Temporal differences in microbial composition of Époisses cheese rinds during ripening and storage.

Époisses is a protected designation of origin smear-ripened cheese from the Burgundy region in France. It has an orange color and a strong flavor, both of which are generated by surface microorganisms. The objective of the present study was to investigate the microbial dynamics at the surface of Époisses cheese during ripening and postmanufacturing storage at low temperatures. Rind samples were analyzed by enumeration on agar plates and by 16S rRNA gene and internal transcribed spacer amplicon sequencing. During most of the ripening process, the counts of yeasts, which corresponded to the species Debaryomyces hansenii and Geotrichum candidum, were higher than those of the aerobic acid-sensitive bacteria. Debaryomyces hansenii reached a level of about 3 × 108 cfu/cm2, and its viability strongly decreased in the late stage of ripening and during storage at 4°C. Two of the inoculated bacterial species, Brevibacterium aurantiacum and Staphylococcus xylosus, did not establish themselves at the cheese surface. At the end of ripening, among the 18 most abundant bacterial species detected by amplicon sequencing, 14 were gram-negative, mainly from genera Psychrobacter, Vibrio, Halomonas, and Mesonia. It was hypothesized that the high moisture level of the Époisses rinds, due the humid atmosphere of the ripening rooms and to the frequent washings of the curds, favored growth of these gram-negative species. These species may be of interest for the development of efficient ripening cultures. In addition, because the orange color of Époisses cheeses could not be attributed to the growth of Brevibacterium, it would be interesting to investigate the type and origin of the pigments that confer color to this cheese.

Safety assessment of Gram-negative bacteria associated with traditional French cheeses.

Twenty Gram-negative bacterial (GNB) strains were selected based on the biodiversity previously observed in French traditional cheeses and their safety was assessed considering various safety criteria. For the majority of tested GNB strains, only gastric stress at pH 2 (vs pH 4) resulted in low survival and no regrowth after an additional simulated gastro-intestinal stress. Presence of milk was shown to be rarely protective. The majority of strains was resistant to human serum and had a low level of adherence to Caco-2 cells. When tested for virulence in Galleria mellonella larvae, GNB strains had LD 50 values similar to that of safe controls. However, four strains, Hafnia paralvei 920, Proteus sp. (close to P. hauseri) UCMA 3780, Providencia heimbachae GR4, and Morganella morganii 3A2A were highly toxic to the larvae, which suggests the presence of potential virulent factors in these strains. Noteworthy, to our knowledge, no foodborne intoxication or outbreak has been reported so far for any of the GNB belonging to the genera/species associated with the tested strains. The role of multiple dynamic interactions between cheese microbiota and GIT barriers could be key factors explaining safe consumption of the corresponding cheeses.

Investigation of associations of Yarrowia lipolytica, Staphylococcus xylosus, and Lactococcus lactis in culture as a first step in microbial interaction analysis.

The interactions that may occur between microorganisms in different ecosystems have not been adequately studied yet. We investigated yeast-bacterium interactions in a synthetic medium using different culture associations involving the yeast Yarrowia lipolytica 1E07 and two bacteria, Staphylococcus xylosus C2a and Lactococcus lactis LD61. The growth and biochemical characteristics of each microorganism in the different culture associations were studied. The expression of genes related to glucose, lactate, and amino acid catabolism was analyzed by reverse transcription followed by quantitative PCR. Our results show that the growth of Y. lipolytica 1E07 is dramatically reduced by the presence of S. xylosus C2a. As a result of a low amino acid concentration in the medium, the expression of Y. lipolytica genes involved in amino acid catabolism was downregulated in the presence of S. xylosus C2a, even when L. lactis was present in the culture. Furthermore, the production of lactate by both bacteria had an impact on the lactate dehydrogenase gene expression of the yeast, which increased up to 30-fold in the three-species culture compared to the Y. lipolytica 1E07 pure culture. S. xylosus C2a growth dramatically decreased in the presence of Y. lipolytica 1E07. The growth of lactic acid bacteria was not affected by the presence of S. xylosus C2a or Y. lipolytica 1E07, although the study of gene expression showed significant variations.

Assessment of the microbial diversity at the surface of Livarot cheese using culture-dependent and independent approaches.

The microbial diversity of the surface of a commercial red-smear cheese, Livarot cheese, sold on the retail market was studied using culture-dependent and independent approaches. Forty yeasts and 40 bacteria from the cheese surface were collected, dereplicated using single-strand conformation polymorphism (SSCP) analysis and identified using rRNA gene sequencing for the culture-dependent approach. The culture-independent approach involved cloning and sequencing of the 16S rRNA gene and SSCP analysis from total DNA extracted from the cheese. The most dominant bacteria were Microbacterium gubbeenense, Leucobacter komagatae and Gram-negative bacteria from the Gamma-Proteobacteria class. Fluorescence in situ hybridization (FISH) analysis was also used to study the cheese microbial diversity with class-level and specific rRNA-targeted probes for bacteria and yeasts, respectively. FISH analysis confirmed that Gamma-Proteobacteria were important microorganisms in this cheese. Four specific FISH probes targeting the dominant yeasts present in the cheese, Candida catenulata, Candida intermedia, Geotrichum spp. and Yarrowia lipolytica, were also designed and evaluated. These probes allowed the detection of these yeasts directly in cheese. The use of the rRNA gene-based approach combined with FISH analysis was useful to investigate the diversity of a surface microbial consortium from cheese.

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese.

AIMS: To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening. METHODS AND RESULTS: The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9.2 log(10) CFU g(-1). In the presence of P. vulgaris, counts of Arthrobacter arilaitensis, Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant. CONCLUSIONS: Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.

An iterative sensory procedure to select odor-active associations in complex consortia of microorganisms: application to the construction of a cheese model.

The aim of this study was to develop and validate an iterative procedure based on odor assessment to select odor-active associations of microorganisms from a starting association of 82 strains (G1), which were chosen to be representative of Livarot cheese biodiversity. A 3-step dichotomous procedure was applied to reduce the starting association G1. At each step, 3 methods were used to evaluate the odor proximity between mother (n strains) and daughter (n/2 strains) associations: a direct assessment of odor dissimilarity using an original bidimensional scale system and 2 indirect methods based on comparisons of odor profile or hedonic scores. Odor dissimilarity ratings and odor profile gave reliable and sometimes complementary criteria to select G3 and G4 at the first iteration, G31 and G42 at the second iteration, and G312 and G421 at the final iteration. Principal component analysis of odor profile data permitted the interpretation at least in part, of the 2D multidimensional scaling representation of the similarity data. The second part of the study was dedicated to 1) validating the choice of the dichotomous procedure made at each iteration, and 2) evaluating together the magnitude of odor differences that may exist between G1 and its subsequent simplified associations. The strategy consisted of assessing odor similarity between the 13 cheese models by comparing the contents of their odor-active compounds. By using a purge-and-trap gas chromatography-olfactory/mass spectrometry device, 50 potent odorants were identified in models G312, G421, and in a typical Protected Denomination of Origin Livarot cheese. Their contributions to the odor profile of both selected model cheeses are discussed. These compounds were quantified by purge and trap-gas chromatography-mass spectrometry in the 13 products and the normalized data matrix was transformed to a between-product distance matrix. This instrumental assessment of odor similarities allowed validation of the choice of G312 as the best 10-strain ecosystem.

Does smearing inoculum reflect the bacterial composition of the smear at the end of the ripening of a French soft, red-smear cheese?

The microbial community composition and dynamics during the production of a French soft, red-smear cheese were investigated. The colonization efficiency of the smearing inoculum was followed, and the parts played by the inoculum used and the resident microflora were tentatively estimated. Single-strand conformation polymorphism analysis (SSCP) was applied to 2 productions of a soft, red-smear cheese produced by the same dairy plant at 4-mo intervals. Microbial composition of the different cheese samples analyzed was found to be reproducible from one production to another. However, the composition of the surface flora of both cheeses at the end of the ripening did not reflect the composition of the smearing inoculum used, qualitatively as well as quantitatively. These results were confirmed by those obtained when assessing the microbial composition of the culturable flora by the spread plate technique. The inoculum used by the industry had low resiliency potentialities against colonization of cheeses by resident organisms. Therefore, fitness and colonization potential of smearing inocula should be carefully assessed by the industry before use. The use of Arthrobacter strains as part of the smearing inoculum should be evaluated.

Assessment of the rind microbial diversity in a farmhouse-produced vs a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methods.

AIMS: The diversity of the surface flora of two French red-smear soft cheeses was examined by cultivation-dependent and cultivation-independent methods to assess their composition and to evaluate the accuracy of both approaches. METHODS AND RESULTS: Culture-independent methods used involved 16S ribosomal DNA gene cloning and sequencing and single-strand conformation polymorphism analysis (SSCP). The culture-dependent method used involved direct culture and macroscopic observation, polymerase chain reaction of the 16S rRNA gene from DNA extracted from single colonies followed by complete sequencing of the gene. Only few species were recovered by both approaches either in the pasteurized and the farmer cheese. A large diversity of isolates or 16S rDNA sequences related to marine bacteria was identified at the surface of both cheeses. CONCLUSIONS: The results indicated that all three techniques were informative and complementary to allow a more accurate representativeness of the cheese surface biodiversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultivation and molecular methods have to be combined in order to obtain an extended view of the bacterial populations of complex ecosystems.