Genomic Characterization of Wild Lactobacillus delbrueckii Strains Reveals Low Diversity but Strong Typicity.

Investigating the diversity of a given species could give clues for the development of autochthonous starter cultures. However, few studies have focused on the intraspecies diversity of Lactobacillus delbrueckii strains, a technologically important lactic acid bacterium for the dairy industry. For this reason, Lactobacillus delbrueckii strains from the Saint-Nectaire Protected Designation of Origin (PDO) area were isolated and characterized. Genetic diversity was determined based on core genome phylogenetic reconstruction and pangenome analysis, while phenotypic assessments encompassed proteolysis and volatile compound production potential. A total of 15 L. delbrueckii ssp. lactis unique new strains were obtained. The genetic analysis and further proteolytic activities measurement revealed low variability among these Saint-Nectaire strains, while substantial genetic variability was observed within the L. delbrueckii ssp. lactis subspecies as a whole. The volatile compound profiles slightly differed among strains, and some strains produced volatile compounds that could be of particular interest for cheese flavor development. While the genetic diversity among Saint-Nectaire strains was relatively modest compared to overall subspecies diversity, their distinct characteristics and pronounced differentiation from publicly available genomes position them as promising candidates for developing autochthonous starter cultures for cheese production.

Genetic and technological diversity of Streptococcus thermophilus isolated from the Saint-Nectaire PDO cheese-producing area.

Streptococcus thermophilus is of major importance for cheese manufacturing to ensure rapid acidification; however, studies indicate that intensive use of commercial strains leads to the loss of typical characteristics of the products. To strengthen the link between the product and its geographical area and improve the sensory qualities of cheeses, cheese-producing protected designations of origin (PDO) are increasingly interested in the development of specific autochthonous starter cultures. The present study is therefore investigating the genetic and functional diversity of S. thermophilus strains isolated from a local cheese-producing PDO area. Putative S. thermophilus isolates were isolated and identified from milk collected in the Saint-Nectaire cheese-producing PDO area and from commercial starters. Whole genomes of isolates were sequenced, and a comparative analysis based on their pan-genome was carried out. Important functional properties were studied, including acidifying and proteolytic activities. Twenty-two isolates representative of the diversity of the geographical area and four commercial strains were selected for comparison. The resulting phylogenetic trees do not correspond to the geographical distribution of isolates. The clustering based on the pan-genome analysis indicates that isolates are divided into five distinct groups. A Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation of the accessory genes indicates that the accessory gene contents of isolates are involved in different functional categories. High variability in acidifying activities and less diversity in proteolytic activities were also observed. These results indicate that high genetic and functional variabilities of the species S. thermophilus may arise from a small (1,800 km2) geographical area and may be exploited to meet demand for use as autochthonous starters.

Protein Sources Alternative to Meat: State of the Art and Involvement of Fermentation.

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.

Modulation of Metabolome and Overall Perception of Pea Protein-Based Gels Fermented with Various Synthetic Microbial Consortia.

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.

Strong effect of Penicillium roqueforti populations on volatile and metabolic compounds responsible for aromas, flavor and texture in blue cheeses.

Studies of food microorganism domestication can provide important insight into adaptation mechanisms and lead to commercial applications. Penicillium roqueforti is a fungus with four genetically differentiated populations, two of which were independently domesticated for blue cheese-making, with the other two populations thriving in other environments. Most blue cheeses are made with strains from a single P. roqueforti population, whereas Roquefort cheeses are inoculated with strains from a second population. We made blue cheeses in accordance with the production specifications for Roquefort-type cheeses, inoculating each cheese with a single P. roqueforti strain, using a total of three strains from each of the four populations. We investigated differences between the cheeses made with the strains from the four P. roqueforti populations, in terms of the induced flora, the proportion of blue color, water activity and the identity and abundance of aqueous and organic metabolites as proxies for proteolysis and lipolysis as well as volatile compounds responsible for flavor and aroma. We found that the population-of-origin of the P. roqueforti strains used for inoculation had a minor impact on bacterial diversity and no effect on the abundance of the main microorganism. The cheeses produced with P. roqueforti strains from cheese populations had a higher percentage of blue area and a higher abundance of the volatile compounds typical of blue cheeses, such as methyl ketones and secondary alcohols. In particular, the Roquefort strains produced higher amounts of these aromatic compounds, partly due to more efficient proteolysis and lipolysis. The Roquefort strains also led to cheeses with a lower water availability, an important feature for preventing spoilage in blue cheeses, which is subject to controls for the sale of Roquefort cheese. The typical appearance and flavors of blue cheeses thus result from human selection on P. roqueforti, leading to the acquisition of specific features by the two cheese populations. These findings have important implications for our understanding of adaptation and domestication, and for cheese improvement.

Sensory Improvement of a Pea Protein-Based Product Using Microbial Co-Cultures of Lactic Acid Bacteria and Yeasts.

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.

Effect of sodium chloride reduction or partial substitution with potassium chloride on the microbiological, biochemical and sensory characteristics of semi-hard and soft cheeses.

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.

Transcription Profiling Reveals Cooperative Metabolic Interactions in a Microbial Cheese-Ripening Community Composed of Debaryomyces hansenii, Brevibacterium aurantiacum, and Hafnia alvei.

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.

Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase from Brevibacterium aurantiacum. Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination.

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 γ-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 α,γ-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 methyl-CpG 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.

Design of microbial consortia for the fermentation of pea-protein-enriched emulsions.

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 lentus) 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.

Transcriptomic response of Debaryomyces hansenii during mixed culture in a liquid model cheese medium with Yarrowia lipolytica.

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.

Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during digestion.

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.

Highlighting the microbial diversity of 12 French cheese varieties.

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.

Draft Genome Sequence of Corynebacterium variabile Mu292, Isolated from Munster, a French Smear-Ripened Cheese.

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.

Investigation of the Activity of the Microorganisms in a Reblochon-Style Cheese by Metatranscriptomic Analysis.

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 ~75 million reads per sample. Except for B. 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 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.

Survival of cheese-ripening microorganisms in a dynamic simulator of the gastrointestinal tract.

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.

Overview of a surface-ripened cheese community functioning by meta-omics analyses.

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.

Growth and adaptation of microorganisms on the cheese surface.

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.

In vitro characterization of the digestive stress response and immunomodulatory properties of microorganisms isolated from smear-ripened cheese.

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 cytokine assay tests showed that the response to yeasts was usually anti-inflammatory, whereas the response to bacteria varied from one strain to another.

Investigation of Geotrichum candidum gene expression during the ripening of Reblochon-type cheese by reverse transcription-quantitative PCR.

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.