Search

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.

Meat represents an important protein source, even in developing countries, but its production is scarcely sustainable, and its excessive consumption poses health issues. An increasing number of Western consumers would replace, at least partially, meat with alternative protein sources. This review aims at: (i) depicting nutritional, functional, sensory traits, and critical issues of single-cell proteins (SCP), filamentous fungi, microalgae, vegetables (alone or mixed with milk), and insects and (ii) displaying how fermentation could improve their quality, to facilitate their use as food items/ingredients/supplements. Production of SCP (yeasts, filamentous fungi, microalgae) does not need arable land and potable water and can run continuously, also using wastes and byproducts. Some filamentous fungi are also consumed as edible mushrooms, and others are involved in the fermentation of traditional vegetable-based foods. Cereals, pseudocereals, and legumes may be combined to offer an almost complete amino acid profile. Fermentation of such vegetables, even in combination with milk-based products (e.g., tarhana), could increase nutrient concentrations, including essential amino acids, and improve sensory traits. Different insects could be used, as such or, to increase their acceptability, as ingredient of foods (e.g., pasta). However, insects as a protein source face with safety concerns, cultural constraints, and a lack of international regulatory framework. info:eu-repo/semantics/publishedVersion

Moving to a more sustainable food system requires increasing the proportion of plant protein in our diet. Fermentation of plant product could thus be used to develop innovative and tasty food products. We investigated the impact of fermentation by synthetic microbial consortia (SMC) on the perception of pea protein-based gels, giving possible keys to better understand the origin of sensory perception (e.g., beany, bitter). Two types of pea gels, containing (i) 100% pea proteins and (ii) 50% pea proteins/50% milk proteins, were fermented with three different SMC. Major species developing in both types of gels were Geotrichum candidum, Lactococcus lactis, and Lactobacillus rhamnosus. In pea gels, sensory analyses revealed that bitterness increased after fermentation, which could be due to hydrophobic amino acids resulting from protein hydrolysis, but also decreased pea note intensity in pea gels. In mixed gels, pea perception was similar whatever the SMC, whereas cheesy perception increased. Olfactometry experiments revealed that some specific “green” aroma compounds, responsible for green off-note, were suppressed/reduced by fermentation. The data presented investigated to which extent the design of SMC, together with gels composition (pea gels versus mixed gels), could modulate sensorial perception and drive consumer acceptability.

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.

International audience; Ripening cultures containing fungi and bacteria are widely used in smear-ripened cheese production processes, but little is known about the biotic interactions of typical ripening microorganisms at the surface of cheese. We developed a lab-scale mini-cheese model to investigate the biotic interactions of a synthetic community that was composed of Debaryomyces hansenii, Brevibacterium aurantiacum, and Hafnia alvei, three species that are commonly used for smear-ripened cheese production. Transcriptomic analyses of cheese samples produced with different combinations of these three species revealed potential mechanisms of biotic interactions concerning iron acquisition, proteolysis, lipolysis, sulfur metabolism, and D-galactonate catabolism. A strong mutualistic interaction was observed between H. alvei and B. aurantiacum. We propose an explanation of this positive interaction in which B. aurantiacum would benefit from siderophore production by H. alvei, and the latter would be stimulated by the energy compounds liberated from caseins and triglycerides through the action of the proteases and lipases secreted by B. aurantiacum. In the future, it would be interesting to take the iron acquisition systems of cheese-associated strains into account for the purpose of improving the selection of the ripening culture components and their association in mixed cultures.

Ripening cultures containing fungi and bacteria are widely used in smear-ripened cheese production processes, but little is known about the biotic interactions of typical ripening microorganisms at the surface of cheese. We developed a lab-scale mini-cheese model to investigate the biotic interactions of a synthetic community that was composed of Debaryomyces hansenii, Brevibacterium aurantiacum, and Hafnia alvei, three species that are commonly used for smear-ripened cheese production. Transcriptomic analyses of cheese samples produced with different combinations of these three species revealed potential mechanisms of biotic interactions concerning iron acquisition, proteolysis, lipolysis, sulfur metabolism, and D-galactonate catabolism. A strong mutualistic interaction was observed between H. alvei and B. aurantiacum. We propose an explanation of this positive interaction in which B. aurantiacum would benefit from siderophore production by H. alvei, and the latter would be stimulated by the energy compounds liberated from caseins and triglycerides through the action of the proteases and lipases secreted by B. aurantiacum. In the future, it would be interesting to take the iron acquisition systems of cheese-associated strains into account for the purpose of improving the selection of the ripening culture components and their association in mixed cultures.

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

International audience; Consumer demands for plant-based products have increased in recent years. However, their consumption is still limited due to the presence of off-flavor compounds, primarily beany and green notes, which are mainly associated with the presence of aldehydes, ketones, furans, and alcohols. To overcome this problem, fermentation is used as a lever to reduce off-flavors. A starter culture of lactic acid bacteria (LAB) was tested in a 4% pea protein solution with one of the following yeasts: Kluyveromyces lactis, Kluyveromyces marxianus, or Torulaspora delbrueckii. The fermented samples were evaluated by a sensory panel. Non-fermented and fermented matrices were analyzed by gas chromatography coupled with mass spectrometry to identify and quantify the volatile compounds. The sensory evaluation showed a significant reduction in the green/leguminous attributes of pea proteins and the generation of new descriptors in the presence of yeasts. Compared to the non-fermented matrix, fermentations with LAB or LAB and yeasts led to the degradation of many off-flavor compounds. Moreover, the presence of yeasts triggered the generation of esters. Thus, fermentation by a co-culture of LAB and yeasts can be used as a powerful tool for the improvement of the sensory perception of a pea protein-based product.

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

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.

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.

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.

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.

Mycelia from the basidiomycete Phanerochaete chrysosporium, producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.

The ability of pellets or immobilized cell cultures of Phanerochaete chrysosporium Burds. wild-type BKM-F-1767 and INA-12 to produce extracellular peroxidases was investigated. Lignin peroxidase (LiP) and Mn(II)-dependent peroxidase (MnP) secretion was increased by immobilizing the fungus on polyurethane foam. Strain INA-12 cultivated as pellets or immobilized cells produced high peroxidase activities more efficiently than strain BKM-F-1767. With immobilized cells, maximal LiP activities of 1296 and 2580 U l-1 were obtained with BKM-F-1767 and INA-12, respectively, after 5 days of cultivation. Other extracellular proteins were stimulated in the same way. Although formed in different ratios, isoenzymes produced by both strains were essentially identical with respect to their physical properties (isoelectric points and molecular weights). Determination of marker enzymes revealed that the enhancement of peroxidase secretion observed with immobilized cells was correlated with a proliferation of endoplasmic reticulum and mitochondria marker enzymes for both strains.

Yarrowia lipolytica is one of the yeasts most frequently isolated from the surface of ripened cheeses. In previous work, it has been shown that this yeast is able to convert l -methionine into various volatile sulfur compounds (VSCs) that may contribute to the typical flavors of several cheeses. In the present study, we show that Y. lipolytica does not assimilate lactate in the presence of l -methionine in a cheeselike medium. Nineteen presumptive genes associated with l -methionine catabolism or pyruvate metabolism in Y. lipolytica were transcriptionally studied in relation to l -methionine degradation. The expression levels of the Yl ARO8 (YALI0E20977g), Yl BAT1 (YALI0D01265g), and Yl BAT2 (YALI0F19910g) genes (confirmed by real-time PCR experiments) were found to be strongly up-regulated by l -methionine, and a greater variety and larger amounts of VSCs, such as methanethiol and its autooxidation products (dimethyl disulfide and dimethyl trisulfide), were released in the medium when Y. lipolytica was grown in the presence of a high concentration of l -methionine. In contrast, other genes related to pyruvate metabolism were found to be down-regulated in the presence of l -methionine; two exceptions were the Yl PDB1 (YALI0E27005g) and Yl PDC6 (YALI0D06930g) genes, which encode a pyruvate dehydrogenase and a pyruvate decarboxylase, respectively. Both transcriptional and biochemical results corroborate the view that transamination is the first step of the enzymatic conversion of l -methionine to VSCs in Y. lipolytica and that the Yl ARO8 , Yl BAT1 , and Yl BAT2 genes could play a key role in this process.

Mn(II) concentrations had little effect on substrate uptake and growth rates, but they profoundly changed the composition of extracellular proteins. At low Mn(II) (0.3 ppm), Phanerochaete chrysosporium produced lignin peroxidase (LiP) isozymes (total activity=760 nmol ml −1 min −1 ), and the levels of Mn(II) peroxidase (MnP) isozymes were very low (130 nmol ml −1 min −1 ), whereas, with high Mn(II) (40 ppm), P. chrysosporium preferentially produced MnP isozymes (950 nmol ml −1 min −1 ), and LiPs were essentially absent. The airlift reactor provided a suitable low-shear environment for enzyme production by free mycelial pellets. This is a simple method to selectively produce LiP and MnP isozymes.

Two families of peroxidases—lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)—are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of 14 CO 2 from 14 C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of 14 CO 2 release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium , two other species of Phanerochaete , three species of Phlebia, Lentinula edodes , and Phellinus pini .

International audience; The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena—which initiate sulfur bearing precursors degradation—leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.

Production of volatile sulphur compounds (VSC) was assessed in culture media supplemented with L-methionine or L-methionine/L-cysteine mixtures, using five cheese-ripening yeasts: Debaryomyces hansenii DH47(8), Kluyveromyces lactis KL640, Geotrichum candidum GC77, Yarrowia lipolytica YL200 and Saccharomyces cerevisiae SC45(3). All five yeasts produced VSC with L-methionine or L-methionine/L-cysteine, but different VSC profiles were found. GC77 and YL200 produced dimethyldisulphide and trace levels of dimethyltrisulphide while DH47(8), KL640 and SC45(3) produced mainly methionol and low levels of methional. S-methylthioacetate was produced by all the yeasts but at different concentrations. DH47(8), KL640 and SC45(3) also produced other minor VSC including 3-methylthiopropyl acetate, ethyl-3-methylthiopropanoate, a thiophenone, and an oxathiane. However, VSC production diminished in a strain-dependent behaviour when L-cysteine was supplemented, even at a low concentration (0.2 g l(-1)). This effect was due mainly to a significant decrease in L-methionine consumption in all the yeasts except YL200. Hydrogen sulphide produced by L-cysteine catabolism did not seem to contribute to VSC generation at the acid pH of yeast cultures. The significance of such results in the cheese-ripening context is discussed.

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSC) is believed to be essential for flavour development in cheese. L-methionine γ-lyase (MGL) can convert L-methionine to methanethiol (MTL) α-ketobutyrate and ammonia. The MGL gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSC. The disruption of the MGL gene, achieved in strain ATCC 9175, resulted in a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via γ-elimination is a key step to form VSC in B. linenes.

Volatile sulfur compounds (VSCs) are of major importance for flavour development in foodstuffs including cheeses. They primarily result from L-methionine degradation to methanethiol (MTL), a precursor for 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 reacting with free thiols—in the upper layer was developed. Thiol production was quantified by measuring the intensity of the yellow-orange colour resulting from DTNB reacting with thiols. Geotrichum candidum strains isolated from cheeses were compared by this method and colour intensity was shown to correlate to the microbial overall VSC production as measured by gas chromatography.

The enzymatic degradation of l -methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. l -Methionine-γ-lyase (MGL) can convert l -methionine to methanethiol (MTL), α-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that l -methionine degradation via γ-elimination is a key step in the formation of VSCs in B. linens .

l-Methionine degradation products and catabolic enzymatic activities involved in methanethiol generation were investigated in Geotrichum candidum GcG. l-Methionine was easily degraded by G. candidum and the transamination product, 4-methylthio-2-oxobutyric acid (KMBA), was found to transiently accumulate. In parallel, considerable l-methionine aminotransferase activity was found in this microorganism. l-Methionine and KMBA demethiolating activities were also detected. The degradation of KMBA corresponded to an overall increase in the production of volatile sulfur compounds. These results show that the transamination pathway is of major importance in the initial breakdown of l-methionine by this cheese-ripening microorganism.

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters – ethylacetate (EA) being the major one – and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA – a common precursor of MTA and EA – but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.