**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 [1]. 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 [12]. 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 [79]. 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 [25]. 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 [21]. But, in certain traditional Greek fermented sausages *Lb. plantarum* and *Lb. plantarum/pentosus* may predominate [82,83].

## **11.3. Lb. brevis**

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

to reduction of biogenic amines in fermented sausages [72].

in the oxidative stress response and in bacteriocin production.

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* [70]. 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 [71]. Suitable starter cultures may contribute

Identification of meat associated LAB is still wideley performed with phenotypic methods only, e.g API 50 CH [73]. These are, however, not always satisfying and may lead to misidentifications [74]. 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 [76].

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 [1]. 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 [12]. 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

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.

**9. Formation of biogenic amines** 

**10. Identification of LAB** 

**11. Important LAB in meats** 

**11.2. Lb. plantarum** 

**11.1. The Lb. sakei/curvatus cluster** 

In combination with *Pc. pentosaceus*, *Lb. brevis* has been used as an indigenous starter culture for a Vietnamese fermented meat product [84]. 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* [85].

### **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 [86]. 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 [90].

### **11.5. Carnobacteria**

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 Role of Lactic Acid Bacteria in Safety and Flavour Development of Meat and Meat Products 143

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 [101]. These authors, employees of a big Canadian food company (then Canada Packers Inc.),

**Figure 6.** Yellow discolourations on prepackaged refrigerated German 'Weisswurst' after targeted

**Figure 7.** Yellow discolourations on pre-packaged meat products produced by *Leuc. gelidum*. A and B, 'Weisswurst' from organic production; C, grill sausage from conventional production; D, sliced cooked

*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

tentatively identified an *Enterococcus* sp. as the causative agent.

inoculation with *Leuc. gelidum* and incubation at 5°C for 14 days [98].

turkey breast from conventional production [98].

[100].

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 alphaketoisocaproic 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 [93].

Positive and negative effects of carnobacteria in the environment and in foods have recently been reviewed [94]. 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 [95]. 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 [96].

### **11.6. Leuconostoc**

*Leuc. gelidum* is a major spoilage organism in Finnish fresh meats [97]. Certain strains of *Leuc. gelidum* may produce yellow discolourations on prepackaged refrigerated German 'Weisswurst' and cold cuts (Figure 6, 7) [98]. Recently, the genome of a plant isolate of *Leuc. gelidum* has been sequenced [99].

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 fatcontaining 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 [100].

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

butanoic acid [93].

*Cb. maltaromaticum* from cheese [96].

*gelidum* has been sequenced [99].

**11.6. Leuconostoc** 

amino acids via the Stickland reaction or from tributyrin hydrolysis.

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

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 alphaketoisocaproic 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

Positive and negative effects of carnobacteria in the environment and in foods have recently been reviewed [94]. 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 [95]. 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

*Leuc. gelidum* is a major spoilage organism in Finnish fresh meats [97]. Certain strains of *Leuc. gelidum* may produce yellow discolourations on prepackaged refrigerated German 'Weisswurst' and cold cuts (Figure 6, 7) [98]. Recently, the genome of a plant isolate of *Leuc.* 

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 fatcontaining 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 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 [101]. These authors, employees of a big Canadian food company (then Canada Packers Inc.), tentatively identified an *Enterococcus* sp. as the causative agent.

**Figure 6.** Yellow discolourations on prepackaged refrigerated German 'Weisswurst' after targeted inoculation with *Leuc. gelidum* and incubation at 5°C for 14 days [98].

**Figure 7.** Yellow discolourations on pre-packaged meat products produced by *Leuc. gelidum*. A and B, 'Weisswurst' from organic production; C, grill sausage from conventional production; D, sliced cooked turkey breast from conventional production [98].

*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 [102]. 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 [55].

The Role of Lactic Acid Bacteria in Safety and Flavour Development of Meat and Meat Products 145

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

Systems biology has become an important approach in LAB microbiology and will become even stronger in the future [108]. It links quantitative microbial physiology with population dynamic modelling and ecological theories. In comparative systems biology of LAB, the socalled "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

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 [21]. The application of probiotic starter microorganisms in dry-fermented sausages remains appealling for the wellnessoriented 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

the complex metabolic interactions of LAB in meats.

processing technologies.

production.
