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International audience; Aims The pea seeds (Pisum sativum) hosts a complex microbial community that is exposed to environmental constraints modulated by biotic, abiotic and anthropogenic factors. The aim of this study was to explore the effect of geographical origins on the composition of the microbial communities associated with pea seeds from storage locations in different countries. These seeds are used by Roquette Freres (R) as source proteins for human food after industrial extraction process of their proteins. Methods The high-throughput sequencing of two phylogenetic markers (V3-V4 regions of the 16S rRNA gene and ITS2 region) was used to evaluate the structure of the microbial community associated with pea seeds. The pea seeds were harvested by different suppliers from three countries (France, Sweden and Canada) with different climates and stored in six cooperatives. Representative microbial strains of dominant species were isolated and characterized using phenotypic methods. Results Although the pea seed microbiota shared a dominant core microbiota at the genus level, seeds showed a species-specific, highly unique microbial signature with variable abundances according to origin. Origin was identified as the main driver of the composition of the pea seed microbiota. Conclusions These results provide an initial insight into the broad factors such as country and cooperative that may determine the assembly of the pea microbiota before processing.

International audience; In order to encourage Western populations to increase their consumption of vegetables, we suggest turning legumes into novel, healthy foods by applying an old, previously widespread method of food preservation: fermentation. In the present study, a total of 55 strains from different microbial species (isolated from cheese or plants) were investigated for their ability to: (i) grow on a emulsion containing 100% pea proteins and no carbohydrates or on a 50:50 pea:milk protein emulsion containing lactose, (ii) increase aroma quality and reduce sensory off-flavors; and (iii) compete against endogenous micro organisms. The presence of carbohydrates in the mixed pea:milk emulsion markedly influenced the fermentation by strongly reducing the pH through lactic fermentation, whereas the absence of carbohydrates in the pea emulsion promoted alkaline or neutral fermentation. Lactic acid bacteria assigned to Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis and Lactobacillus casei species grew well in both the pea and pea:milk emulsions. Most of the fungal strains tested (particularly those belonging to the Mucor and Geotrichum genera) were also able to grow on both emulsions. Although most Actinobacteria and Proteobacteria did not compete with endogenous microbiota (Bacillus), some species such as Hafnia alvei, Acinetobacter johnsonii and Glutamicibacter arilaitensis grew strongly and appeared to restrict the development of the endogenous microbiota when the pea emulsion was inoculated with a combination of three to nine strains. In the mixed emulsions, lactic fermentation inhibited Actinobacteria and Proteobacteria (e.g. Brevibacterium casei, Corynebacterium casei, Staphylococcus lentos) to the greatest extent but also inhibited Bacillus (e.g. Bacillus subtilis and Bacillus licheniformis). Overall, this procedure enabled us to select two microbial consortia able to colonize pea-based products and positively influence the release of volatile compounds by generating a roasted/grilled aroma for the 100% pea emulsion, and a fruity, lactic aroma for the 50:50 pea:milk emulsion. Moreover, the fermentation in the pea-based emulsions reduced the level of hexanal, which otherwise leads to an undesired green pea aroma. Our present results show how the assembly of multiple microbial cultures can help to develop an innovative food product.

The study of food microorganism domestication can bring important insights on adaptation mechanisms and have industrial applications. The Penicillium roqueforti mold is divided into four main populations, with two populations domesticated for blue-cheese making and two populations thriving in other environments. While most blue cheeses worldwide are made with the same P. roqueforti clonal lineage, the emblematic Roquefort cheeses are inoculated with a specific population. To study the differences among P. roqueforti populations in the context of domestication for cheesemaking, we compared blue cheeses made with the four fungal populations following Roquefort-type production specifications. We found that the P. roqueforti populations had a minor impact on the cheese bacterial diversity and none on the main microorganism abundance. The cheese P. roqueforti populations produced cheeses with higher percentages of blue area and with different sets and higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, which was related due to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results show the strong influence of P. roqueforti populations on several important aspects of cheese safety, appearance and flavour. The typical appearance and flavours of blue cheeses are therefore the result of human selection on P. roqueforti , thus constituting domestication, and the two cheese populations have acquired specificities. This has important implications for our understanding of adaptation and domestication processes as well as for improving cheese production. Importance Several fungi have been domesticated for food fermentation, with selection for traits beneficial for food production. The mold used for making blue cheeses, Penicillium roqueforti , is subdivided into four genetically different populations, two being found in cheese, one being specific of the Roquefort protected designation of origin, and two in other environments. The cheese P. roqueforti populations produced bluer cheeses with higher quantities of desired volatile compounds. The Roquefort P. roqueforti population in particular produced higher quantities of positive aromatic compounds in cheeses, in relation to its most efficient proteolysis and lipolysis, and also produced cheeses with lower water activity, thus restricting spoiler microorganisms. Our results support that the blue cheese typical aspect and flavors are the result of a selection by humans and show the strong influence of P. roqueforti populations for several important aspects of cheese safety, aspect and flavor, paving the way for improving cheese production.

International audience; The rheological and microstructural properties of pure pea, pure milk, and mixed pea/milk gels were investigated and compared. Three gelation techniques were used: a thermal treatment, an acid treatment employing glucono delta-lactone, and an enzyme treatment employing chymosin and transglutaminase. Considerable differences in the gels' rheological and microstructural properties were observed. The thermally induced pure pea and mixed gels had comparable rheological and microstructural properties, suggesting the existence of interactions between pea and whey proteins. The enzyme-induced gels had a higher strain at rupture, a property that was associated with the presence of coarse aggregates. Within this gel type, mixed gels displayed a lower storage modulus than did pure pea or pure milk gels. Among acid-induced gels, pure pea gels were much stiffer than pure milk or mixed gels, probably because their final pH was lower. The addition of fat strengthens the mechanical properties of all pea gels studied, in particular in the mixed gels obtained by acid and enzyme gelation. The microstructural differences were consistent with the rheological differences. These results provide insight into the formation of gels via these gelation techniques and could inform the formulation of new food products that are enriched in plant protein.

Methionine deprivation induces growth arrest and death of cancer cells. To eliminate l-methionine we produced, purified, and characterized the recombinant pyridoxal 5'-phosphate (PLP)-dependent l-methionine

Sodium reduction in the human diet is currently one of the main concerns for public health agencies and, consequently, has become a challenge for the food industries. In this study, the impact of reduced sodium chloride content (20%) or its partial substitution with potassium chloride in soft (“Camembert”-type) and semi-hard (“Reblochon”-type) cheeses was evaluated. Analyses included physicochemical and biochemical composition, microbial counts, 16S rRNA gene metabarcoding and metatranscriptomic analysis, volatile aroma compounds and sensory analysis. Regarding soft cheeses, the salt content of cheeses affected proteolysis at 21 days of ripening. RNA sequencing revealed that the relative activity of G. candidum increased, whereas that of P. camemberti decreased in reduced salt cheeses in comparison to the controls. Higher global intensity of odor and taste was observed in cheeses with reduced salt content, consistent with higher levels of alcohol and ester components. Regarding semi-hard cheeses, modifications of salt content did not significantly affect either their biochemical parameters and sensory characteristics or their technological microbial composition at day 21 of ripening. Finally, no impact of salt content was observed on the growth of the spoiler Yarrowia lipolytica in soft cheeses. In contrast, reducing salt content increased spoiler growth in semi-hard cheeses, as highlighted by a greater development of Pseudomonas that led to an increase in cheese proteolysis and lipolysis. In conclusion, the effect of reducing salt content is highly dependent on the cheese type. This factor should thus be taken into account by the dairy industry when the reduction of salt content is being considered. Moreover, the quality of raw products, in particular, the level of spoiler microorganisms, must be controlled before use during dairy processes.

International audience; Methionine deprivation induces growth arrest and death of cancer cells. To eliminate L-methionine we produced, purified, and characterized the recombinant pyridoxal 59-phosphate (PLP)-dependent L-methionine g-lyase (MGL) BL929 from the cheese-ripening Brevibacterium aurantiacum. Transformation of an Escherichia coli strain with the gene BL929 from B. aurantiacum optimized for E. coli expression led to production of the MGL-BL929. Elimination of L-methionine and cytotoxicity in vitro were assessed, and methylation-sensitive epigenetics was explored for changes resulting from exposure of cancer cells to the enzyme. A bioreactor was built by encapsulation of the protein in human erythrocytes to achieve sustained elimination of L-methionine in extracellular fluids. Catalysis was limited to alpha, gamma-elimination of L-methionine and L-homocysteine. The enzyme had no activity on other sulfur-containing amino acids. Enzyme activity decreased in presence of serum albumin or plasma resulting from reduction of PLP availability. Elimination of L-methionine induced cytotoxicity on a vast panel of human cancer cell lines and spared normal cells. Exposure of colorectal carcinoma cells to the MGL-BL929 reduced methylCpG levels of hypermethylated gene promoters including that of CDKN2A, whose mRNA expression was increased, together with a decrease in global histone H3 dimethyl lysine 9. The MGL-erythrocyte bioreactor durably preserves enzyme activity in vitro and strongly eliminates L-methionine from medium.

Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L’Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production., The ability of Geotrichum candidum to produce volatile compounds is strain dependent and inversely proportional to its ability to store metabolites inside the cells

This study evaluated the ability of dairy matrices, different in composition (with and without fat) and structure (liquid and gel), to enhance microorganisms survival through digestion. The viability of three dairy microorganisms Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei was measured during in vitro and in vivo digestion. S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. LIVE/DEAD confocal microscopy's images, probed the effect of low pH on microorganisms survival. However, in vivo analyses (16S rRNA gene metabarcoding) failed to confirm in vitro observations since tested microorganisms were not detected. Despite of the different evolutions during digestion on buffer capacity, lipolysis, and rheological characteristics, we did not observe any protective effect of the dairy matrices on microorganisms survival.

on line novembre 2017paper : janvier 2018; Yeasts play a crucial role in cheese ripening. They contribute to the curd deacidification, the establishment of acid-sensitive bacterial communities, and flavour compounds production via proteolysis and catabolism of amino acids (AA). Negative yeast-yeast interaction was observed between the yeast Yarrowia lipolytica 1E07 (YL1E07) and the yeast Debaryomyces hansenii 1L25 (DH1L25) in a model cheese but need elucidation. YL1E07 and DH1L25 were cultivated in mono and co-cultures in a liquid synthetic medium (SM) mimicking the cheese environment and the growth inhibition of DH1L25 in the presence of YL1E07 was reproduced. We carried out microbiological, biochemical (lactose, lactate, AA consumption and ammonia production) and transcriptomic analyses by microarray technology to highlight the interaction mechanisms. We showed that the DH1L25 growth inhibition in the presence of YL1E07 was neither due to the ammonia production nor to the nutritional competition for the medium carbon sources between the two yeasts. The transcriptomic study was the key toward the comprehension of yeast-yeast interaction, and revealed that the inhibition of DH1L25 in co-culture is due to a decrease of the mitochondrial respiratory chain functioning.

International audience; The use of pea protein is limited in food by the persistence of off-flavours. Fermentation by microbial consortia could be a way to circumvent this problem. The aim of our study was to investigate the adaptation potential and metabolic features of consortia as a function of the matrix composition (pea and/or milk proteins). Three designed consortia were investigated for their ability (i) to grow in gels containing 100% pea proteins, 50% pea-50% milk proteins and 100% milk proteins; (ii) to reduce off-flavours and increase aroma perception. VEGAN is suited for the pea gel (PG): it better colonises than in the mixed (PMG) and milk gels (MG), except for lactic acid bacteria that grow better with lactose. In PG, aromatic notes are mainly smoked/onion/garlic, while those in MG and PMG are perceived as dairy/cheese. MEGAN is adapted to MG and PG: it did not mask "green notes" in PMG and generates cheesy and fruity notes in MG. The ExEco consortium is not suited in PG: it does not mask "green notes". Cheese, yogurt and fruits (apple, apricot) notes were perceived in PMG and MG. Our study shows that fermentation could be applied to develop pea-based products with diversified sensory characteristics.

Chapter 38; International audience; Smear-ripened cheeses develop a viscous, red-orange smear on their surfaces during ripening. For this reason, they are also called red-smear cheeses or bacterial surface-ripened cheeses. During the past decade, tremendous progress has been made in the understanding of the microbiology of the surface microbiota of these cheeses. This progress stems from the knowledge of their microbial diversity gained through the use of culture-dependent and independent methods including high-throughput sequencing, the genomics of the microorganisms composing the smear microbiota with the sequencing of yeast and bacterial species of technological interest, the functions of these microorganisms and their interactions. In this chapter, we will review the current knowledge on the smear cheese microbiota including the factors affecting ripening of smear cheeses, their microbial diversity, the sources of these microorganisms, their genomic features and functions and the pathogens associated with smear cheeses and their control.

The yeast Yarrowia lipolytica has to develop dynamic metabolic adaptation mechanisms to survive within the cheese habitat. The availability of amino acids (AAs) is of major importance for microbial development and/or aroma production during cheese ripening. Using 2-D protein gel electrophoresis, we analyzed the adaptation mechanisms of Y. lipolytica for AAs limitation or supplementation in a batch culture containing lactate as a carbon source. Proteome analyses allow the identification of 34 differentially expressed proteins between the culture conditions. These analyses demonstrated that prior to the AAs addition, mainly proteins involved in the oxidative stress of the yeast were induced. Following the AAs addition, yeast cells reorganize their metabolism toward AAs catabolism and also generate a higher induction of proteins related to carbon metabolism and proteins biosynthesis. Using real-time reverse transcription PCR, we re-evaluated the expression of genes encoding proteins involved in these processes. The expression levels of the genes were in accordance with the proteomic results, with the up-regulation of genes encoding a branched-chain amino transferase BAT2, a pyruvate decarboxylase PDC6 and an Hsp70 protein SSZ1 involved in protein biosynthesis. A volatile compound analysis was also performed, and increased production of dimethyldisulfide from methionine and 3-methyl-butanal from leucine was observed in media supplemented with AAs.

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 9AE2 log10 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.

With the view to investigate the presence of thiols in cheese, the use of different methods of preparation and extraction with an organomercuric compound ( p-hydroxymercuribenzoate) enabled the isolation of a new compound. The analysis of cheese extracts by gas chromatography coupled with pulse flame photometry, mass spectrometry, and olfactometry detections led to the identification of ethyl 3-mercaptopropionate in Munster and Camembert cheeses. This compound, described at low concentrations as having pleasant, fruity, grapy, rhubarb, and empyreumatic characters, has previously been reported in wine and Concord grape but was never mentioned before in cheese. A possible route for the formation of this compound in relation with the catabolism of sulfur amino acids is proposed.

The effect of cysteine on the ability of smear cheese-ripening bacteria (Brevibacterium linens and Arthrobacter spp) to produce volatile sulphur compounds (VSC) from methionine was studied. These bacteria were cultivated in a synthetic medium supplemented with various cysteine concentrations with or without methionine. Cultures with only cysteine showed slightly lower levels of VSC produced and an unpleasant odour like rotten eggs, resulting from hydrogen sulphide production. The levels and profiles of VSC produced with supplemented methionine-cysteine mixtures had strain-dependant behaviours. However, the highest levels of dimethyl disulfide, dimethyl trisulfide and dimethyl tetrasulfide were observed when increasing the cysteine concentration from 0.2 to 1.0 gl(-1) at the same methionine concentration (1.0 gl(-1)). In contrast, production levels of thioesters, especially S-methylthio acetate, were reduced by 50 and 80% under such conditions. An initial sensory approach showed that such an effect could have a strong impact on the global odour of ripened cheeses.

Many health authorities have targeted salt reduction in food products as a means to reduce dietary sodium intake due to the harmful effects associated with its excessive consumption. In the present work, we evaluated the effect of reducing sodium chloride (NaCl) content on the microbiological and biochemical characteristics of an experimental surface-ripened cheese. A control cheese (1.8% NaCl) and a cheese with a reduced NaCl content (1.3% NaCl) were sampled weekly over a period of 27d. Reducing NaCl content induced microbial perturbations such as the lesser development of the yeast Debaryomyces hansenii and the greater development of the gram-negative bacterium Hafnia alvei. This was accompanied by changes in proteolytic kinetics and in profiles of volatile aroma compounds and biogenic amine production. Finally, the development of the spoilage microorganism Pseudomonas fragi was significantly higher in the cheese with a reduced salt content.

Surface-ripened cheeses host complex microbial communities responsible for the transformation of milk into cheese as well as the development of important properties in terms of texture, color and sensory perception. In this study, we used high-throughput amplicon sequencing to decipher the bacterial and fungal diversity of 60 cheeses belonging to 12 popular French cheese varieties. Using this approach, 76 bacterial and 44 fungal phylotypes were identified. Major differences were observed between rind and core samples and also according to cheese varieties and manufacturing processes. Occurrence analysis revealed the presence of widespread taxa as well as operational taxonomic units (OTUs) specific to one or several cheese varieties. Finally, we observed patterns specific to the cheese production facility, supporting the importance of indigenous microorganisms for the microbial assemblage of cheese microbiota.

Here, we report the draft genome sequence of Corynebacterium variabile Mu292, which was originally isolated from the surface of Munster, a French smear-ripened cheese. This genome investigation will improve our knowledge on the molecular determinants potentially involved in the adaptation of this strain during the Munster-type cheese manufacturing process.

The microbial communities in cheeses are composed of varying bacteria, yeasts, and molds, which contribute to the development of their typical sensory properties. In situ studies are needed to better understand their growth and activity during cheese ripening. Our objective was to investigate the activity of the microorganisms used for manufacturing a surface-ripened cheese by means of metatranscriptomic analysis. The cheeses were produced using two lactic acid bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), one ripening bacterium (Brevibacterium aurantiacum), and two yeasts (Debaryomyces hansenii and Geotrichum candidum). RNA was extracted from the cheese rinds and, after depletion of most ribosomal RNA, sequencing was performed using a short-read sequencing technology that generated approximately 75 million reads per sample. Except for Brevibacterium aurantiacum, which failed to grow in the cheeses, a large number of CDS reads were generated for the inoculated species, making it possible to investigate their individual transcriptome over time. From day 5 to day 35, G. candidum accounted for the largest proportion of CDS reads, suggesting that this species was the most active. Only minor changes occurred in the transcriptomes of the lactic acid bacteria. For the two yeasts, we compared the expression of genes involved in the catabolism of lactose, galactose, lactate, amino acids and free fatty acids. During ripening, genes involved in ammonia assimilation and galactose catabolism were down-regulated in the two species. Genes involved in amino acid catabolism were up-regulated in G. candidum from day 14 to day 35, whereas in D. hansenii, they were up-regulated mainly at day 35, suggesting that this species catabolized the cheese amino acids later. In addition, after 35 days of ripening, there was a down-regulation of genes involved in the electron transport chain, suggesting a lower cellular activity. The present study has exemplified how metatranscriptomic analyses provide insight into the activity of cheese microbial communities for which reference genome sequences are available. In the future, such studies will be facilitated by the progress in DNA sequencing technologies and by the greater availability of the genome sequences of cheese microorganisms.

International audience; Twelve bacterial strains belonging to eight taxonomic groups: Brevibacterium linens, Microbacterium foliorum, Arthrobacter arilaitensis, Staphylococcus cohnii, Staphylococcus equorum, Brachybacterium sp., Proteus vulgaris and Psychrobacter sp., isolated from different surface-ripened French cheeses, were investigated for their abilities to generate volatile aroma compounds. Out of 104 volatile compounds, 54 volatile compounds (identified using dynamic headspace technique coupled with gas chromatography-mass spectrometry [GC-MS]) appeared to be produced by the different bacteria on a casamino acid medium. Four out of eight species used in this study: B. linens, M. foliorum, P. vulgaris and Psychrobacter sp. showed a high flavouring potential. Among these four bacterial species, P. vulgaris had the greatest capacity to produce not only the widest varieties but also the highest quantities of volatile compounds having low olfactive thresholds such as sulphur compounds. Branched aldehydes, alcohols and esters were produced in large amounts by P. vulgaris and Psychrobacter sp. showing their capacity to breakdown the branched amino acids. This investigation shows that some common but rarely mentioned bacteria present on the surface of ripened cheeses could play a major role in cheese flavour formation and could be used to produce cheese flavours.

A practical adaptation of the methylene blue reaction for hydrogen sulfide quantification was developed to perform microbial selection. Closed plate flasks containing a zinc-agar layer above the liquid microbial culture are proposed as a trap system where the H2S can be retained and then quantified by the methylene blue reaction. Using this quantitative method, the ability to produce H2S was studied in several cheese-ripening microorganisms. Our aim was to select strains that produce the highest quantities of H2S as the main product of l -cysteine catabolism. Thirty seven yeast and bacteria strains were cultivated with or without l -cysteine. The separation between the growth medium and the H2S trapping layer displayed good performance: all the studied strains grew efficiently and only negligible loss of H2S was observed during culturing. The strains displayed large differences in their H2S production capabilities: yeast strains were greater producers of H2S than bacteria with production strain-related in both cases. Furthermore, the relationship between H2S production and l -cysteine consumption was analyzed, which made it possible for us to select microorganisms with high capacity in l -cysteine degradation. The production of volatile sulfur compounds was also studied and the possible effect of culture pH and metabolic differences between strains are discussed.

This paper provides new information about the metabolism of various fatty acids and gamma-decalactone production by yeast. An analysis of the fatty acid composition of the yeast Sporidiobolus salmonicolor during batch production of lactone with ricinoleic acid methyl ester as a precursor showed an accumulation of the gamma-decalactone precursor inside the cells. Electron microscopy of the yeasts showed the presence of large internal inclusions leading to membrane and organelle lysis and, consequently, death of the yeast. S. salmonicolor cultivated with methyl oleate did not produce gamma-decalactone and is viable during the whole culture. Analysis of the long chain fatty acid fraction showed incorporation of methyl oleate.

International audience; Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow-orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.

Thirty-six microorganisms (twenty-one bacteria, twelve yeasts and three fungi) were isolated from surface-ripened cheeses and subjected to in vitro digestive stress. The approach mimicked gastric and/or duodenal digestion. Lactobacillus rhamnosus GG, Escherichia coli Nissle 1917 and Saccharomyces boulardii were used as reference strains. We studied the microorganisms grown separately in culture medium and then included (or not) in a rennet gel. The microorganisms' immunomodulatory abilities were also assessed by profiling cytokine induction in human peripheral blood mononuclear cells (PBMCs). The loss of viability was less than 1 log CFU/mL for yeasts under all conditions. In contrast, Gram-negative bacteria survived gastric and/or duodenal stress well but most of the Gram-positive bacteria were more sensitive (especially to gastric stress). Inclusion of sensitive Gram-positive bacteria in rennet gel dramatically improved gastric survival, when compared with a non-included cultured (with a 4 log CFU/mL change in survival). However, the rennet gel did not protect the bacteria against duodenal stress. The PBMC cytoldne assay tests showed that the response to yeasts was usually anti-inflammatory, whereas the response to bacteria varied from one strain to another.

Cet article a été publié une nouvelle fois dans le numéro 362 http://femsle.oxfordjournals.org/content/362/1/1.20#sec-1; Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to the development of the typical color, flavor and texture of the final product. In situ measurements of gene expression are increasingly being used to improve our understanding of the microbial flora activity in cheeses. The objective of the present study was to investigate the physiology and metabolic activity of Geotrichum candidum during the ripening of Reblochon-type cheeses by quantifying mRNA transcripts at various ripening times. The expression of 80 genes involved in various functions could be quantified with a correct level of biological repeatability using a set of three stable reference genes. As ripening progresses, a decrease in expression was observed for genes involved in cell wall organization, translation, vesicular mediated transport, and in cytoskeleton constituents and ribosomal protein genes. There was also a decrease in the expression of mitochondrial F1F0 ATP synthase and plasma membrane H(+) ATPase genes. Some genes involved in the catabolism of lactate, acetate and ethanol were expressed to a greater extent at the beginning of ripening. During the second part of ripening, there was an increased expression of genes involved in the transport and catabolism of amino acids, which could be attributed to a change in the energy source. There was also an increase in the expression of genes involved in autophagy and of genes possibly involved in lifespan determination. Quantification of mRNA transcripts may also be used to produce bioindicators relevant for cheesemaking, for example when considering genes encoding enzymes involved in the catabolism of amino acids.

Although large numbers of viable microorganisms are ingested in ripened cheese, little is known about the microbial ecosystems' influence on the host's immune responses. We designed experimental smear-ripened cheeses with bacteria and yeasts that have opposite immune effects and evaluated their impact in the dextran sulphate sodium (DSS) and trinitrobenzene sulphonic acid (TNBS) colitis mouse models. Mice were fed with a control diet, a milk matrix or with lab-designed, 28-day-ripened prototype soft cheeses A and B (CheA and CheB) from cow milk that respectively hosted consortia of immuno-enhancing and immuno-modulatory microbial strains. Inflammatory markers and transcriptional signatures were evaluated in healthy mice colitic mice. In the DSS colitis model, there were no differences between CheA and CheB in terms of the inflammatory read-outs. In contrast, CheA (but not CheB) exacerbated weight loss and colon lesions in the TNBS model suggesting that designer cheeses may provide opportunities for diet management. (C) 2014 Elsevier Ltd. All rights reserved.

International audience; A mixture of nine microorganisms (six bacteria and three yeasts) from the microflora of surface-ripened cheeses were subjected to in vitro digestive stress in a three-compartment "dynamic gastrointestinal digester" (DIDGI). We studied the microorganisms (i) grown separately in culture medium only (ii) grown separately in culture medium and then mixed, (iii) grown separately in culture medium and then included in a rennet gel and (iv) grown together in smear-ripened cheese. The yeasts Geotrichum candidum, Kluyveromyces lactis and Debaryomyces hansenii, were strongly resistant to the whole DIDGI process (with a drop in viable cell counts of less than < 1 log CFU mL(-1)) and there were no significant differences between lab cultures and cheese-grown cultures. Ripening bacteria such as Hafnia alvei survived gastric stress less well when grown in cheese (with no viable cells after 90 min of exposure of the cheese matrix, compared with 6 CFU mL(-1) in lab cultures). The ability of Corynebacterium casei and Staphylococcus equorum to withstand digestive stress was similar for cheese and pure culture conditions. When grow in a cheese matrix, Brevibacterium aurantiacum and Arthrobacter arilaitensis were clearly more sensitive to the overall digestive process than when grown in pure cultures. Lactococcus lactis displayed poorer survival in gastric and duodenal compartments when it had been grown in cheese. In vivo experiments in BALB/c mice agreed with the DIDGI experiments and confirmed the latter's reliability.

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus luteus and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds including methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.

Several yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, and Saccharomyces cerevisiae, were studied for their ability to generate volatile sulfur compounds. These yeasts were cultivated on a synthetic culture medium supplemented with a biosynthetic precursor. With S-methylmethionine, dimethylsulfide (DMS) was the major sulfur compound produced. l-methionine promoted the synthesis of a wider spectrum of volatile sulfur compounds, methanethiol (MTL), DMS, dimethyldisulfide (DMDS), dimethyltrisulfide (DMTS) and S-methylthioacetate (MTA). Enzymatic activities possibly involved in sulfur compound synthesis were also investigated. Important l-methionine-transaminase activities, and also l-methionine-demethiolase activities were detected in cellular extracts. Their possible role in the generation of sulfur compounds and related biosynthetic pathways are also discussed.

The synthesis of short chain S-methyl thioesters was investigated in Geotrichum candidum strain GcG. The results indicated the involvement of an enzymatic reaction in this microorganism that led to the synthesis of S-methyl thioacetate (MTA) when methanethiol and acetyl-CoA were used as substrates. MTA was generated from these substrates by enzymatic or spontaneous reactions, whose relative importance depended largely on pH and temperature. For longer chain acyl-CoA compounds (C3 to C6), thioester synthesis was primarily spontaneous. Short chain fatty acid activation by a CoA residue probably is a prerequisite for the synthesis of S-methyl thioesters.

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with l -phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ( 14 C- and 13 C-labelled l -phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that l -phenylalanine was deaminated to trans -cinnamic acid by a phenylalanine ammonia lyase and trans -cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from l -phenylalanine to aryl metabolites. Additionally, trans -cinnamic acid can also be transformed via β-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a β-oxidation degradation intermediate. To our knowledge, this is the first time that a β-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from l -phenylalanine is proposed.

Bjerkandera adusta produced aromatic compounds such as benzaldehyde (bitter almond aroma), benzyl alcohol and benzoic acid from L-phenylalanine (3 g kg−1). Two supports for the fungus, wheat bran (organic support) and Perlite (mineral support), gave optimal production with water contents of 66% and 60%, respectively. Benzyl alcohol (4.53 g kg−1) and benzaldehyde (1.56 g kg−1) were produced after 4 days on wheat bran respectively with 20 and 30 g L-phenylalanine kg−1. Aryl alcohol oxidase activity, which oxidises benzyl alcohol to benzaldehyde, was only detected when the fungus was grown on wheat bran, the support which promotes the most benzaldehyde production. Results are compared with those obtained in submerged liquid cultures.

Within the genus Sporidiobolus, S. odorus has been widely reported as having the ability to convert castor oil or its derivatives to γ-decalactone, but other species have also shown potential for lactone production. In this work, the bioconversion of ricinoleic acid methyl ester to γ-decalactone was studied with four species of Sporidiobolus: S. salmonicolor, S. ruinenii, S. johnsonii, and S. pararoseus. With 4.1 g/l of ricinoleic acid methyl ester, only S. salmonicolor and S. ruinenii were able to produce γ-decalactone (12 and 40 mg/l respectively). S. johnsonii and S. pararoseus did not produce any lactone in spite of consuming the precursor. The four strains showed different sensitivities to lactone. Between S. salmonicolor and S. ruinenii, the latter was the best biocatalyst. During four successive batch cultivations in a 7-l bioreactor, 5.5 g/l of γ-decalactone was produced with S. ruinenii in each 10-d run. This was essentially due to its ability to produce the open form of γ-decalactone (i.e., 4-hydroxy-decanoic acid), which is far less toxic than the lactone.

A total of 108 strains of Trichoderma sp. were compared for their ability to produce 6-pentyl-α-pyrone (6PP), a coconut-like aroma. Among them, one strain, Trichoderma viride TSP2, demonstrated a good ability to produce 6PP from several commercial vegetable oils including castor oil (C18:1, OH n-7=89.3%), hazelnut oil (C18:1=78.6%), grapeseed oil (C18:2=66%) and linseed oil (α-C18:3=46.6%). By increasing the oil concentration from 5 to 20%, 1.2 g l−1 of 6PP was achieved after 6 days of cultivation. In comparison, 80 mg l−1 of 6PP were produced when glucose (30 g l−1) was used as the carbon source.

Resting cells of Nocardia paraffinaeCBS 255 58 anaerobically converted oleic acid to 10-hydroxystearic acid (optimum activity at 35°C and pH 7). Addition of oleic acid (0.22 mg/ml of medium), as an inducer, improved the hydroxylating activity. The biocatalyst was inactivated by sonication, lyophilization and heating, but kept 65% of its initial activity after five freeze-thawing treatments. This activity appeared to be located either in the cells or bound to the plasmic membrane.

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei , Brevibacterium aurantiacum , and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed.

Yarrowia lipolytica , located at the frontier of hemiascomycetous yeasts and fungi, is an excellent candidate for studies of metabolism evolution. This yeast, widely recognized for its technological applications, in particular produces volatile sulfur compounds (VSCs) that fully contribute to the flavor of smear cheese. We report here a relevant global vision of sulfur metabolism in Y. lipolytica based on a comparison between high- and low-sulfur source supplies (sulfate, methionine, or cystine) by combined approaches (transcriptomics, metabolite profiling, and VSC analysis). The strongest repression of the sulfate assimilation pathway was observed in the case of high methionine supply, together with a large accumulation of sulfur intermediates. A high sulfate supply seems to provoke considerable cellular stress via sulfite production, resulting in a decrease of the availability of the glutathione pathway's sulfur intermediates. The most limited effect was observed for the cystine supply, suggesting that the intracellular cysteine level is more controlled than that of methionine and sulfate. Using a combination of metabolomic profiling and genetic experiments, we revealed taurine and hypotaurine metabolism in yeast for the first time. On the basis of a phylogenetic study, we then demonstrated that this pathway was lost by some of the hemiascomycetous yeasts during evolution.

Due to their importance in flavouring capacities (e.g. production of volatile sulphur compounds (VSCs)), their role in redox regulation (e.g. production of thiols) and their possible role in microbial interactions, sulphur compounds play a key role in traditional fermented products. A better knowledge and control of their microbial production is therefore of major interest not only to maintain the high quality of these foodstuffs but also to get generic knowledge on sulphur metabolism in the microbial world. Yarrowia lipolytica is a commonly found yeast in numerous cheeses, and its enzymatic feature gives this microorganism a competitive superiority over other yeast species found in cheese. Several studies have shown that this yeast could efficiently degrade sulphur amino acids, leading to VSC production.