furthermore, it has been shown that *S. cerevisiae* var. *boulardii* also has a role in the maintenance of epithelial barrier integrity; during bacterial infection the tight junc‐ tions are disrupted and this yeast enhances the ability of intestinal epithelial cells to restore the

tight-junction structure and the barrier permeability [106].

fungaemia [112, 113].

538 Food Industry

The benefits from ingesting yeasts do not stop here, many other have been reported as: se‐ lectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon [107], *e.g.,* fructooligosaccharides [108]; decreasing the serum cholesterol levels [109], in the treatment of diabetes (regulation of insulin levels) and chronic acne, reducing the ap‐ petite, for healthy hair and nails [70, 110, 111] and also for promoting the bioavailability of minerals through the hydrolysis of phytate, folate biofortification and detoxification of my‐ cotoxins due to surface binding to the yeast cell wall [106]. Yet, still are some concerns about public health safety as cause of crescent reports associating the intake of yeast with cases of

Since *S. cerevisiae* var. *boulardii* is recognised as a member of the species *S. cerevisiae*, it is most likely that also other strains within *S. cerevisiae* might show probiotics properties. So far, great efforts have been placed on utilising the probiotic effects of especially LAB, where‐ as rather limited emphasis has been placed on the beneficial effects offered by yeast. How‐ ever, yeasts offer several advantages compared to LAB. They have a more diverse enzymatic profile and appear to have a more versatile effect on the immune system. They also provide protection against pathogenic bacteria and toxic compounds by surface binding and appear to be better suited for nutritional enrichment and delivery of bio-active molecules. Besides, yeast is much more robust than LAB and therefore easier to produce and to distribute, espe‐ cially in less developed areas [106]. Furthermore and though there is still much room for im‐ provement, also the encapsulation technology applied to probiotics has shown benefits, *e.g.*, *S. cerevisiae* var. *boulardii* in microspheres protect the yeast from destruction in the gastroin‐ testinal tract and therefore increase intestinal delivery of the viable probiotic [114].

## **Acknowledgements**

Authors would like to acknowledge Hugh S. Johnson for the several critical readings of the manuscript regarding proper English usage and Maria Manuel Azevedo for reviewing the manuscript and for valuable suggestions. Fábio Faria-Oliveira is supported by a PhD grant from FCT-SFRH/BD/45368/2008. This work was financed by FEDER through COMPETE Programme (Programa Operacional Factores de Competitividade) and national funds from FCT (Fundação para a Ciência e a Tecnologia) project PEst-C/BIA/UI4050/2011.

## **Author details**

Fábio Faria-Oliveira, Sónia Puga and Célia Ferreira

University of Minho/ CBMA (Centre of Molecular and Environmental Biology), Portugal

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**Chapter 24**

ton/year; of this amount only 50% is

**Valorisation of Cheese Whey, a By-Product from the**

Whey is the by-product of cheese or casein production, it is of relative importance in the dai‐ ry industry due to the large volumes produced and the nutritional composition. Worldwide

processed [1]. Approximately 50% of worldwide cheese-whey (CW) production is treated and transformed into various food and feed products. About half of this amount is used di‐ rectly in liquid form, 30% as powdered cheese-whey, 15% as lactose and its byproducts and

plus of whey is 13×106 tons, containing about 619,250 tons of lactose. Nowadays this surplus is not utilized for further production of lactose; consequently, whey disposal represents a se‐ rious problem from both an economical and an environmental point of view. On the contra‐ ry, recovery of whey components and/or use of whey as fermentation medium may be

Whey contains more than half of the solids present in the original whole milk, including whey proteins (20% of the total protein) and most of the lactose, water-soluble vitamins and minerals. Consequently, whey can be considered a valuable by-product with several appli‐

From a valorization point of view, two different options in CW management can be consid‐ ered: the first one is based on the application of technologies to recover valuable compounds such as proteins and lactose. Currently, valorization processes applied to CW constitute the preferential option to treat this by-product, only exceeded by the production of powdered CW. The second option relies on the application of fermentation processes to obtain value

advantageous not only for the environment but also for a sustainable economy [4,5].

tons/year of whey is produced in the European Union [3]; the annual sur‐

© 2013 Mollea et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Chiara Mollea, Luca Marmo and Francesca Bosco

Additional information is available at the end of the chapter

whey production is estimated at around 180 to 190×106

the rest as cheese whey- protein concentrates [2].

cations in the food and pharmaceutical industries.

**Dairy Industry**

http://dx.doi.org/10.5772/53159

**1. Introduction**

A total of 40×106
