**3. The fungi relevance on food industry**

The living world is classified in three domains such as the *Bacteria*, the *Archaea* and the *Eukaryota* (Woese, 1990). Some authors argued that these species represent separate lines of

Electrochemical Behaviour of AISI 304 Stainless Steel

halogens Oxidising agents

**4.2 Legislative framework of using biocide** 

1999 Peroxydes

cell lysis

ATPase

Table 1. Biocides types in food processing and action mode

Membrane destabiliser,

Chlorhexidine specifically inhibits membrane – bound

Fig. 2. Action mode of biocide entry into a microorganism type (adapted from Russell, 2003).

During the past decades, the food production has become more complex; the production volume is larger, the operations are more mechanical, the food is more processed and the time and distance between production and consumption are longer. The new trends in food production and consumption lead to an increased need for efficient safety practices in the food processing industry. Currently, the biocides industry as a whole is governed by endindustry growth, technological developments, regulatory changes and the growing use of biocides as an aid in the hygiene processes. Science 2009 the world biocide demand is estimated to grow to 6,880 million dollars (5.4% annual growth from 2004) and by 2014 to 9,050 million dollars (5.6% annual growth from 2009). Accordingly, emerging EU (European Union) regulations such as Biocidal Products Directive 98/8/EC (BPD) (from May 14th 2000) and Registration, Evaluation and Authorisation and Restriction of Chemicals (REACH), strive to increase the safety and the eco-efficiency of chemical products and production processes (BPD, 1998; REACH, 2006). This directive has as objectives: (i) to harmonise the

effect (Russell, 2003).

Chlorine compounds and

Quaternary Ammonium

Compounds

Biguanides

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 103

the presence of organic matters, the concentration and the age of the microorganisms, the microorganisms ability to transform the biocide into inactive forms (McDonnell & Russell, 1999). The biocides have a broad spectrum of antimicrobial activity and generally act to several targets sites in microbial cells (Figure 2) and damage them as a results of the biocidal

**Biocides Action mode References**  Alcohols Membrane damage, cell lysis Larson & Morton, 1991

McDonnell & Russell,

97

Breslin & Tharp, 2001

descent that diverged early from an ancestral colony of organisms (Woese & Gupta, 1981; Woese, 1998). The Eukaryota domain include three major multicellular groups: animals, plants and fungi. Fungi including yeasts and molds are unicellular or multicellular eukaryotic microrganisms (Tournas et al., 2006; Pommerville, 2010). The fungal cells are larger than bacteria and structurally more complex than other microorganisms (Hugo et al., 2004). Some strains are important in food biotechnology as starter cultures with the ability to modify food characteristics and in industrial biotechnology to produce antibiotics and other beneficial by-products, such as enzymes, vitamins, organic acids (Deacon, 1997; Maier et al., 2009; Gautam et al., 2011) and others. *Saccharomyces cerevisiae* plays an important role in various fermentation processes to produce enzymes, antioxidants and vitamins. *Candida mycoderma* and *Aspergillus niger* have a major role to forming the citric acid (Tisnadjaja et al., 1996; Papagianni, 2007). Moreover, *Aspergillus niger* is important as a producer of proteins and a great variety of enzymes such as catalase, celullase, endoglucanase, glucosoxidase, invertase and pectinase (Lu et al., 2010). On the other hand, the food spoilage by fungi raises an economic issues and it is estimated that annually between 5% and 10% of the world's food production is lost due to fungal biodeterioration (Pitt & Hocking, 1997). The risk of health problems can appear due to mycotoxins produced as secondary metabolites by fungi during the stationary phase of growth in some specific physico-chemical conditions. The control of fungal spoilage of food products is therefore an essential and decisive matter to prevent different biological safety risks.

#### **4. Biocides**

Biocides represent a very diversified group of chemical substances with a crucial role in pharmaceutical and food industry and this role is becoming increasingly significant (Zani et al., 1997; Bridier et al., 2011). In food industry biocides are commonly used to disinfect the processing areas, equipments, containers, surfaces or pipes associated with the production, transport and storage of food or drink, including drinking water.

#### **4.1 Biocides effectiveness**

Biocides act in different ways (sometimes several biocides are combined in a product to increase the overall effectiveness) to destroy, render harmless, prevent the action, or otherwise exert a controlling effect on any harmful organism by chemical or biological means (Table 1). The biocides effectiveness is limited and much dependent on application conditions (Bessems 1998). The efficacy of biocides greatly depend on contact time, concentration temperature, *p*H and microorganisms type (Russell and McDonnell, 2000; Russel, 2003; Kitis 2004).

All biocides show varying degrees of activity against bacteria, bacterial spores, fungi, viruses and protozoa and at least some have algicidal activity (Russell, 2003). The biocides efficacy depends on a large of intrinsic and extrinsic factors. The intrinsic factors are characterized by the biocide type and its concentration, contact time and application (Russell & McDonnell, 2000). Furthermore, the relationship between contact time and concentration biocides determines the microbial reduction. The stability of the active biocide compounds in the environment also has an influence on the microbial action. The temperature is important, as most substances have a lower efficiency at low temperature. The contact mode also influences the biocides efficacy, as well as the contact time (mechanical effects) and the *p*H which plays an important role. Extrinsic factors consist in

descent that diverged early from an ancestral colony of organisms (Woese & Gupta, 1981; Woese, 1998). The Eukaryota domain include three major multicellular groups: animals, plants and fungi. Fungi including yeasts and molds are unicellular or multicellular eukaryotic microrganisms (Tournas et al., 2006; Pommerville, 2010). The fungal cells are larger than bacteria and structurally more complex than other microorganisms (Hugo et al., 2004). Some strains are important in food biotechnology as starter cultures with the ability to modify food characteristics and in industrial biotechnology to produce antibiotics and other beneficial by-products, such as enzymes, vitamins, organic acids (Deacon, 1997; Maier et al., 2009; Gautam et al., 2011) and others. *Saccharomyces cerevisiae* plays an important role in various fermentation processes to produce enzymes, antioxidants and vitamins. *Candida mycoderma* and *Aspergillus niger* have a major role to forming the citric acid (Tisnadjaja et al., 1996; Papagianni, 2007). Moreover, *Aspergillus niger* is important as a producer of proteins and a great variety of enzymes such as catalase, celullase, endoglucanase, glucosoxidase, invertase and pectinase (Lu et al., 2010). On the other hand, the food spoilage by fungi raises an economic issues and it is estimated that annually between 5% and 10% of the world's food production is lost due to fungal biodeterioration (Pitt & Hocking, 1997). The risk of health problems can appear due to mycotoxins produced as secondary metabolites by fungi during the stationary phase of growth in some specific physico-chemical conditions. The control of fungal spoilage of food products is therefore an essential and decisive matter to

Biocides represent a very diversified group of chemical substances with a crucial role in pharmaceutical and food industry and this role is becoming increasingly significant (Zani et al., 1997; Bridier et al., 2011). In food industry biocides are commonly used to disinfect the processing areas, equipments, containers, surfaces or pipes associated with the production,

Biocides act in different ways (sometimes several biocides are combined in a product to increase the overall effectiveness) to destroy, render harmless, prevent the action, or otherwise exert a controlling effect on any harmful organism by chemical or biological means (Table 1). The biocides effectiveness is limited and much dependent on application conditions (Bessems 1998). The efficacy of biocides greatly depend on contact time, concentration temperature, *p*H and microorganisms type (Russell and McDonnell, 2000;

All biocides show varying degrees of activity against bacteria, bacterial spores, fungi, viruses and protozoa and at least some have algicidal activity (Russell, 2003). The biocides efficacy depends on a large of intrinsic and extrinsic factors. The intrinsic factors are characterized by the biocide type and its concentration, contact time and application (Russell & McDonnell, 2000). Furthermore, the relationship between contact time and concentration biocides determines the microbial reduction. The stability of the active biocide compounds in the environment also has an influence on the microbial action. The temperature is important, as most substances have a lower efficiency at low temperature. The contact mode also influences the biocides efficacy, as well as the contact time (mechanical effects) and the *p*H which plays an important role. Extrinsic factors consist in

transport and storage of food or drink, including drinking water.

prevent different biological safety risks.

**4.1 Biocides effectiveness** 

Russel, 2003; Kitis 2004).

**4. Biocides** 

96

the presence of organic matters, the concentration and the age of the microorganisms, the microorganisms ability to transform the biocide into inactive forms (McDonnell & Russell, 1999). The biocides have a broad spectrum of antimicrobial activity and generally act to several targets sites in microbial cells (Figure 2) and damage them as a results of the biocidal effect (Russell, 2003).


Table 1. Biocides types in food processing and action mode

Fig. 2. Action mode of biocide entry into a microorganism type (adapted from Russell, 2003).

#### **4.2 Legislative framework of using biocide**

During the past decades, the food production has become more complex; the production volume is larger, the operations are more mechanical, the food is more processed and the time and distance between production and consumption are longer. The new trends in food production and consumption lead to an increased need for efficient safety practices in the food processing industry. Currently, the biocides industry as a whole is governed by endindustry growth, technological developments, regulatory changes and the growing use of biocides as an aid in the hygiene processes. Science 2009 the world biocide demand is estimated to grow to 6,880 million dollars (5.4% annual growth from 2004) and by 2014 to 9,050 million dollars (5.6% annual growth from 2009). Accordingly, emerging EU (European Union) regulations such as Biocidal Products Directive 98/8/EC (BPD) (from May 14th 2000) and Registration, Evaluation and Authorisation and Restriction of Chemicals (REACH), strive to increase the safety and the eco-efficiency of chemical products and production processes (BPD, 1998; REACH, 2006). This directive has as objectives: (i) to harmonise the

Electrochemical Behaviour of AISI 304 Stainless Steel

measurements, using the Metrohm 712 Conductometer.

*SS* immersed in *Neo* and also in different fungal suspensions.

(2), *Candida mycoderma* (3) and *Saccharomyces cerevisiae* (4).

**5.2 Results and discussions** 

**5.2.1 Electroactivity of working solutions** 

**5.1.4 Electrochemical measurements** 

Immersed in Mixtures Consisting by Biocide and Fungal Suspensions 105

99

Tests were performed on the corrosion behaviour of samples of *SS* in *Neo* with and without fungal suspensions. All electrochemical measurements were carried out in a glass electrochemical-cell (Metrohm, Switzerland) equipped with three electrodes, at room temperature (22±1ºC).The working electrode was AISI 304 Stainless Steel type, the counter electrode (CE) was a Platinum foil and as the reference electrode (RE) was a saturated calomel electrode (SCE). The entire three-electrode assembly was placed in a Faraday cage to limit the noise disturbance and then connected to potentiostat-galvanostat Bio-Logic SP-150 (France). The investigations are carried out using EC-Lab® Express v 9.46 software. The electrochemical measurements were carried out using the Linear Polarization Technique (LP). The polarization measurements were initiated after 60 seconds immersions to access an equilibrium open potential on the sample surface. In the preliminary study the potential was tested for different ranges, as ±2V; ±1.0 V (SCE) a variation of scan rate between 100÷10 mV/s. There were no large modifications of the results in the polarization curves tested. The following measurements recorded for a potential of ±1.5V (SCE) at the 20 mVs-1 were take into consideration because of larger scan rate appeared in the distorsion of the curves also for higher potential range. At the same time all solutions were examined for conductivity

The electrochemical behaviour of *SS* samples in *Neo* with and without fungal suspensions was investigated at room temperature. Figure 3 shows the polarization curves obtained for

Fig. 3. Polarization curves of SS in *Neo* (1) and different fungal suspensions: *Aspergillus niger* 

A larger range passivation of the processes could be observed in both electrochemical reactions (anodic and cathodic). From the polarization curves (Fig. 3, curves 2-4), that the potential *Ecorr* values show a more negative range for all fungal suspensions tested, whereas for *Neo Ecorr* the value is situated at a positive potential range (Fig. 3, curve 1). Electrochemical parameters for *Neo* (Fig. 3, curve 1) are *Ecorr +*390 mV and corrosion current

European market for biocidal products and their active substances such that product authorisation content in one Member State can be recognized in other Member States; (ii) to provide a high level of protection for people, animals and the environment (from the use of biocidal products) through risk assessment. These objectives requires the submission and evaluation of data relating to substances' chemistry, toxicity to humans, and toxicity and fate in the environment and (iii) to ensure products are sufficiently effective against the target species. The Registration REACH - a new chemical regulation was implemented by the EU on June 1st 2007 and is being implemented in stages to be completed by 1 st June 2018. The main objective of REACH is a high level of protection for human health and the environment, while maintaining the competitiveness and innovation of EU chemicals industry. REACH provides a single regulatory framework for the control of chemicals, replacing the previous patchwork of controls, and ensures that information on the properties of chemicals is transmitted down the supply chain, thus enabling them to be safely handled.
