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

International audienceThe 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 cultureindependent 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 rRNAtargeted 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

The Arthrobacter arilaitensis Re117 Genome Sequence Reveals Its Genetic Adaptation to the Surface of Cheese

Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final product. The A. arilaitensis Re117 genome is composed of a 3,859,257 bp chromosome and two plasmids of 50,407 and 8,528 bp. The chromosome shares large regions of synteny with the chromosomes of three environmental Arthrobacter strains for which genome sequences are available: A. aurescens TC1, A. chlorophenolicus A6 and Arthrobacter sp. FB24. In contrast however, 4.92% of the A. arilaitensis chromosome is composed of ISs elements, a portion that is at least 15 fold higher than for the other Arthrobacter strains. Comparative genomic analyses reveal an extensive loss of genes associated with catabolic activities, presumably as a result of adaptation to the properties of the cheese surface habitat. Like the environmental Arthrobacter strains, A. arilaitensis Re117 is well-equipped with enzymes required for the catabolism of major carbon substrates present at cheese surfaces such as fatty acids, amino acids and lactic acid. However, A. arilaitensis has several specificities which seem to be linked to its adaptation to its particular niche. These include the ability to catabolize D-galactonate, a high number of glycine betaine and related osmolyte transporters, two siderophore biosynthesis gene clusters and a high number of Fe3+/siderophore transport systems. In model cheese experiments, addition of small amounts of iron strongly stimulated the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with abilities to catabolize substrates such as lactic acid, lipids and amino acids

Microbial interactions in cheese: implications for cheese quality and safety

International audienceThe cheese microbiota, whose community structure evolves through a succession of different microbial groups, plays a central role in cheese-making. The subtleties of cheese character, as well as cheese shelf-life and safety, are largely determined by the composition and evolution of this microbiota. Adjunct and surface-ripening cultures marketed today for smear cheeses are inadequate for adequately mimicking the real diversity encountered in cheese microbiota. The interactions between bacteria and fungi within these communities determine their structure and function. Yeasts play a key role in the establishment of ripening bacteria. The understanding of these interactions offers to enhance cheese flavour formation and to control and/or prevent the growth of pathogens and spoilage microorganisms in cheese

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

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Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses

Extraction of RNA from Cheese without Prior Separation of Microbial Cells▿

In situ gene expression studies are promising approaches for improving our understanding of the cheese microbial flora. This requires efficient RNA extraction methods, but studies of cheeses are scarce. The objective of the present study was to determine whether RNA samples compatible with quantitative mRNA transcript analyses can be obtained without separating the cells from the cheese matrix. In the method that we describe, the cellular processes are stopped at the very beginning of the procedure. When cheeses were produced with Lactococcus lactis LD61 as the only starter microorganism, the integrity of the purified RNA was good, even for 2-week-old cheeses that had been incubated at 30°C. In addition, the RNA samples did not contain any traces of RNases, and the amount of genomic DNA was negligible. A good level of reproducibility could also be achieved. When real-time reverse transcription-PCR analyses were normalized to the total RNA concentration, the amounts of 16S and 23S rRNA transcripts were constant during the 2-week incubation period, whereas the amount of tuf mRNA transcripts decreased substantially. RNA samples obtained using the method described in this study were compared to samples obtained using the method described by Ulvé et al. (J. Appl. Microbiol., in press), which is based on separation of the cells from the cheese matrix. For most of the 29 genes investigated, the transcript abundance was the same for both types of samples. Differences were observed mainly for genes whose expression has previously been shown to be modified by heat, acid, or osmotic stresses, such as busAA and glnQ

Les préférences des consommateurs pour de nouveaux produits alimentaires fermentés qui mélangent des sources de protéines animales et végétales

International audienceConsumers are being encouraged to increase the proportion of plant protein in their diets to enhance the sustainability of food systems. One approach is to develop plant-protein-rich foods that are acceptable to consumers. This study examined French people’s reactions to cheese alternatives—new fermented products that mixed animal and plant protein sources. We conducted experimental sessions with 240 French participants to assess their responses to three fermented products containing different percentages of yellow pea and cow’s milk. First, we asked the participants to blind-taste the three products and solicited hedonic scores of products. We then provided the participants with simple information about the products’ composition and asked them to taste and score the liking of the products a second time. We also asked consumers to estimate their willingness to pay (WTP) for each product before and after revealing additional information about the nutritional or environmental benefits of consuming pea-based foods. The product with the lowest percentage of pea and the highest percentage of milk received the highest hedonic scores, and WTP was correlated with the hedonic scores. The additional information about the nutritional and environmental benefits of pea-based foods led to significant increases in WTP for two of the fermented products, but not for the least preferred product, namely the one with the highest percentage of pea. This finding suggests that participant reactions to information depended on hedonic preferences.Les consommateurs sont encouragés à augmenter la proportion de protéines végétales dans leur alimentation afin d'améliorer la durabilité des systèmes alimentaires. Une approche consiste à développer des aliments riches en protéines végétales acceptables pour les consommateurs. Cette étude examine les réactions des Français face aux alternatives au fromage - de nouveaux produits fermentés mélangeant des sources de protéines animales et végétales. Nous avons mené des sessions expérimentales avec 240 participants français pour évaluer leurs réponses à trois produits fermentés contenant différents pourcentages de pois jaunes et de lait de vache. Tout d'abord, nous avons demandé aux participants de goûter à l'aveugle les trois produits, et nous avons sollicité leur jugement hédonique. Nous avons ensuite fourni aux participants des informations simples sur la composition des produits et leur avons demandé de goûter à nouveau, en notant le goût des produits une seconde fois. Nous avons également demandé aux consommateurs d'estimer leur consentement à payer (CAP) pour chaque produit avant, et après avoir révélé des informations supplémentaires sur les avantages nutritionnels ou environnementaux de la consommation d'aliments à base de pois. Le produit avec le pourcentage le plus bas de pois et le pourcentage le plus élevé de lait a reçu les scores hédoniques les plus élevés, et les CAP étaient corrélés aux scores hédoniques. Les informations supplémentaires sur les avantages nutritionnels et environnementaux des aliments à base de pois ont conduit à des augmentations significatives du CAP pour deux des produits fermentés, mais pas pour le produit le moins préféré, à savoir celui avec le pourcentage le plus élevé de pois. Cette découverte suggère que les réactions des participants à l'information dépendent des préférences hédoniques