Characterization of a wild, novel nisin a-producing Lactococcus strain with an L. lactis subsp. cremoris genotype and an L. lactis subsp. lactis phenotype, isolated from Greek raw milk.

Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis(+)) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijasic, and M. C. Montel, J. Food Prot. 72:783-790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897(T) strain, while they were distant from strains of the lactis genotype, including the LMG 6890(T) strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890(T) strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.

Cow teat skin, a potential source of diverse microbial populations for cheese production.

The diversity of the microbial community on cow teat skin was evaluated using a culture-dependent method based on the use of different dairy-specific media, followed by the identification of isolates by 16S rRNA gene sequencing. This was combined with a direct molecular approach by cloning and 16S rRNA gene sequencing. This study highlighted the large diversity of the bacterial community that may be found on teat skin, where 79.8% of clones corresponded to various unidentified species as well as 66 identified species, mainly belonging to those commonly found in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea, Aerococcus, and Staphylococcus). Several of them, such as nonstarter lactic acid bacteria (NSLAB), Staphylococcus, and Actinobacteria, may contribute to the development of the sensory characteristics of cheese during ripening. Therefore, teat skin could be an interesting source or vector of biodiversity for milk. Variations of microbial counts and diversity between the farms studied have been observed. Moreover, Staphylococcus auricularis, Staphylococcus devriesei, Staphylococcus arlettae, Streptococcus bovis, Streptococcus equinus, Clavibacter michiganensis, Coprococcus catus, or Arthrobacter gandavensis commensal bacteria of teat skin and teat canal, as well as human skin, are not common in milk, suggesting that there is a breakdown of microbial flow from animal to milk. It would then be interesting to thoroughly study this microbial flow from teat to milk.

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses.

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.

Impact of Gram-negative bacteria in interaction with a complex microbial consortium on biogenic amine content and sensory characteristics of an uncooked pressed cheese.

The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines (BA) in the core of an uncooked pressed type model cheese was investigated in the presence of a defined complex microbial consortium. Eleven strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA-production ability, were individually tested in a model cheese. Four out of 6 strains of Enterobacteriaceae (Citrobacter freundii UCMA 4217, Klebsiella oxytoca 927, Hafnia alvei B16 and Proteus vulgaris UCMA 3780) reached counts close to 6 log CFU g⁻¹ in the model cheese. In core of cheeses inoculated with Gram-negative bacteria, only slight differences were observed for microbial counts (Enterococcus faecalis or Lactobacillus plantarum count differences below 1 log CFU g⁻¹), acetate concentration (differences below 200 mg kg⁻¹) and texture (greater firmness) in comparison to control cheeses. Cheese core colour, odour and volatile compound composition were not modified. Although ornithine, the precursor of putrescine, was present in all cheeses, putrescine was only detected in cheeses inoculated with H. alvei B16 and never exceeded 2.18 mmol kg⁻¹ cheese dry matter. Cadaverine was only detected in cheeses inoculated with H. alvei B16, K. oxytoca 927, Halomonas venusta 4C1A or Morganella morganii 3A2A but at lower concentrations (<1.05 mmol kg⁻¹ cheese dry matter), although lysine was available. Only insignificant amounts of the detrimental BA histamine and tyramine, as well as isopentylamine, tryptamine or phenylethylamine, were produced in the cheese model by any of the Gram-negative strains, including those which produced these BA at high levels in vitro.

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese.

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community.

The response of Staphylococcus aureus growth inhibition by Lactococcus garvieae to catalase and milk lactoperoxidase, and its efficiency in raw milk cheese were evaluated. S. aureus and L. garvieae were co-cultivated in broth buffered at pH 6.8, and in raw, pasteurized and microfiltered milk, in presence and absence of catalase. Although H2O2 production by L. garvieae was detected only in agitated broth, the inhibition of S. aureus by L. garvieae was reduced by catalase both in static and shaking cultures by 2.7 log, pasteurized milk (approximately 0.7 log), microfiltered milk (approximately 0.6 log) and raw milk (approximately 0.2 log). The growth of S. aureus alone in microfiltered milk was delayed compared with that in pasteurized milk and inhibition of S. aureus by L. garvieae was stronger in microfiltered milk. The inhibition of coagulase-positive staphylococci (CPS) by L. garvieae in raw milk cheese was similar to that in raw milk (approximately 0.8 log), but weaker than that in pasteurized and microfiltered milks. L. garvieae also had an early antagonistic effect on the growth of several other microbial groups, which lastingly affected populations levels and balance during cheese ripening.

Traditional cheeses: rich and diverse microbiota with associated benefits.

The risks and benefits of traditional cheeses, mainly raw milk cheeses, are rarely set out objectively, whence the recurrent confused debate over their pros and cons. This review starts by emphasizing the particularities of the microbiota in traditional cheeses. It then describes the sensory, hygiene, and possible health benefits associated with traditional cheeses. The microbial diversity underlying the benefits of raw milk cheese depends on both the milk microbiota and on traditional practices, including inoculation practices. Traditional know-how from farming to cheese processing helps to maintain both the richness of the microbiota in individual cheeses and the diversity between cheeses throughout processing. All in all more than 400 species of lactic acid bacteria, Gram and catalase-positive bacteria, Gram-negative bacteria, yeasts and moulds have been detected in raw milk. This biodiversity decreases in cheese cores, where a small number of lactic acid bacteria species are numerically dominant, but persists on the cheese surfaces, which harbour numerous species of bacteria, yeasts and moulds. Diversity between cheeses is due particularly to wide variations in the dynamics of the same species in different cheeses. Flavour is more intense and rich in raw milk cheeses than in processed ones. This is mainly because an abundant native microbiota can express in raw milk cheeses, which is not the case in cheeses made from pasteurized or microfiltered milk. Compared to commercial strains, indigenous lactic acid bacteria isolated from milk/cheese, and surface bacteria and yeasts isolated from traditional brines, were associated with more complex volatile profiles and higher scores for some sensorial attributes. The ability of traditional cheeses to combat pathogens is related more to native antipathogenic strains or microbial consortia than to natural non-microbial inhibitor(s) from milk. Quite different native microbiota can protect against Listeria monocytogenes in cheeses (in both core and surface) and on the wooden surfaces of traditional equipment. The inhibition seems to be associated with their qualitative and quantitative composition rather than with their degree of diversity. The inhibitory mechanisms are not well elucidated. Both cross-sectional and cohort studies have evidenced a strong association of raw-milk consumption with protection against allergic/atopic diseases; further studies are needed to determine whether such association extends to traditional raw-milk cheese consumption. In the future, the use of meta-omics methods should help to decipher how traditional cheese ecosystems form and function, opening the way to new methods of risk-benefit management from farm to ripened cheese.

Ecological and aromatic impact of two Gram-negative bacteria (Psychrobacter celer and Hafnia alvei) inoculated as part of the whole microbial community of an experimental smear soft cheese.

The impact of the growth of two Gram-negative bacteria, Psychrobacter celer and Hafnia alvei, inoculated at 10(2) and 10(6) cfu/g, on the dynamics of a multispecies community as well as on volatile aroma compound production during cheese ripening was investigated. Results showed that P. celer was able to successfully implant itself in cheese, regardless of its inoculation level. However, when it was inoculated at a high level, the bacterial biodiversity was drastically lowered from day 25 to the end of ripening. Overall, the presence of P. celer led to the higher production of volatile aroma compounds such as aldehydes, ketones and sulfur compounds. Regardless of its inoculation level, H. alvei barely affected the growth of the bacterial community and was subdominant at the end of ripening. It influenced total volatile aroma compound production with volatile sulfur compounds being the most abundant. Overall, these two bacteria were able to implant themselves in a cheese community and significantly contributed to the aromatic properties of the cheese. Their role in flavoring and their interactions with the technological microorganisms must be considered during cheese ripening and should be further investigated.