Milk fat composition modifies the texture and appearance of Cantal-type cheeses but not their flavor.

Although the effects of cow diet on cheese sensory properties have been well documented, the putative interactions between the biochemical and microbial milk components and their respective roles in the development of the sensory properties of cheeses have yet to be explored in depth. The aim of this study was to evaluate the specific contribution of milk fat composition to the formation of cheese sensory properties. Two creams with different fat compositions were obtained from cows fed either pasture or maize silage. Cheeses were manufactured from the same skim milk (identical chemical and microbial composition) with either the pasture- or maize silage-origin pasteurized cream added. The gross composition and microbial composition of milks did not vary with cream origin. In milks and cheeses, the fatty acid (FA) profiles were modified by the origin of the cream. The concentrations of C18:0 and unsaturated FA such as cis-9 C18:1, trans-11 C18:1, C18:3n-3, total conjugated linoleic acids, and mono- and polyunsaturated FA were higher in milks and cheeses with the pasture-origin cream than in those with the maize-origin cream. In contrast, the maize milks and cheeses had higher concentrations of short- and medium-chain saturated FA, C16:0, and C18:2n-6. The level of lipolysis was 11% in the cheese rind and only 0.30% in the cheese core. The rind of pasture cheeses had a higher concentration of free C18:0 and C18:3n-3 and a lower concentration of free C14:0 and free C16:0 than the rind of maize cheeses. The levels of major microbial groups were similar in pasture and maize cheeses at different stages of ripening. The pasture cheeses had a more elastic and creamier texture, a yellower color, and a thinner rind than the maize cheeses, but the odor and aroma of cheeses were not affected by the origin of the cream, despite a few modifications in the balance of volatile compounds from FA catabolism. Based on these results, we conclude that milk fat composition modulated by cow diet had a direct role in the texture of the cheese but no effect on flavor. The high degree of lipolysis in cheese rind, along with the higher concentration of long-chain unsaturated free FA in pasture cheeses may be responsible for antimicrobial activity, which could explain differences in the appearance of cheese rind.

Fluorescence spectroscopy coupled with independent components analysis to monitor molecular changes during heating and cooling of Cantal-type cheeses with different NaCl and KCl contents.

BACKGROUND: Reduction of NaCl content of cheeses has received considerable attention by research during the past decades because of its health effects. Nonetheless, NaCl reduction is a challenge since it plays an important role in cheese quality, such as structure, texture and functional properties. Several methods were used to evaluate the effect of NaCl on these attributes. In this study, Cantal-type cheeses with different salts (NaCl and KCl) were analyzed for their structure at a molecular level and rheological properties during heating (20-60 °C) and cooling (60-20 °C). The structure was investigated by synchronous fluorescence spectroscopy (SFS) and the rheological properties by small-amplitude oscillatory test. RESULTS: Independent components analysis (ICA) gave three independent components that were attributed to coenzyme/Maillard reaction products (IC1), tryptophan (IC2) and vitamin A (IC3). Signal proportions of each IC depicted information regarding the changes in those fluorophores with salts, heating and cooling. In addition, canonical correlation analysis (CCA) of the IC proportions and rheological measurements related modifications at a molecular level evaluated by fluorescence to cheese texture (0.34 < R2 < 0.99). CONCLUSION: This study demonstrated that SFS can monitor and characterize modification of Cantal-type cheeses at a molecular level, based on the analysis of the fluorescence spectra by ICA. The nature of correlation between signal proportions and the rheological parameters depicted that rheological attributes of cheeses observed at the macroscopic level can be derived from fluorescence spectra. © 2017 Society of Chemical Industry.

Control of Shigatoxin-producing Escherichia coli in cheese by dairy bacterial strains.

Bio-preservation could be a valuable way to control Shigatoxin-producing Escherichia coli (STEC) in cheese. To this end, 41 strains were screened for their inhibitory potential on model cheese curd and on pasteurized and raw milk uncooked pressed cheeses. Strains of Lactococcus lactis, Lactococcus garvieae, Leuconostoc pseudomesenteroides, Leuconostoc citreum, Lactobacillus sp, Carnobacterium mobile, Enterococcus faecalis, Enterococcus faecium, Macrococcus caseolyticus and Hafnia alvei reduced STEC O26:H11 counts by 1.4-2.5 log cfu g(-1) and to a lesser extent STEC O157:H7 counts in pasteurized milk cheeses. Some strains can act in synergy to inhibit STEC in raw milk uncooked pressed cheeses. Inhibitory associations had no adverse effect on the sensory characteristics of these cheeses. The association of H. alvei, Lactobacillus plantarum and Lc. lactis was the most inhibitory: after inoculation of this consortium into milk, STEC O26:H11 and O157:H7, inoculated at 2 log cfu ml(-1), were reduced by up to 3 log cfu g(-1) in ripened cheese. Inhibition in cheese cannot be predicted from H2O2 production in BHI medium, decreased pH or milk reduction. It is not clear what role the rapid decrease in pH during the first 6 h may play in the inhibition. Further studies will be needed to determine the nature of the inhibition.

Staphylococcus aureus transcriptomic response to inhibition by H2O2-producing Lactococcus garvieae.

Growth of the foodborne pathogen Staphylococcus aureus can be inhibited in milk and in cheese by the hydrogen peroxide-producing Lactococcus garvieae N201 dairy strain. Transcriptomic responses of two S. aureus strains, the S. aureus SA15 dairy strain and the MW2 human pathogenic strain, to this growth inhibition were investigated in Brain-Heart Infusion broth under a high or a low aeration level. We demonstrated that S. aureus MW2 had a higher resistance to L. garvieae inhibition under the high aeration level: this correlated to a higher survival under hydrogen peroxide exposure. Conversely, the two strains were similarly inhibited under the low aeration level. Expression of S. aureus genes involved in response to H2O2 or other stresses as well as in cell division was generally repressed by L. garvieae. However, differential expressions between the two S. aureus strains were observed, especially under the high aeration level. Additionally, expression of virulence-related genes (enterotoxins, regulatory genes) was modulated by L. garvieae depending on the aeration level and on the S. aureus strain. This study led to new insights into potential molecular mechanisms of S. aureus inhibition by Lactic Acid Bacteria via H2O2 production.

Construction of a dairy microbial genome catalog opens new perspectives for the metagenomic analysis of dairy fermented products.

BACKGROUND: Microbial communities of traditional cheeses are complex and insufficiently characterized. The origin, safety and functional role in cheese making of these microbial communities are still not well understood. Metagenomic analysis of these communities by high throughput shotgun sequencing is a promising approach to characterize their genomic and functional profiles. Such analyses, however, critically depend on the availability of appropriate reference genome databases against which the sequencing reads can be aligned. RESULTS: We built a reference genome catalog suitable for short read metagenomic analysis using a low-cost sequencing strategy. We selected 142 bacteria isolated from dairy products belonging to 137 different species and 67 genera, and succeeded to reconstruct the draft genome of 117 of them at a standard or high quality level, including isolates from the genera Kluyvera, Luteococcus and Marinilactibacillus, still missing from public database. To demonstrate the potential of this catalog, we analysed the microbial composition of the surface of two smear cheeses and one blue-veined cheese, and showed that a significant part of the microbiota of these traditional cheeses was composed of microorganisms newly sequenced in our study. CONCLUSIONS: Our study provides data, which combined with publicly available genome references, represents the most expansive catalog to date of cheese-associated bacteria. Using this extended dairy catalog, we revealed the presence in traditional cheese of dominant microorganisms not deliberately inoculated, mainly Gram-negative genera such as Pseudoalteromonas haloplanktis or Psychrobacter immobilis, that may contribute to the characteristics of cheese produced through traditional methods.

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.

Behavior of different Shiga toxin-producing Escherichia coli serotypes in various experimentally contaminated raw-milk cheeses.

Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log(10) CFU · g(-1)) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.

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.

Differential effect of cheese fatty acid composition on blood lipid profile and redox status in normolipidemic volunteers: a pilot study.

The present study investigated the effect of the consumption of two cheese varieties differing for fat quality on blood lipid profile and redox status biomarkers in 30 selected healthy volunteers, consuming either the experimental cheese (from milk produced by cows fed a grass and maize silage based diet with 5% of linseed oil added) or the control cheese (from normal cows' milk) for 4 weeks according to a crossover design. The experimental cheese had a lower content of medium-chain saturated fatty acids and a higher content of stearic acid and polyunsaturated fatty acids; its consumption led to higher levels of vitamins C and E and stearic acid in blood, while myristic acid and oxidized low-density lipoprotein concentrations were significantly lower. As myristic acid and oxidized low-density lipoprotein are highly correlated with increased atherogenic risk and vitamins C and E with antioxidant activity, the enrichment of cows' diet with linseed oil could provide a dietary option to prevent cardiovascular diseases risk.

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.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

Do milking practices influence the bacterial diversity of raw milk?

The link between milk production practices and bacterial diversity of 67 raw milks from dairy farms in the Savoie and Haute-Savoie regions of France was studied by Single Strand Conformation Polymorphism (SSCP) analysis. The milking practices and the cleanliness of different parts of the cow housing were evaluated. The SSCP bacterial profiles allow to classify the 67 milks into three groups: group A was characterised by a majority of Gram-positive non-lactic acid bacteria (Corynebacterineae and Micrococcaceae) and a high level of hygiene in milking practices. The SSCP profiles of groups B and C were close but different from those of group A: they were both dominated by lactic acid bacteria and by a less intensive hygiene practices. The group B milks were characterised by the dominance of Gram-negative bacteria and Lactococcus lactis species while those of group C were dominated by Brevibacterium linens and Leuconostoc mesenteroides. The variation of balance between bacterial populations can be associated with differences in hygienic milking production practices.

Bacterial community assembly from cow teat skin to ripened cheeses is influenced by grazing systems.

The objectives of this study were to explore bacterial community assembly from cow teat skin to raw milk cheeses and to evaluate the role of farming systems on this assembly using 16S rRNA gene high-throughput sequencing. The two grazing systems studied (extensive vs. semi-extensive) had a greater effect on the microbiota of cow teat skin than on that of raw milks and cheeses. On teat skin, the relative abundance of several taxa at different taxonomic levels (Coriobacteriia, Bifidobacteriales, Corynebacteriales, Lachnospiraceae, Atopobium, and Clostridium) varied depending on the grazing system and the period (early or late summer). In cheese, the abundance of sub-dominant lactic acid bacteria (LAB) varied depending on the grazing system. Overall, 85% of OTUs detected in raw milks and 27% of OTUs detected in ripened cheeses were also found on cow teat skin. Several shared OTUs were assigned to taxa known to be involved in the development of cheese sensory characteristics, such as Micrococcales, Staphylococcaceae, and LAB. Our results highlight the key role of cow teat skin as a reservoir of microbial diversity for raw milk, and for the first time, that cow teat skin serves as a potential source of microorganisms found in raw-milk cheeses.

Milk Fat Globules Hamper Adhesion of Enterohemorrhagic Escherichia coli to Enterocytes: In Vitro and in Vivo Evidence.

Enterohemorrhagic Escherichia coli (EHEC; E. coli) are food-borne agents associated with gastroenteritis, enterocolitis, bloody diarrhea and the hemolytic-uremic syndrome (HUS). Bovine milk glycans have been shown to contain oligosaccharides which are similar to host epithelial cell receptors and can therefore prevent bacterial adhesion. This study aimed to describe interactions between EHEC O157:H7 EDL933 and O26:H11 21765 and milk fat globules (MFGs) in raw milk and raw milk cheese, and the impact of MFGs on EHEC strains adhesion to the intestinal tract in vitro and in vivo. Both EHEC serotypes clearly associated with native bovine MFGs and significantly limited their adhesion to a co-culture of intestinal cells. The presence of MFGs in raw milk cheese had two effects on the adhesion of both EHEC serotypes to the intestinal tracts of streptomycin-treated mice. First, it delayed and reduced EHEC excretion in mouse feces for both strains. Second, the prime implantation site for both EHEC strains was 6 cm more proximal in the intestinal tracts of mice fed with contaminated cheese containing less than 5% of fat than in those fed with contaminated cheese containing 40% of fat. Feeding mice with 40% fat cheese reduced the intestinal surface contaminated with EHEC and may therefore decrease severity of illness.

Stability of microbial communities in goat milk during a lactation year: molecular approaches.

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.

New Insights into the Anti-pathogenic Potential of Lactococcus garvieae against Staphylococcus aureus Based on RNA Sequencing Profiling.

The bio-preservation potential of Lactococcus garvieae lies in its capacity to inhibit the growth of staphylococci, especially Staphylococcus aureus, in dairy products and in vitro. In vitro, inhibition is modulated by the level of aeration, owing to hydrogen peroxide (H2O2) production by L. garvieae under aeration. The S. aureus response to this inhibition has already been studied. However, the molecular mechanisms of L. garvieae underlying the antagonism against S. aureus have never been explored. This study provides evidence of the presence of another extracellular inhibition effector in vitro. This effector was neither a protein, nor a lipid, nor a polysaccharide, nor related to an L-threonine deficiency. To better understand the H2O2-related inhibition mechanism at the transcriptome level and to identify other mechanisms potentially involved, we used RNA sequencing to determine the transcriptome response of L. garvieae to different aeration levels and to the presence or absence of S. aureus. The L. garvieae transcriptome differed radically between different aeration levels mainly in biological processes related to fundamental functions and nutritional adaptation. The transcriptomic response of L. garvieae to aeration level differed according to the presence or absence of S. aureus. The higher concentration of H2O2 with high aeration was not associated with a higher expression of L. garvieae H2O2-synthesis genes (pox, sodA, and spxA1) but rather with a repression of L. garvieae H2O2-degradation genes (trxB1, ahpC, ahpF, and gpx). We showed that L. garvieae displayed an original, previously undiscovered, H2O2 production regulation mechanism among bacteria. In addition to the key factor H2O2, the involvement of another extracellular effector in the antagonism against S. aureus was shown. Future studies should explore the relation between H2O2-metabolism, H2O2-producing LAB and the pathogen they inhibit. The nature of the other extracellular effector should also be determined.

Application of SSCP-PCR fingerprinting to profile the yeast community in raw milk Salers cheeses.

Bacteria and yeasts are important sensory factors of raw-milk cheeses as they contribute to the sensory richness and diversity of these products. The diversity and succession of yeast populations in three traditional Registered Designation of Origin (R.D.O.) Salers cheeses have been determined by using phenotypic diagnoses and Single-Strand Conformation Polymorphism (SSCP) analysis. Isolates were identified by phenotypic tests and the sequencing of the D1-D2 domains of the 26S rRNA gene. Ninety-two percent of the isolates were identified as the same species in both tests. Yeast-specific primers were designed to amplify the V4 region of the 18S rRNA gene for SSCP analysis. The yeast species most frequently encountered in the three cheeses were Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida zeylanoides and Debaryomyces hansenii. Detection of less common species, including Candida parapsilosis, Candida silvae, Candida intermedia, Candida rugosa, Saccharomyces unisporus, and Pichia guilliermondii was more efficient with the conventional method. SSCP analysis was accurate and could be used to rapidly assess the proportions and dynamics of the various species during cheese ripening. Each cheese was clearly distinguished by its own microbial community dynamics.