**3. Development of new starter cultures for cheese processing**

Traditional raw-milk cheeses are highly valued for their flavors, while large-scale products are often perceived by the consumer as ''boring'' (Law, 2001) – a consequence of the elimination by pasteurization, of the flora that has a key role in flavor development; and this puts the food industry under pressure to look for alternative LAB cultures capable of improving products flavor (Leroy & De Vuyest, 2004).

Today, the increased understanding of the genomics and metabolomics of food microbes opens up new perspectives for starter-cultures improvements and through genetic engineering it is now possible to express their desirable properties or suppress undesirable features (Del- cour, De Vuyst, & Shortt, 1999; Law, 2001; Mogensen, 1993).

Originally, starter cultures for the cheese industry were maintained by daily propagation, and later, they became available as frozen concentrates and dried or lyophilised preparations, produced on an industrial scale, some of them allowing direct vat inoculation (Sandine, 1996). Because the original starter cultures were mixtures of several undefined microbes, the daily propagation, eventually led to shifts of the ecosystem resulting in the disappearance of certain strains. Because some important metabolic traits in LAB are plasmid-encoded, there was a risk that they would be lost during propagation (Weerkamp et al., 1996). Lactococci are generally used as starter cultures in the production of industrial cheeses and cultured milk products. In traditional cheeses the natural starter cultures may harbor many different species and strains.

12 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

increasing inhospitable conditions inside the cheese.

by pasteurization.

During processing, the pH history of the cheese is a good indicator of the actual product safety. For example a 'slow vat' allows more time at high pH for undesirable bacteria to grow, while during cheese ripening, unwanted bacteria may grow due to an acidity neutralization resulting from secondary microflora growth such as moulds. For most ripened varieties the combination of a low pH and ripening time, which leads to moisture decrease in the cheese, will in general cause a gradual decline of all groups of bacteria due to

The pH history of a cheese and the hygienic practices applied in its manufacture are thus key factors to guarantee safe products. Thus, the isolation of autochthonous LAB intend to be used for development of specific starter cultures with improved acid production and other antimicrobial activities may be an excellent way towards reaching the goals of

Nowadays, western consumers still enjoy artisan cheeses thanks to their outstanding gastronomic qualities; however, in most industrialized countries the large-scale cheese processing is the most important branch of the food industry. In such cases, there is a strong need to control the fermentation process towards maximum efficiency in terms of yields and standardization of the end product. This, and the need to fulfill the safety assurance of the final product, is usually achieved by, among other improvements, adding a high dosage of pure LAB selected starter cultures, commercially available (today's world starter culture market is more than US\$1 billion), as well as by heat treating the raw milk, most commonly

Traditional raw-milk cheeses are highly valued for their flavors, while large-scale products are often perceived by the consumer as ''boring'' (Law, 2001) – a consequence of the elimination by pasteurization, of the flora that has a key role in flavor development; and this puts the food industry under pressure to look for alternative LAB cultures capable of

Today, the increased understanding of the genomics and metabolomics of food microbes opens up new perspectives for starter-cultures improvements and through genetic engineering it is now possible to express their desirable properties or suppress undesirable

Originally, starter cultures for the cheese industry were maintained by daily propagation, and later, they became available as frozen concentrates and dried or lyophilised preparations, produced on an industrial scale, some of them allowing direct vat inoculation (Sandine, 1996). Because the original starter cultures were mixtures of several undefined microbes, the daily propagation, eventually led to shifts of the ecosystem resulting in the disappearance of certain strains. Because some important metabolic traits in LAB are plasmid-encoded, there was a risk that they would be lost during propagation (Weerkamp et al., 1996). Lactococci are generally used as starter cultures in the production of industrial

simultaneously obtaining safe traditional cheeses, still bearing their unique flavors.

**3. Development of new starter cultures for cheese processing** 

features (Del- cour, De Vuyst, & Shortt, 1999; Law, 2001; Mogensen, 1993).

improving products flavor (Leroy & De Vuyest, 2004).

On the other hand, cheeses manufactured in a standard (large-scale) processing manner, are considered as safer because of the application of pasteurization and following the standard hygienic practices, including the HACCP. Traditional cheeses have their own specific processing methods, namely the common use of raw milk, however the hygienic procedures and HACCP approaches adapted to their specificities should be applied as well.


**Table 3.** Main bacteria associated with cheeses or other fermented products (From: Broome et al., 2003).

As previously stated, LAB are only a part of the complete microflora of raw milk (Kongo et al, 2007) and this, associated to other technological methods such as pressing, allows the production of a diversity of traditional cheeses (Parguel, 2011). This raw-milk microflora represents the contamination from the environment (air, utensils, the animal skin), and the load and its diversity will thus vary with local, season and livestock type, influenced by temperature.

These microbial mixes have an interdependent activity when together in their ecosystem and therefore their physiological properties may differ when the biodiversity is disrupted. In fact, it has been shown that certain microbial associations reveal a higher protecting effect against pathogens such as listeria, than when their association diversity is disrupted, (Montel 2010) see Figure 9.

Bacteriocinogenic probiotic bacteria could be beneficial when used as starter cultures in cheese, as they may prolong the shelf-life of the products, while simultaneously providing the consumer with a healthy advantage at a low cost (Gomes et al. 1998). The presence of bacteriocins in foods is, in general, seen as safe for consumers because bacteriocins are inactivated by pancreatic or gastric enzymes (Liu et al., 2011).

Low level of *L. monocytogenes* in cheeses prepared with consortium associating lactic acid bacteria (species) and non lactic acid bacteria.

Highest level of *L. monocytogenes* in cheeses with *S.thermophilus* and without lactic acid bacteria in the consortium

Lactic Acid Bacteria as Starter-Cultures for Cheese Processing:

factors has increased the interest of food industry to use LAB polysaccharides. Research has also shown that EPS+ LAB can enhance the functional properties of low fat cheese and that the excellent water- binding properties and moisture retention of EPS can improve the melting properties of low fat Mozzarella cheese. These properties show that EPS have wide technical potentials for development of novel and improved food products with enhanced texture, mouth-feel, taste perception and stability, representing potential sources for

EPS have also the potential to be used as surface carriers of bacteriocins or bacteriocinproducing LAB, and species such as *Leuconostoc mesenteroides*, *Streptococcus mutans* and several lactobacilli (Lactobacillus brevis, *Lactococcus lactis* subsp. *lactis*, *L*. *lactis* subsp. *cremoris*, *Lactobacillus casei*, *Lb*. *sake*, *Lb. rhamnosus*,) and thermophilic (*Lb*. *acidophilus, Lb. delbrueckii*  subsp. *bulgaricus*, *Lb.helveticus* and *S. thermophilus*) are known to produce EPS. The isolation and characterization of EPS from new wild LAB species, which are ubiquitous in traditional

Finally, cheese ripening is a lengthy and costly process. Therefore, attenuated starter cultures with high autolysis are being sought towards increasing the amount of endogenous peptides, thus accelerating the cheese ageing process as well as enhancing flavour and texture. These cultures may be obtained via application of several techniques such as pulsed

Thus, the cheese industry in looking for new types of LAB starter-cultures bearing several properties: – cultures that increase microbial safety or offer one or more organoleptic, technological, nutritional (enzymes, or polyunsaturated fatty acids - PUFAs) or health advantages such as probiotic properties, starter cultures with increased resistance to bacteriophage, (recall that high product loss, especially in cheese manufacturing, is often

cheeses, is a key strategy towards finding strains with optimized production of EPS.

electric field, heat treatment, freeze–thawing and lysozyme treatment (Briggs, 2003).

**Figure 10.** Antilisterial activity of LAB isolates from a traditional cheese.

economic gains for the dairy industry.

Past, Present and Future Developments 15

**Figure 9.** Level of *L. monocytogenes* in the core of Saint-Nectaire type cheese (28d) (Adapted from Montel & Samelis, (2010).

### **3.1. EPS-producing cultures and acceleration of cheese ripening**

Many LAB produce exopolysaccharides (EPS), which may provide viscosifying, stabilizing, and water-binding effects in cheeses. The growing demand for all-natural, healthy food products, foods with low fat or sugar content and low levels of additives, as well as cost factors has increased the interest of food industry to use LAB polysaccharides. Research has also shown that EPS+ LAB can enhance the functional properties of low fat cheese and that the excellent water- binding properties and moisture retention of EPS can improve the melting properties of low fat Mozzarella cheese. These properties show that EPS have wide technical potentials for development of novel and improved food products with enhanced texture, mouth-feel, taste perception and stability, representing potential sources for economic gains for the dairy industry.

14 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

inactivated by pancreatic or gastric enzymes (Liu et al., 2011).

Low level of *L. monocytogenes* in cheeses prepared with consortium associating lactic acid bacteria

(species) and non lactic acid bacteria.

Montel & Samelis, (2010).

temperature.

(Montel 2010) see Figure 9.

As previously stated, LAB are only a part of the complete microflora of raw milk (Kongo et al, 2007) and this, associated to other technological methods such as pressing, allows the production of a diversity of traditional cheeses (Parguel, 2011). This raw-milk microflora represents the contamination from the environment (air, utensils, the animal skin), and the load and its diversity will thus vary with local, season and livestock type, influenced by

These microbial mixes have an interdependent activity when together in their ecosystem and therefore their physiological properties may differ when the biodiversity is disrupted. In fact, it has been shown that certain microbial associations reveal a higher protecting effect against pathogens such as listeria, than when their association diversity is disrupted,

Bacteriocinogenic probiotic bacteria could be beneficial when used as starter cultures in cheese, as they may prolong the shelf-life of the products, while simultaneously providing the consumer with a healthy advantage at a low cost (Gomes et al. 1998). The presence of bacteriocins in foods is, in general, seen as safe for consumers because bacteriocins are

> Highest level of *L. monocytogenes* in cheeses with *S.thermophilus* and without lactic acid bacteria in

the consortium

**Figure 9.** Level of *L. monocytogenes* in the core of Saint-Nectaire type cheese (28d) (Adapted from

Many LAB produce exopolysaccharides (EPS), which may provide viscosifying, stabilizing, and water-binding effects in cheeses. The growing demand for all-natural, healthy food products, foods with low fat or sugar content and low levels of additives, as well as cost

**3.1. EPS-producing cultures and acceleration of cheese ripening** 

EPS have also the potential to be used as surface carriers of bacteriocins or bacteriocinproducing LAB, and species such as *Leuconostoc mesenteroides*, *Streptococcus mutans* and several lactobacilli (Lactobacillus brevis, *Lactococcus lactis* subsp. *lactis*, *L*. *lactis* subsp. *cremoris*, *Lactobacillus casei*, *Lb*. *sake*, *Lb. rhamnosus*,) and thermophilic (*Lb*. *acidophilus, Lb. delbrueckii*  subsp. *bulgaricus*, *Lb.helveticus* and *S. thermophilus*) are known to produce EPS. The isolation and characterization of EPS from new wild LAB species, which are ubiquitous in traditional cheeses, is a key strategy towards finding strains with optimized production of EPS.

Finally, cheese ripening is a lengthy and costly process. Therefore, attenuated starter cultures with high autolysis are being sought towards increasing the amount of endogenous peptides, thus accelerating the cheese ageing process as well as enhancing flavour and texture. These cultures may be obtained via application of several techniques such as pulsed electric field, heat treatment, freeze–thawing and lysozyme treatment (Briggs, 2003).

**Figure 10.** Antilisterial activity of LAB isolates from a traditional cheese.

Thus, the cheese industry in looking for new types of LAB starter-cultures bearing several properties: – cultures that increase microbial safety or offer one or more organoleptic, technological, nutritional (enzymes, or polyunsaturated fatty acids - PUFAs) or health advantages such as probiotic properties, starter cultures with increased resistance to bacteriophage, (recall that high product loss, especially in cheese manufacturing, is often associated with bacteriophages (Parente and Cogan, 2004), cultures that produce EPS and cultures that accelerate cheese ripening.

Lactic Acid Bacteria as Starter-Cultures for Cheese Processing:

**Figure 11.** Proteolytic acitivity of a Lactobacillus ssp isolate on milk agar.

(Kongo et al., 2007)

addressed.

Molecular biology (genotypic) methods (Figure 12) on the other hand - largely DNA-based techniques - offer much greater discriminatory power, all the way to differentiation of individual strains (Aymerich et al., 2006, Cocolin et al., 2004, Furet et al., 2004, Prabhakar et al., 2011). Thus, a combination of both phenotypic and genotypic identification techniques (so called polyphasic approach) is preferred (Temmerman et al., 2004, Aquilanti et al., 2006).

**Figure 12.** Ribotyping as molecular biology technique for identification of LAB to type or strain level.

Finally, it should be mentioned that there are concerns today that commensal bacterial populations from food and the gastrointestinal tract (GIT) of humans and animals, such as LAB, could act as a reservoir for antibiotic resistance genes, and therefore, be transferred to possibly pathogenic bacterial species, complicating the treatment of a disease or infection and leading to the spread of antibiotic-resistant bacteria (Ammor et al., 2007). Thus, before using new isolates as starter cultures or as probiotics, the antibiotic resistance must be

The European Food Safety Agency (EFSA) proposed a system for a pre-market safety assessment of selected groups of microorganisms, leading to granting a "Qualified Presumption of Safety (QPS)". Therefore, EFSA proposed that a safety assessment of a

Past, Present and Future Developments 17

### **3.2. Methods used to characterize LAB for starter cultures development**

To characterize new LAB isolates, phenotypic methods relying on physiological or biochemical criteria have been widely applied (Montel, Talon, Fournaud, & Champomier, 1991, Kongo et al., 2007). These phenotypic profiling methods are very important - especially related to finding the technological features, such as the acidification, proteolytic and lipolytic activity, of a new isolate (see Tables 3 and 4, and Figure 11) and have the advantage of requiring less sophisticated equipment. In most of the cases however, these tests are insufficient for accurate species identification due to the great number of different LAB species with similar phenotypic characteristics (Temmerman et al. 2004).


**Table 4.** Phenotypic characteristics for discrimination of common LAB for dairy processing (modified from Batt, 1995).


**Table 5.** Enzyme profiling of 14 representative LAB isolates found in Sao Jorge traditional cheese (from Kongo et al., 2007).

**Figure 11.** Proteolytic acitivity of a Lactobacillus ssp isolate on milk agar.

cultures that accelerate cheese ripening.

from Batt, 1995).

Kongo et al., 2007).

associated with bacteriophages (Parente and Cogan, 2004), cultures that produce EPS and

To characterize new LAB isolates, phenotypic methods relying on physiological or biochemical criteria have been widely applied (Montel, Talon, Fournaud, & Champomier, 1991, Kongo et al., 2007). These phenotypic profiling methods are very important - especially related to finding the technological features, such as the acidification, proteolytic and lipolytic activity, of a new isolate (see Tables 3 and 4, and Figure 11) and have the advantage of requiring less sophisticated equipment. In most of the cases however, these tests are insufficient for accurate species identification due to the great number of different LAB

Characteristics *Lactobacillus Enterococcus Lactococcus Leuconostoc Pediococcus Streptococcus*  CO2 from glucose +/- - - + - - Growth at 10 ºC +/- + + + +/- - Growth at 45 ºC +/- + - - +/ +/- Growth at 6.5% NaCl +/- + - +/- +/ - Growth at 18% NaCl - - - - - - Growth at pH 4.4. +/- + +/- +/- + - Growth at pH 9.6 - + - - - - Type of lactic acid D, L, DL L L D L, DL L **Table 4.** Phenotypic characteristics for discrimination of common LAB for dairy processing (modified

**Table 5.** Enzyme profiling of 14 representative LAB isolates found in Sao Jorge traditional cheese (from

**3.2. Methods used to characterize LAB for starter cultures development** 

species with similar phenotypic characteristics (Temmerman et al. 2004).

Molecular biology (genotypic) methods (Figure 12) on the other hand - largely DNA-based techniques - offer much greater discriminatory power, all the way to differentiation of individual strains (Aymerich et al., 2006, Cocolin et al., 2004, Furet et al., 2004, Prabhakar et al., 2011). Thus, a combination of both phenotypic and genotypic identification techniques (so called polyphasic approach) is preferred (Temmerman et al., 2004, Aquilanti et al., 2006).

**Figure 12.** Ribotyping as molecular biology technique for identification of LAB to type or strain level. (Kongo et al., 2007)

Finally, it should be mentioned that there are concerns today that commensal bacterial populations from food and the gastrointestinal tract (GIT) of humans and animals, such as LAB, could act as a reservoir for antibiotic resistance genes, and therefore, be transferred to possibly pathogenic bacterial species, complicating the treatment of a disease or infection and leading to the spread of antibiotic-resistant bacteria (Ammor et al., 2007). Thus, before using new isolates as starter cultures or as probiotics, the antibiotic resistance must be addressed.

The European Food Safety Agency (EFSA) proposed a system for a pre-market safety assessment of selected groups of microorganisms, leading to granting a "Qualified Presumption of Safety (QPS)". Therefore, EFSA proposed that a safety assessment of a

defined taxonomic group, such as a genus or group of related species could be made based on establishing identity, body of knowledge, possible pathogenicity and end use (European Commission 2007). The 33 *Lactobacillus* species shown in Table 6 are the ones that in 2007 EFSA stated could be considered to have QPS-status. In addition to *Lactobacillus* species, also other LAB species have been granted QPS –status. They include three leuconostocs, (*Ln. citreum, Ln. lactis* and *Ln. mesenteroides*), three pediococci (*P. acidilactici, P. dextrinicus* and *P. pentosaceus*), *Lc. lactis* and *Streptococcus thermophilus.* 

Lactic Acid Bacteria as Starter-Cultures for Cheese Processing:

**Figure 13.** Result of a screening for antibiotic resistance of a *Lactobacillus paracasei* isolate

LAB are important in cheese processing because (i) they increase food safety through the release of lactic acid and bacteriocins, (ii) produce aromas and flavor and accelerate the maturation process of cheese via their proteolytic and lipolytic activities, bringing economic advantages to the industry, (iii) bring about desirable food textures via release of polysaccharides that increase the viscosity and firmness, and reduce susceptibility to syneresis, (iv) they may be used to deliver polyunsaturated fatty acids (PUFA) and vitamins, leading to dairy products with increased nutritional value, (v) specific probiotic strains contribute to liberation of health-enhancing bioactive peptides improving absorption in the intestinal tract, stimulating the immune system, exerting antihypertensive, antithrombotic

Novel insights arising from use of Bioinformatics, Systems Biology and Bioengeneering approaches will offer perspectives for the application of a new generation of starter cultures for cheese-making, having enhanced functional features and offering several health, marketing, and technological advantages, contributing to the development of small and medium sized enterprises on the one hand, and product diversification of large companies

However, there are still many developments to be achieved towards fully realizing the many foreseen potential of LAB or their products. For example extraction and purification of bacteriocins is still difficult as they form micelles or clumps with the nitrogen sources already in the growth medium. On the other hand while genetic engineering may offer many solutions related to optimal use of LAB, they may not be easily allowed by food

**4. Concluding remarks** 

on the other.

legislation.

effects, or functioning as carriers for minerals.

Past, Present and Future Developments 19


**Table 6.** *Lactobacillus* (Lb) species with QPS- status according to EFSA (from Korhonen, 2010).

Lactobacilli are generally susceptible to antibiotics inhibiting the synthesis of proteins, such as chloramphenicol, erythromycin, clindamycin and tetracycline, and more resistant to aminoglycosides (neomycin, kanamycin, streptomycin and gentamicin. While some species show a high level of resistance to glycopeptides (vancomycin and teicoplanin), susceptibility to bacitracin will vary greatly (Ammor et al, 2007; Coppola et al., 2005).


Key: IR, intrinsically resistant

**Table 7.** Microbiological break points (g mL-1) categorizing some LAB species as resistant (Adapted from Ammor et al., 2007)

**Figure 13.** Result of a screening for antibiotic resistance of a *Lactobacillus paracasei* isolate
