Detection of probiotic cultures in probiotic raw fermented sausages from retail .
Lactic acid bacteria (LAB) are widespread in nature and commonly occur on all kind of plant materials, on mucous membranes, in saliva and, in feces. Consequently and unavoidably they are part of the contamination flora of fresh meats after slaughter. Under certain conditions, e.g. in packaged refrigerated meats or raw sausage meats, they are able to compete efficiently with accompanying microorganisms for nutrients and may reach substantial viable counts. Their metabolic activities may ultimately result in either a desired preservative effect due to the repression of pathogenic and spoilage microorganisms, a desired tasty meat product, such as raw fermented sausage, or in meat spoilage through undesired transformations of raw and cooked meats. Heterofermentative LAB of the Carnobacterium, Leuconostoc and Weissella genera are usually more involved in meat spoilage than the homofermentative Lactobacillus and Pediococcus genera. Therefore, commercially available meat starter cultures for dry-fermented sausage production exclusively belong to the latter two. Homofermentative LAB produce almost exclusively lactic acid from fermentable carbohydrates present in meats, which is relatively mild and palatable, while heterofermentative species produce significant amounts of less desirable fermentation end products, such as CO2 gas, ethanol, acetic acid, butanoic acid and acetoin. However, under certain conditions Lactobacillus spp. may also produce significant amounts of acetic acid, ropy slime and, discolouration (greening) of meats [1,2].
In food industry starter and protective cultures are currently used in a number of products to safeguard the microbial and sensory quality. Lactic acid bacteria (LAB) are the main players in the natural transformation of agricultural primary products into safe, delicious and shelf stable foods for human consumption. In meat products there are three basic fields of application for the targeted use of such cultures: raw fermented sausages, raw cured hams, and pasteurised, sliced prepackaged meats (cold cuts) [3-8]. The use of protective cultures in prepackaged, refrigerated sliced Bologna-type sausage and cooked ham against pathogenic listeria is a much discussed, sustainable technology for improving the microbial safety and quality of these products. It helps to avoid chemical preservatives, such as sodium lactate/potassium acetate additives, or repasteurisation in package after slicing and packaging, which both have a negative impact on sensory product quality leaving a numb mouthfeel or warmed-over flavour, resp. [9,10].
2. Meat and meat products
2.1. Raw fermented sausages
The importance of starter and protective cultures for the manufacturing of safe and high-quality fermented sausages has been known for a long time and, lactobacilli play an important role in their production [11,5]. Lb. sakei and Lb. curvatus are quite often the predominant LAB in dry-fermented sausage while other lactobacilli, such as Lb. versmoldensis, Lb. plantarum, Lb. brevis, Lb. farciminis, Lb. alimentarius, Weissella species, pediococci, and leuconostocs, usually occur in significantly lower numbers . This has recently been also shown for different traditional salamis from North Italy [13-15]. However, other recipes and ripening conditions may promote other LAB as well. LAB isolated from dry spontaneously fermented sausages from 15 different producers in Spain included mainly Lb. sakei (66%), Lb. curvatus (26%), and Lb. plantarum (8%) . For dry fermented Spanish 'chorizo' sausage Lb. sakei (69%), Lb. curvatus (16%) and Pediococcus (9%) have been reported . From naturally fermented Greek dry salami about 50% of the isolates belonged to Lb. sakei/curvatus, 30% to the Weissella genus, 10% to Lb. plantarum and 3% each to Lb. farciminis and Enterococcus (Ec.) faecium . In "Alheira", a fermented sausage produced in Portugal, Lb. plantarum and Ec. faecalis prevailed while other LAB, such as Lb. paraplantarum, Lb. brevis, Lb. rhamnosus, Lb. sakei, Lb. zeae, Lb. paracasei, Leuconostoc (Leuc.) mesenteroides, Pediococcus (Pc.) pentosaceus, Pc. acidilactici, Weissella (Ws.) cibaria, Ws. viridescens and Ec. faecium, occurred in lower numbers .
The main role of LAB is to convert fermentable sugars in the sausage batter to lactic acid, thereby contributing to product safety by creating unfavourable conditions for pathogens and spoilage organisms. The production of lactic acid has also a direct impact on sensory product quality by providing a mild acidic taste, and by supporting the drying process which requires a sufficient decline in pH. Furthermore, LAB influence the sensory characteristics of the fermented sausages by the production of small amounts of acetic acid, ethanol, acetoin, pyruvic acid, carbon dioxide, and their ability to initiate the production of aromatic substances from proteinaceous precursors [20-22]. The selection criteria for lactic acid bacteria to be used in the production of fermented sausage include (i) fast production of lactic acid (ii) good growth at different temperatures, (iii) homofermentative metabolism, (iv) persistence over the whole fermentation and ripening process, (v) nitrate reduction, (vi) ability to express catalase, (vii) no fermentation of lactose, (viii) formation of flavour, (ix) no formation of peroxide, (x) no formation of biogenic amines, (xi) no formation of ropy slime, (xii) tolerance or even synergy to other microbial components of the starter, (xiii) antagonism against pathogens, (xiv) antagonism against technologically undesirable microorganisms, (xv) improvement of the nutritional value of the sausage and, (xvi) economic factors . Many homofermentative LAB associated with cured meat products are quite resistant to nitrite up to 200 ppm . A new starter culture for raw sausages, ‘BITEC Advance LD-20’ from Frutarom Savory Solutions, containing Lb. sakei and S. carnosus is marketed as consistently providing a ‘pleasant mild taste’ while rapidly deminishing the pH value of the sausage batter. Rapid acidification is important for product safety while a high competitiveness against the spontaneous lactic flora is important for product quality. The culture can be used for firm and fresh raw sausages as well as sausage spreads.
The use of homofermentative lactic acid bacteria is desirable because acetic acid has an unpleasant taste as compared with lactic acid .
It must be kept in mind, however, that, although lactic acid production and pH reduction by LAB provide quite unfavorable conditions for pathogenic bacteria thereby preventing them from growing and contributing to their reduction, several pathogenic microorganisms are able to survive in fermented sausages under certain conditions for extended periods, especially during refrigerated storage of sparsely dried sausages. Pathogenic strains of Escherichia (E.) coli, Listeria (Li.) monocytogenes and Yersinia (Y.) enterocolitica are inactivated better after the initial fermentation and ripening stage if stored at ambient rather than at refrigeration temperature. Inclusion of a maturation period above refrigeration temperatures before distribution may increase the safety of these products [26-29].
2.2. Dry-cured hams
Currently there are only a few publications which clearly substantiate the advantages of starter and protective cultures during raw cured ham production. On the other hand, starter cultures have been more and more implemented by meat industry into the production of dry-cured hams since the early 1980s [6,30]. These cultures are expected to be active under the harsh manufacturing conditions (low temperatures, high salt, lack of oxygen, presence of nitrite). LAB contribute to a moderate pH decrease which promotes the microbial stability as well as product texture, reduce stickiness and pH variations of the raw material. As an example, FSC-111 BactofermR from Chr.-Hansen A/S contains, besides a staphylococcal strain, also a strain of Lb. sakei.
The LAB induced acidification is usually more pronounced with injected or compound meats than with dry-salted ones. Modern turkey hams are produced by squeezing turkey breast over the screw of an extruder in the presence of (g/kg) nitrite curing salt (35), diphosphate (2,5), dextrose (2), water (100), starter culture and a spice compound, and subsequent tumbling until protein release. This mixture is then stuffed into fiber casings and left for 5 days at 2°C. This is followed by a fermentation step of around 16 hours at 22°C and 92-94% relative humidity until a pH below 5.4 is reached. Finally, the product is heated in a cabinet at 47 °C to a core temperature of 40°C. The desired result is a fresh looking product with a slightly hyaline appearance with an optimum safety against undesired and pathogenic microorganisms .
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 . 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 . 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 .
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) . 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) .
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 .
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].
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 .
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 . 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 . 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 . 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 , 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 wellness-oriented 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 .
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.
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 . The concept of probiotics requires the intake of relevant amounts by the consumer of living probiotic microorgansims,
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 .
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 .
6. Functional starter cultures
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’ .
7. Bacteriocin production
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,
|Type||soft, quickly ripened, smoked salami||very hard, air-dried salami||quickly ripened, thin-calibre, smoked sausage||smoked, dry-fermented salami with 30% weightloss|
|Characteristics||pH 4.7; aw 0.954||pH 5.6||pH 4.9||n. d.|
|Claims||’probiotic poultry salami with three probiotic cultures (Bifidus, Lb. casei, Lb. acidophilus)’||‚probiotic’,|
with beef and pork
|‘probiotic culture in high numbers (5x108 cfu/g)’,|
with beef and pork
|’Probiotic !!!, naturally ripened’,|
with beef and vegetable fat (no lard)
|advertised culture detectable||no||not labeled||yes||not labeled|
|detected po-tentially pro-biotic culture||Lb. rhamnosus||Lb. paracasei subsp. paracasei||Lb. paracasei subsp. paracasei||Lb. paracasei subsp. paracasei|
|Viable counts (cfu/g) of pro-biotic culture||1-6 x 107||4-9 x 107||3 x 107||1 x 107|
bacteriocin-producing LAB with anti-listerial activity naturally occur on a wide range of ready-to-eat foods, including meats [63-65]. From a meat point of view the sakacins of Lb. sakei are the most interesting because of the high competitivity of this species in the meat environment [22,49]. The pediocin producer Pc. acidilactici is commonly used by the Spanish meat industry as a starter culture .
8. Hydrogen peroxide production
The demonstration of hydrogen peroxide formation by meat-borne lactic acid bacteria is of considerable importance for the characterization of individual strains, the selection of suitable starter and protective cultures for various applications for meat and meat products as well as for the search of potential microbiological causes for undesired sensory deviations (discolourations/'greening', rancidity). Many LAB are able to form hydrogen peroxide as a by-product of O2-dependent metabolic pathways. Dependent on the environment, this trait may be desired or undesired [1,23,67,69,104].
In foods and feed it may contribute to the inhibition of an undesired accompanying microbiota . The H2O2 formed by LAB acts bacteriostatic on GRAM-positive bacteria and bactericidal on Gram-negatives [12,68].
In a recent study a novel agar medium (‘Prussian Blue’ (PB) agar) was applied for the first time to lactic acid bacteria relevant to meat and meat products . The PB agar detects H2O2 through the formation of Prussian Blue (Figure 5). It principally delivers similar results as the traditional manganese dioxide agar. However, it is more sensitive and, it is also more easily prepared and delivers results more quickly. A representative number of strains was used in the evaluation of the new medium (Table 2).
As to the production of H2O2, the study revealed large differences within the Lb. sakei/curvatus group. The bacteriocin producers frequently seemed to be relatively weak peroxide producers, while many commercial starter cultures were recognized as more or less strong peroxide producers. More recent field isolates of Lb. sakei/curvatus from prepackaged sliced Bologna-type sausage gave an essentially similar picture. In this case, however, only one of ten isolates of Lb. curvatus gave rise to a positive reaction.
|Lb. sakei (Lb. bavaricus)||DSM 20494||-||nd|
|Lb. sakei ssp. carnosus (Lb. curvatus ssp. melibiosus)||DSM 15740||+||++|
|Lb. sakei ssp. carnosus||Lb1047||-||+|
|Lb. sakei ssp. carnosus||DSM 15831T||++||++|
|Lb. sakei ssp. carnosus (ssp. sakei)||23K||+++||++|
|Lb. sakei ssp. carnosus||Lb790||+++||++|
|Lb. sakei ssp. sakei||DSM 20017T||+++||+|
|Lb. brevis||DSM 20054T||++||-|
|Lb. farciminis||DSM 20180T||-||nd|
|Lb. hilgardii||DSM 20176T||-||-|
|Weissella paramesenteroides||DSM 20288T||+++||nd|
|Weissella minor||DSM 20014T||+++||nd|
9. Formation of biogenic amines
Several LAB may produce biogenic amines by decarboxylation of amino acids, e.g. Lb. buchneri, Lb. brevis, Lb. curvatus, Lb. hilgardii, Cb. maltaromaticum, Cb. divergens . Examples are such as tyramine and histamine during sausage fermentation. Strains of Lb. plantarum, Lb. brevis and Lb. casei/paracasei, and Ec. faecium and Ec. faecalis were identified as tyramine/histamine producers in the sausages . Suitable starter cultures may contribute to reduction of biogenic amines in fermented sausages .
10. Identification of LAB
Identification of meat associated LAB is still wideley performed with phenotypic methods only, e.g API 50 CH . These are, however, not always satisfying and may lead to misidentifications . Nowadays, the application of PCR-DGGE and 16S rRNA gene sequencing allow the identification of a large number of strains in a quick and fast way [21,75]. Also various genomic fingerprinting methods are available. Nevertheless, conventional approaches remain important, especially when dealing with previously unknown species. Modern identification procedures rely on polyphasic approaches, integrating several lines of evidence to obtain a comprehensive description of a new species or of a microbiota .
11. Important LAB in meats
11.1. The Lb. sakei/curvatus cluster
In his 1983 review on lactic acid bacteria of meat and meat products Egan mentions that according to recent findings of Kandler and co-workers Lb. sakei (then Lb. sake) and Lb. curvatus were very common on German meat products . Presently, two subspecies of Lb. sakei are known of which ssp. carnosus is the one characteristic for meats. It is common in fermented meat products, and is regularly found in vacuum-packaged meat and fermented plant material (sauerkraut). The subspecies sakei has been isolated from the Japanese sake starter and is regularly found in fermented meat products, vacuum-packaged meat, fermented plant material (sauerkraut), and human feces. The two subspecies can not be separated based on their physiological and biochemical characteristics . The genomes of Lb. sakei 23K from a French dry-fermented sausage and Lb. curvatus CRL705 from an Argentinean artisanal fermented sausage have been sequenced [77,78]. Both genomes are highly similar. Lb. curvatus CRL705 lacks several genes present in Lb. sakei such as those related to fatty acid biosynthesis FASII, sucrose utilization, the arginine deiminase pathway, and citrate metabolism. The ones unique in Lb. curvatus CRL705 include genes for proteins and enzymes involved in the metabolism of carbohydrates, DNA, and fatty acids, as well as in the oxidative stress response and in bacteriocin production.
11.2. Lb. plantarum
The LAB species Lb. plantarum displays a high flexibility and versatility, and is able to colonize several ecological niches such as vegetables, meats, fish, milk substrates, and the human GI tract. This is the basis of many applications in the food and health areas. As a starter culture for salamis Lb. plantarum is used since decades. More recently also probiotic strains have been described. With a size of 3.3 Mb its genome is one of the largest of LAB. A recent study on the phenetic and genetic diversity of the species revealed a high phenetic diversity which generally correlated with the origin of the isolates, e.g. from meat fermentations, kimchi, sourdough, egg plants and cheese. Four main clusters were determined: (i) meat, (ii) vegetable, (iii) sourdough, (iv) mixed sources with high meat content. On the genome level there were seven main clusters. The core genome contains more than 2000 genes, 121 genes being specific for L. plantarum. None of the strains could grow in milk, or at 4°C, or in the presence of 10% NaCl. A limited number grew at 17°C, or at 6% NaCl . One of the earliest and most successful starter cultures for raw fermented sausages on the German market, “DuploFerment 66”, contains a strain of Lb. plantarum. This is also the case for the “Saga II” starter from the US. In contrast to the first one, the latter strain does not grow at 10°C. Both strains are homofermentative for lactate and grow at 42°C but not at 8°C . They provide rapid acidification of the raw sausage batter. On the other hand, Lb. plantarum is not very well adapted to meat and fails to maintain sufficiently high cell numbers to outcompete indigenous LAB. Sometimes it even does not grow in the meat batter [80,81]. In Italian natural fermented sausage the initial dominant populations of Lb. plantarum were accompanied by Lb. sakei and Lb. curvatus from the 10th day of fermentation and were finally competed out by the latter . But, in certain traditional Greek fermented sausages Lb. plantarum and Lb. plantarum/pentosus may predominate [82,83].
11.3. Lb. brevis
In combination with Pc. pentosaceus, Lb. brevis has been used as an indigenous starter culture for a Vietnamese fermented meat product . While Lb. brevis strongly acidifies the product, Pc. pentosaceus acts as a mild acidifier. The combination of both species resulted in a product with an intermediate taste (not too mild and not too sour) preferred by the sensory panel. Meat isolates of Lb. brevis may produce bacteriocins with antagonistic activity against Li. monocytogenes .
11.4. Lb. versmoldensis
This species was first reported in 2003 as the dominant LAB in some German raw fermented poultry salamis. The species was present in high numbers and frequently dominated the lactic acid bacteria (LAB) populations of the products . Later, the species has been isolated also from Scandinavian fermented meats, Egyptian Domiati cheese and Japanese traditional fermented fish products [87-89]. There are no studies to date on the general behaviour of this species in meat ecosystems. The genome of strain KCTC 3814, an isolate from poultry salami, has been recently sequenced by the Korea Research Institute of Bioscience & Biotechnology .
Carnobacteria are non-aciduric and, therefore, are preferentially isolated from meats with elevated pH. Cb. divergens and Cb. maltaromaticum frequently constitute a major component of the microflora of packaged raw meats as well as of refrigerated, prepackaged, sliced cooked deli meats. Meat spoilage by Cb. maltaromaticum has been associated with “dairy”, “spoiled-meat”, and “mozarella cheese” perception [31,91,92]. The major volatiles on meat, acetoin, 1-octen-3-ol and butanoic acid, are volatile organic compounds with low sensory impacts. Butanoic acid in stored beef was also associated with Cb. divergens. It has a rancid cheese-like odor and can derive from leucine metabolism, microbial consumption of free amino acids via the Stickland reaction or from tributyrin hydrolysis.
The metabolites from leucine degradation are involved in dry fermented sausage aroma. The catabolism of leucine by a strain of Cb. maltaromaticum was studied directly in the growth medium with H-3-labelled leucine to investigate the effect of five parameters: phase of growth, pH, oxygen, glucose and alpha-ketoisocaproic acid. Leucine catabolism was most important during the exponential phase of growth. The addition of alpha-ketoisocaproic acid at 1%, glucose at levels of 0.5% to 2% and shaking of the growth medium increased leucine catabolism. At pH 5.4 and 7.2, the main metabolites detected were 3-methyl butanal, 3-methyl butanol and alpha-ketoisocaproic acid. At pH 6.5, the leucine catabolism was maximum and was characterised by a high production of 3-methyl butanoic acid .
Positive and negative effects of carnobacteria in the environment and in foods have recently been reviewed . Because Cb. divergens and Cb. maltaromaticum show good growth in refrigerated meats and some of the strains produce potent anti-listerial bacteriocins, they may have some role as bioprotectants in meat environments. However, carnobacteria are associated with unpleasant spoilage metabolites in meats, such as acetic and butanoic acid as well as gas production in vacuum packed beef. An undesirable trait is also their ability to produce the biogenic amine tyramine from tyrosine. Carnobacteria are not regarded as human pathogens, but Cb. maltaromaticum is a well known fish pathogen and catagorised as a safety-level-2 microorganism. The genome of Cb. maltaromaticum ATCC 35586 carries putative virulence genes which probably play a role in fish pathogenesis . Since carnobacteria are inhibited by acetate they do not grow well on routine LAB media such as MRS. A selective enumeration medium using a combination of three antibiotics (gentamicin, nalidixic acid, vancomycin) and an alkaline pH value (8.8) has recently been proposed for Cb. maltaromaticum from cheese .
Leuc. gelidum is a major spoilage organism in Finnish fresh meats . Certain strains of Leuc. gelidum may produce yellow discolourations on prepackaged refrigerated German ‘Weisswurst' and cold cuts (Figure 6, 7) . Recently, the genome of a plant isolate of Leuc. gelidum has been sequenced .
The responsible pigment for the intensive ’neon-like’ yellow discolouration is a bacterial carotenoid, the non-polar C30-carotenoid 4,4’-di-apo-7,8,11,12-tetra-hydro-lycopene. On fat-containing substrates this compound does not only stain the bacterial cells but also the substrate and, in the case of ’Weisswurst’ does stain the natural casing (porc intestine) of the sausage as well as the sausage surface beneath. This triterpenoid is an intermediate in the microbial synthesis of 4,4’-diaponeurosporene which represents the main carotenoid in pigmented enterococci, Leuc. citreum and Lb. plantarum. Identification of the pigment was achieved by using UV-VIS spectroscopy in combination with available data from literature .
A report from Canada also described the yellow discolouration phenomenon on cooked sliced meats which had been stored for an extended time period under refrigeration . These authors, employees of a big Canadian food company (then Canada Packers Inc.), tentatively identified an Enterococcus sp. as the causative agent.
Leuc. gasicomitatum has been recognized as a specific spoilage organism in cold-stored Finnish MAP meats. It emerged as a spoilage problem of tomato-marinated, raw broiler meat strips. Due to CO2 production the packages already showed clear bulging more than a week before the expected shelf life . It is a psychrotrophic species and, because of its dominance in marinated meats and fish as well as in vegetable sausages, probably of plant origin. But, it was also detected in minced meat and high-oxygen modified-atmosphere packaged raw, beef steaks injected with sugar-salt solutions, so-called moisture-enhanced or value-added meats [97,103]. Recently, the genome of the type strain Leuc. gasicomitatum LMG 18811T has been sequenced .
Weissella spp. are heterofermenters producing CO2, ethanol and/or acetate from glucose. The species Ws. viridescens, Ws. halotolerans and Ws. hellenica have been associated with meat and meat products. Ws. viridescens is considered as heat resistant and may cause green discolouration in cured meats . This species is frequently isolated from refrigerated sliced cooked meats  and was reported to produce cavities in the muscles of hams after cooking .
The homofermentative pediococci are mostly applied for rapid and strong acidification at elevated temperature, especially in US summer sausage fermentation. Usually Pc. acidilactici and Pc. pentosaceus are the species involved. Pediococcus sp. are among the most common starter cultures in the US [11,21]. A pediocin producing Pc. acidilactici is also commonly used by the Spanish meat industry as a starter culture .
In mediterranian traditional dry-fermented sausages enterococci are found in relevant numbers and are believed to contribute to the characterisic product flavor. Ec. faecalis, e.g., is common in Portuguese ‘alheira’ .
On the other hand, the presence of enterococci in foods is debatable, since some strains carry antibiotic resistances and virulence determinants relevant in human medicine [22,106]. Also, Ec. faecium and Ec. faecalis were identified as tyramine/histamine producers in the sausages . The use of Ec. faecium strains has been suggested to control the growth of undesirable microorganisms such as listeria on material and environmental surfaces in meat plants .
Meat and meat products provide a concentrated source of protein of high biological value and can make a valuable contribution to human diets. However, they are also highly perishable commodities which rapidly spoil and may even allow the growth of food-borne pathogenic microorganisms if no suitable preservative actions are taken. Meat fermentation involving beneficial LAB has become an important and sustainable preservation technology, and today a number of suitable species and strains are successfully applied as starter and protective cultures in various fermented meats all over the world. These cultures not only prevent the growth of common food pathogens but also of undesirable food spoilage bacteria, including heterofermentative LAB. The answer to the question which strains we should use for which products largely depends on consumer expectations and technological needs. Much has been learned over the years, however, we are still far from understanding the complex metabolic interactions of LAB in meats.
Systems biology has become an important approach in LAB microbiology and will become even stronger in the future . It links quantitative microbial physiology with population dynamic modelling and ecological theories. In comparative systems biology of LAB, the so-called ”omics”-techniques (”genomics”, ”proteomics”, ”transcriptomics”, ”metabolomics”) and mathematical and statistical methods are of crucial importance [109, 110]. Comparative analyses between various species is expected to deliver understandable models of the metabolism of these species. Whole genome sequencing has made a quantum leap in the past few years and it is likely that very soon all genomes of meat associated LAB species and even of different strains will be available for comparative studies. Diversity and differences within each of the species at the strain level will have to be considered. The ripening, packaging and storage of meats could benefit from improved systems knowledge of the diverse meat microcosms with respect to microbial survival and growth, as well as desired and unwanted microbial transformations of meat components to ensure high-quality, healthy, safe and tasty products. The beneficial aspects of LAB in meat preservation could be explored using systems techniques and will decrease our dependence on chemical preservatives. Likewise, the impact of microbes on meat spoilage could be better managed with a systems understanding of the interplay of microbes, raw materials, additives and processing technologies.
In a global perspective, the role of starter and protective cultures for the safety and quality of meats is expected to increase. Although the chemical preservatives currently applied to prevent the growth of pathogens and spoilage bacteria in deli meats perfectly serve this purpose, there is an increasing consumer demand for more natural products. This is in part reflected by the so-called clean label strategies of the big manufacturers. Many chemical additives not only contribute to the sodium burden of the meats, but also leave an undesirable numb mouthfeel which negatively effects the sensory perception of the meat aroma. Innovations in fermented meat production will benefit from an improved knowledge of systems microbiology of LAB in the various meat environments on the one hand, and the gastrointestinal environment on the other. A future challenge will be to link intraspecies diversity to a specific sensory profile . The application of probiotic starter microorganisms in dry-fermented sausages remains appealling for the wellness-oriented consumers even if immediate health claims should be difficult to establish. In this sense beneficial LAB will vitally contribute to a sustainable and diversified food production.