**2.3. Fresh meats**

In chilled vacuum-packaged beef, even close to the freezing point, psychrotrophic LAB are able to attain high population densities. At -1.5 °C LAB grew to 8-9 log10 cfu ml-1 drip in 16 weeks with maximum doubling times of around 2-4 days [31]. In this study, *Cb. divergens*, *Leuc. mesenteroides* and *Lb. delbruckii* dominated the LAB flora after 4, 8-12 and 16 weeks, respectively. At 2 °C other workers have reported *Lb. sakei, Lb. curvatus, Carnobacterium (Cb.) divergens, Cb. maltaromaticum, Leuconostoc spp.* and *Lactococcus raffinolactis* as relevant LAB with td of around 19 hours and less [32]. After 25 days maximum LAB numbers of around 7-8 log10 cfu cm-2 were reached and after 8 weeks the the meat odour immediately after opening the bags was regarded "definitely off" ("slightly off" between 4-6 weeks).

LAB may be useful as protective cultures during the ripening of vacuum-packaged raw beef and, bioprotective cultures may also help to reduce *E. coli* O157:H7 in frozen ground-beef patties [33,34]. Peptides generated by LAB have been suggested as sensorial and hygienic biomarkers in meat conditioning and fermentation [35].

Today, meat industry is forced to produce meats with a shelf life long enough to fulfill logistic, retail sale and consumer demands. Besides general hygienic considerations, including appropriate temperature control modified-atmosphere packaging (MAP) with 30- 40% CO2 is used to prevent early spoilage. While Gram-negative spoilage bacteria are suppressed, psychrotrophic LAB are not [36-38].

### **2.4. Cooked meats**

Cooked, sliced and prepackaged meat products are popular convenience foods. They are retailed under refrigeration with varying shelf lifes, e.g. at 5 to 7 °C for 14 to 28 days. During slicing and packaging the slices may be contaminated with microorganisms from the production environment. Especially certain psychrotrophic LAB may then attain high cell counts during cold storage and impair the sensory quality of the products [39-42]. More than 2/3 of the refrigerated sliced cooked meats from the German retail market contained LAB counts above 7 log10 cfu g-1 one week past the indicated shelf life (Figure 1) [43]. The LAB flora on Bologna-type sausage is mostly dominated by the *Lb. sakei/curvatus* cluster while *Leuc. carnosum* frequently dominates on cooked ham. Occasionally, also *Ws. viridescens*, *Cb. maltaromaticum* and *Leuc. mesenteroides* ssp. *mesenteroides* may occur in higher numbers. Independent from dominant occurrence, eight LAB species have been identified in German retail samples. The number of samples (n) out of 50 in which these species occurred were *Lb. sakei* (40), *Leuc. carnosum* (22), *Lb. curvatus* (18), *Ws. viridescens* (11), *Leuc. mesenteroides* ssp. *mesenteroides* (8), *Cb. maltaromaticum* (4), *Lactobacillus* sp. (4), *Lactococcus* sp. (4), *Cb. divergens* (2), *Leuc. gelidum* (1), *Leuconostoc* sp. (1) (Figure 2) [43].

**Figure 1.** Distribution of samples of refrigerated sliced cooked meats from the German retail market with respect to different LAB counts one week past the indicated shelf life [43].

**Figure 2.** Abundancy of different LAB species in refrigerated sliced cooked meats from German retail (n=50). sak, *Lb. sakei*; carn, *Leuc. carnosum*; curv, *Lb. curvatus*; viri, *Ws. viridescens*; mes, *Leuc. mesenteroides*; malt, *Cb. maltaromaticum*; Lb, *Lactobacillus* sp.; La, *Lactococcus* sp.; div, *Cb. divergens*; gel, *Leuc. gelidum*; Lc, *Leuconostoc* sp. [43].

### **3. Biopreservation**

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

biomarkers in meat conditioning and fermentation [35].

suppressed, psychrotrophic LAB are not [36-38].

(1), *Leuconostoc* sp. (1) (Figure 2) [43].

pathogenic microorganisms [30].

**2.3. Fresh meats** 

**2.4. Cooked meats** 

with a slightly hyaline appearance with an optimum safety against undesired and

In chilled vacuum-packaged beef, even close to the freezing point, psychrotrophic LAB are able to attain high population densities. At -1.5 °C LAB grew to 8-9 log10 cfu ml-1 drip in 16 weeks with maximum doubling times of around 2-4 days [31]. In this study, *Cb. divergens*, *Leuc. mesenteroides* and *Lb. delbruckii* dominated the LAB flora after 4, 8-12 and 16 weeks, respectively. At 2 °C other workers have reported *Lb. sakei, Lb. curvatus, Carnobacterium (Cb.) divergens, Cb. maltaromaticum, Leuconostoc spp.* and *Lactococcus raffinolactis* as relevant LAB with td of around 19 hours and less [32]. After 25 days maximum LAB numbers of around 7-8 log10 cfu cm-2 were reached and after 8 weeks the the meat odour immediately after

LAB may be useful as protective cultures during the ripening of vacuum-packaged raw beef and, bioprotective cultures may also help to reduce *E. coli* O157:H7 in frozen ground-beef patties [33,34]. Peptides generated by LAB have been suggested as sensorial and hygienic

Today, meat industry is forced to produce meats with a shelf life long enough to fulfill logistic, retail sale and consumer demands. Besides general hygienic considerations, including appropriate temperature control modified-atmosphere packaging (MAP) with 30- 40% CO2 is used to prevent early spoilage. While Gram-negative spoilage bacteria are

Cooked, sliced and prepackaged meat products are popular convenience foods. They are retailed under refrigeration with varying shelf lifes, e.g. at 5 to 7 °C for 14 to 28 days. During slicing and packaging the slices may be contaminated with microorganisms from the production environment. Especially certain psychrotrophic LAB may then attain high cell counts during cold storage and impair the sensory quality of the products [39-42]. More than 2/3 of the refrigerated sliced cooked meats from the German retail market contained LAB counts above 7 log10 cfu g-1 one week past the indicated shelf life (Figure 1) [43]. The LAB flora on Bologna-type sausage is mostly dominated by the *Lb. sakei/curvatus* cluster while *Leuc. carnosum* frequently dominates on cooked ham. Occasionally, also *Ws. viridescens*, *Cb. maltaromaticum* and *Leuc. mesenteroides* ssp. *mesenteroides* may occur in higher numbers. Independent from dominant occurrence, eight LAB species have been identified in German retail samples. The number of samples (n) out of 50 in which these species occurred were *Lb. sakei* (40), *Leuc. carnosum* (22), *Lb. curvatus* (18), *Ws. viridescens* (11), *Leuc. mesenteroides* ssp. *mesenteroides* (8), *Cb. maltaromaticum* (4), *Lactobacillus* sp. (4), *Lactococcus* sp. (4), *Cb. divergens* (2), *Leuc. gelidum*

opening the bags was regarded "definitely off" ("slightly off" between 4-6 weeks).

Biopreservation of meats refers to the control of pathogenic and spoilage microorganisms by a competitive microflora of desired indigenous microorganisms or so-called starter and protective cultures. The development of starter cultures for meats is tightly coupled with the industrialisation of the traditional artisanal processes. The production of safe and tasty fermented sausages by traditional technologies requires expert knowledge and continous attention to guide the fermentation into the desired direction, i.e. to promote the development of the desired microorganisms and to suppress the development of undesired microorganisms. Mistakes are heavily paid for by dangerous and/or low quality outcomes.

Starter cultures, added at the beginning of fermentation, allow a standardization of the product quality and considerably reduce the risk of product defects. However, it should be kept in mind that starter cultures can not replace good manufacturing practice which besides the selection of the appropriate raw materials with acceptable hygienic parameters also includes the implementation and control of appropriate processing conditions. This is especially true with respect to the health risks associated with enterohaemorrhagic *E. coli*. Because of its increased acid tolerance and low infective dose for human infection, additional hurdles besides starter cultures have become very important for the production of safe raw fermented sausages. The hurdles principle for controlling undesired microorganisms in raw sausage fermentation has been illustrated by LEISTNER [27,44,45] and, in the meantime the implementation of HACCP (hazard analyis critical control point) concepts have become mandatory in food production [46].

The Role of Lactic Acid Bacteria in Safety and Flavour Development of Meat and Meat Products 135

possible free of chemical preservatives [8], processing aids and allergenic additives, and which are not overly treated by physical processes, such as heat, high pressure and irradiation. Nevertheless, many consumers also simply do not care, as long as the product is safe and affordable. Thus, protective cultures may be interesting for health and wellnessoriented consumers in countries with higher living standards. But less developed countries could also benefit, especially where cold-chain management is difficult and high-tech processing aids are not readily available. The challenge simply is to find the right LAB

**4. Sensory acceptance of bioprotective cultures on prepackaged cold cuts** 

As already mentioned, the application of bioprotective microbial cultures to prepackaged cold cuts is a much discussed innovative and sustainable technology for improving the microbiological safety and overall quality of these products. It could be an alternative to chemical preservatives or to a second pasteurisation step after packaging which both have a negative sensory impact. Although quite a number of lactic acid bacteria (LAB) have been suggested as protective cultures for sliced cooked meats, there is basically no information on consumer perception of products with added LAB. At the International Green Week Berlin 2010 the concept was introduced for the first time to a broader public and visitors

Bologna-type sausages in 70 mm fiber casings were produced and stored at 2 °C until slicing. On the day of packaging the casings were removed and the sausages were briefely submersed in an aqueous suspension of a protective culture consisting of *Lb. sakei* strain Lb674 (sakacin P positive) and containing 8.5 log10 LAB ml-1. Subsequently, the sausages were sliced, vacuum-packaged in polyethylene bags and kept refrigerated at 5°C until presentation to interested visitors. The consumers reacted predominantly positive on the possibility of safeguarding cold cuts with bioprotectants. Up to day 15 after packaging the inoculated samples reached a relative preference score (achieved points versus achievable points) of more than 45% (max. 60%) as compared to 60-70% for the freshly sliced samples without added LAB. Thereafter, the overall liking of the inoculated prepackaged sausage gradually decreased (Figure 3). The results indicate a potential market for more natural, microbiologically safe and sound cold cuts as a specialized segment of the convenience sector. As stated above, a mild acidic note may not be completely avoided when using protective cultures. But, this 'disadvantage' should be balanced against the risk of an uncontrolled growth of listeria on the one hand and the demand of many consumers for less

The steeply increasing business in the industrialised countries with health and wellness oriented foods in the 1990s, starting with probiotics in dairy products, has also raised interest in the development of probiotic meat products [56]. The concept of probiotics requires the intake of relevant amounts by the consumer of living probiotic microorgansims,

cultures for the particular product.

were asked to participate in a sensory preference test [7].

chemical preservatives or thermal treatments on the other hand.

**5. Probiotics** 

Protective cultures may be distinquished from starter cultures by their lack of, or their reduced product transformation capabilities. Protective cultures my be used for a number of applications with the main focus on pathogen control, especially of *Li. monocytogenes*, but also of spoilage organisms such as LAB involved in the spoilage of deli meats, or of *Brochothrix thermosphacta* and *Clostridium estertheticum* in vacuum-packaged raw meats [47-50].

Of special interest are strains which excrete powerful anti-listerial bacteriocins *in situ* and, which at the same time have no or only a very weak spoilage potential [21, 51].

A strain of *Lactococcus (Lc.) lactis*, marketed as Bactoferm® Rubis by Chr.-Hansen A/S, is offered as a protective culture to be used instead of chemicals to preserve/stabilize the normal colour of vacuum packed or controlled atmosphere packaged, sliced, cured meat products [52].

The big retail chains and the official food control authorities look at high microbial counts in deli meats, regardless of the responsible microflora, usually with suspicion. The German Society for Hygiene and Microbiology (DGHM), e.g., recommends a maximum of 5x106 cfu g-1 [53]. In reality, however, many of the prepackaged sliced cold cuts display 10-100 times higher counts at the the end of their indicated shelf lives without being recognized as spoiled by sensory panels. On the other hand, unpleasant tastes and smells (not fresh, sour) are often associated with high LAB counts [54]. But a high count *per se* does not tell how long the product has been exposed to this high count already. Protective LAB cultures are looked at with suspicion because they have to be added in high numbers and, if metabolically too active, may reduce shelf life. Some authors generally view psychrotrophic LAB as spoilage organisms, regardless of their generally moderate role in spoilage [55]. There is no doubt that cold-cuts with protective cultures will differ from products without protective culture. But, as long as this difference is only manifested in a minor sour taste this kind of sensory deviation may be a reasonable price to pay for an increased food safety, especially with respect to *Li. monocytogenes*, without chemical preservatives and the control of more striking spoilage organisms, e.g. such as *Brochothrix thermosphacta*. Food preferences are changing, and presently many consumers tend to prefer products which are as much as possible free of chemical preservatives [8], processing aids and allergenic additives, and which are not overly treated by physical processes, such as heat, high pressure and irradiation. Nevertheless, many consumers also simply do not care, as long as the product is safe and affordable. Thus, protective cultures may be interesting for health and wellnessoriented consumers in countries with higher living standards. But less developed countries could also benefit, especially where cold-chain management is difficult and high-tech processing aids are not readily available. The challenge simply is to find the right LAB cultures for the particular product.

## **4. Sensory acceptance of bioprotective cultures on prepackaged cold cuts**

As already mentioned, the application of bioprotective microbial cultures to prepackaged cold cuts is a much discussed innovative and sustainable technology for improving the microbiological safety and overall quality of these products. It could be an alternative to chemical preservatives or to a second pasteurisation step after packaging which both have a negative sensory impact. Although quite a number of lactic acid bacteria (LAB) have been suggested as protective cultures for sliced cooked meats, there is basically no information on consumer perception of products with added LAB. At the International Green Week Berlin 2010 the concept was introduced for the first time to a broader public and visitors were asked to participate in a sensory preference test [7].

Bologna-type sausages in 70 mm fiber casings were produced and stored at 2 °C until slicing. On the day of packaging the casings were removed and the sausages were briefely submersed in an aqueous suspension of a protective culture consisting of *Lb. sakei* strain Lb674 (sakacin P positive) and containing 8.5 log10 LAB ml-1. Subsequently, the sausages were sliced, vacuum-packaged in polyethylene bags and kept refrigerated at 5°C until presentation to interested visitors. The consumers reacted predominantly positive on the possibility of safeguarding cold cuts with bioprotectants. Up to day 15 after packaging the inoculated samples reached a relative preference score (achieved points versus achievable points) of more than 45% (max. 60%) as compared to 60-70% for the freshly sliced samples without added LAB. Thereafter, the overall liking of the inoculated prepackaged sausage gradually decreased (Figure 3). The results indicate a potential market for more natural, microbiologically safe and sound cold cuts as a specialized segment of the convenience sector. As stated above, a mild acidic note may not be completely avoided when using protective cultures. But, this 'disadvantage' should be balanced against the risk of an uncontrolled growth of listeria on the one hand and the demand of many consumers for less chemical preservatives or thermal treatments on the other hand.

## **5. Probiotics**

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

concepts have become mandatory in food production [46].

raw meats [47-50].

products [52].

Starter cultures, added at the beginning of fermentation, allow a standardization of the product quality and considerably reduce the risk of product defects. However, it should be kept in mind that starter cultures can not replace good manufacturing practice which besides the selection of the appropriate raw materials with acceptable hygienic parameters also includes the implementation and control of appropriate processing conditions. This is especially true with respect to the health risks associated with enterohaemorrhagic *E. coli*. Because of its increased acid tolerance and low infective dose for human infection, additional hurdles besides starter cultures have become very important for the production of safe raw fermented sausages. The hurdles principle for controlling undesired microorganisms in raw sausage fermentation has been illustrated by LEISTNER [27,44,45] and, in the meantime the implementation of HACCP (hazard analyis critical control point)

Protective cultures may be distinquished from starter cultures by their lack of, or their reduced product transformation capabilities. Protective cultures my be used for a number of applications with the main focus on pathogen control, especially of *Li. monocytogenes*, but also of spoilage organisms such as LAB involved in the spoilage of deli meats, or of *Brochothrix thermosphacta* and *Clostridium estertheticum* in vacuum-packaged

Of special interest are strains which excrete powerful anti-listerial bacteriocins *in situ* and,

A strain of *Lactococcus (Lc.) lactis*, marketed as Bactoferm® Rubis by Chr.-Hansen A/S, is offered as a protective culture to be used instead of chemicals to preserve/stabilize the normal colour of vacuum packed or controlled atmosphere packaged, sliced, cured meat

The big retail chains and the official food control authorities look at high microbial counts in deli meats, regardless of the responsible microflora, usually with suspicion. The German Society for Hygiene and Microbiology (DGHM), e.g., recommends a maximum of 5x106 cfu g-1 [53]. In reality, however, many of the prepackaged sliced cold cuts display 10-100 times higher counts at the the end of their indicated shelf lives without being recognized as spoiled by sensory panels. On the other hand, unpleasant tastes and smells (not fresh, sour) are often associated with high LAB counts [54]. But a high count *per se* does not tell how long the product has been exposed to this high count already. Protective LAB cultures are looked at with suspicion because they have to be added in high numbers and, if metabolically too active, may reduce shelf life. Some authors generally view psychrotrophic LAB as spoilage organisms, regardless of their generally moderate role in spoilage [55]. There is no doubt that cold-cuts with protective cultures will differ from products without protective culture. But, as long as this difference is only manifested in a minor sour taste this kind of sensory deviation may be a reasonable price to pay for an increased food safety, especially with respect to *Li. monocytogenes*, without chemical preservatives and the control of more striking spoilage organisms, e.g. such as *Brochothrix thermosphacta*. Food preferences are changing, and presently many consumers tend to prefer products which are as much as

which at the same time have no or only a very weak spoilage potential [21, 51].

The steeply increasing business in the industrialised countries with health and wellness oriented foods in the 1990s, starting with probiotics in dairy products, has also raised interest in the development of probiotic meat products [56]. The concept of probiotics requires the intake of relevant amounts by the consumer of living probiotic microorgansims,

The Role of Lactic Acid Bacteria in Safety and Flavour Development of Meat and Meat Products 137

and the strains under consideration have to cope with and survive in the presence of nitrite, sodium chloride, reduced pH and water activity, various processing steps and, eventually, long-term storage. Due to the manufacturing process raw fermented sausages contain high numbers of lactic acid bacteria which, however, are not regarded as probiotics. On the other hand, most of the known probiotic bacteria are unable to establish themselves in the raw sausage environment. Exceptions thereof are microbial cultures belonging to the *Lactobacillus plantarum group* and to the *Lactobacillus casei* group [57-59]. The use of protective and probiotic cultures may be a useful and effective strategy to prevent or reduce pathogens

Within a project investigating the possibilities for manufacturing high quality and microbiologically sound products from meat of mother sheep, salami-type raw fermented sausages were produced with added conventional (*Lb. sakei*, *Lb. plantarum*) and probiotic lactic starter cultures (*Lb. paracasei*). The products were subjected to microbiological and sensory evaluation for up to nine months. All sausage batches with added cultures resulted in microbiological safe and sensory appealing products. The *Lb. sakei* culture survived during the whole storage period on a high level (> 108 cfu/g) while the two other cultures (*Lb. plantarum, Lb. paracasei* ) partly reached the threshold of 106 cfu g-1 already after 3 months and were replaced by indigenous lactic acid bacteria of the *Lb. sakei / curvatus* group. For some batches, however, an acceptable number of probiotic bacteria could still be detected after nine months. Overall, *Lb. paracasei* showed a better survival in the ripened sausage than *Lb. plantarum* [7].

One problem for official authorities involved in consumer protection is to verify the presence of the indicated probiotics at sufficiently high levels. In the absence of simple and relyable identification procedures this may be a challenging task. In such cases genetic fingerprinting of isolates recovered on suitable agar media at relevant dilutions is the method of choice (Figure 4) [57, 60]. In the past, *Lb. rhamnosus* and *Lb. paracasei* ssp. *paracasei* have been used in fermented sausages, and labelling was quite confusing (Table 1). As can be seen, *Lb. paracasei* survived in relatively high numbers even in very dry salami. More recently, also other LAB species have been suggested as probiotics, and microencapsulation of strains has been used to overcome survival problems in the sausage environment. Still,

In fermented sausage production classical starter cultures are usually also protective cultures, especially with respect to the acid-sensitive microflora. Modern cultures may provide additional protective action, e.g. by producing bacteriocins inhibitory to listeria and/or undesired LAB, or they may possess an additional probiotic functionality. Strains

Strains from many LAB species excrete anti-listerial bacteriocins, of which nisin produced by *Lc. lactis* and pediocin produced by *Pc. acidilactici* are the most wellknown. Besides,

human verification studies for probiotic administration are quite rare [61].

combining these traits have been termed also 'functional starter cultures' [62].

**6. Functional starter cultures** 

**7. Bacteriocin production** 

in the food chain, improve food safety and consumer health.

**Figure 3.** Consumer preference of vacuum-packaged Bologna-type sausage with *Lb. sakei* protective culture (bio) in comparison to non-packaged, sliced on-the-spot sausages without (nat) and with chemical (chem) preservatives presented at the International Green Week Berlin 2010. n, number of responses [7].

**Figure 4.** Genetic fingerprints of probiotic LAB and related reference strains using BOX-PCR [57].

and raw fermented sausages were considered as an appropriate vehicle for these probiotics. However, these environments are quite different from the human gastrointestinal (GI) tract, and the strains under consideration have to cope with and survive in the presence of nitrite, sodium chloride, reduced pH and water activity, various processing steps and, eventually, long-term storage. Due to the manufacturing process raw fermented sausages contain high numbers of lactic acid bacteria which, however, are not regarded as probiotics. On the other hand, most of the known probiotic bacteria are unable to establish themselves in the raw sausage environment. Exceptions thereof are microbial cultures belonging to the *Lactobacillus plantarum group* and to the *Lactobacillus casei* group [57-59]. The use of protective and probiotic cultures may be a useful and effective strategy to prevent or reduce pathogens in the food chain, improve food safety and consumer health.

Within a project investigating the possibilities for manufacturing high quality and microbiologically sound products from meat of mother sheep, salami-type raw fermented sausages were produced with added conventional (*Lb. sakei*, *Lb. plantarum*) and probiotic lactic starter cultures (*Lb. paracasei*). The products were subjected to microbiological and sensory evaluation for up to nine months. All sausage batches with added cultures resulted in microbiological safe and sensory appealing products. The *Lb. sakei* culture survived during the whole storage period on a high level (> 108 cfu/g) while the two other cultures (*Lb. plantarum, Lb. paracasei* ) partly reached the threshold of 106 cfu g-1 already after 3 months and were replaced by indigenous lactic acid bacteria of the *Lb. sakei / curvatus* group. For some batches, however, an acceptable number of probiotic bacteria could still be detected after nine months. Overall, *Lb. paracasei* showed a better survival in the ripened sausage than *Lb. plantarum* [7].

One problem for official authorities involved in consumer protection is to verify the presence of the indicated probiotics at sufficiently high levels. In the absence of simple and relyable identification procedures this may be a challenging task. In such cases genetic fingerprinting of isolates recovered on suitable agar media at relevant dilutions is the method of choice (Figure 4) [57, 60]. In the past, *Lb. rhamnosus* and *Lb. paracasei* ssp. *paracasei* have been used in fermented sausages, and labelling was quite confusing (Table 1). As can be seen, *Lb. paracasei* survived in relatively high numbers even in very dry salami. More recently, also other LAB species have been suggested as probiotics, and microencapsulation of strains has been used to overcome survival problems in the sausage environment. Still, human verification studies for probiotic administration are quite rare [61].

## **6. Functional starter cultures**

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

1 2 3 4 5 6 7 8 9 10 11 12 13 14

responses [7].

**Figure 3.** Consumer preference of vacuum-packaged Bologna-type sausage with *Lb. sakei* protective culture (bio) in comparison to non-packaged, sliced on-the-spot sausages without (nat) and with chemical (chem) preservatives presented at the International Green Week Berlin 2010. n, number of

1 100-bp marker

ProbioTecTM)

*4 Lb. rhamnosus* DSM 20021T

6 Lambda EcoRI/HindIII marker

fermented sausage, CH)

*12 Lb. casei* SHIROTA (Yakult)

*2 Bifidobacterium animalis subsp. lactis* DSM 10140T *3 Bifidobacterium lactis* BB-12 (FD-DVS BB-12®

*5 Lb. rhamnosus* (from probiotic poultry salami, D)

*7 Lb. paracasei subsp. paracasei* (from probiotic dry-

*8 Lb. paracasei subsp. paracasei* (from probiotic thincalibre raw fermented sausages, D) *9 Lb. paracasei subsp. paracasei* DSM 5622T *10 Lb. casei* DSM 20011T, ATCC 393T

*11 Lb. casei* 01 (FD-DVS Lb. casei 01 nutrish® )

*13 Lb. paracasei subsp. tolerans* DSM 20258T

**n, 55 108 85 59 67 94 64 123 108 54**

**Figure 4.** Genetic fingerprints of probiotic LAB and related reference strains using BOX-PCR [57].

and raw fermented sausages were considered as an appropriate vehicle for these probiotics. However, these environments are quite different from the human gastrointestinal (GI) tract,

14 100-bp Marker

In fermented sausage production classical starter cultures are usually also protective cultures, especially with respect to the acid-sensitive microflora. Modern cultures may provide additional protective action, e.g. by producing bacteriocins inhibitory to listeria and/or undesired LAB, or they may possess an additional probiotic functionality. Strains combining these traits have been termed also 'functional starter cultures' [62].
