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.

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.

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.

Quantification of yeast and bacterial gene transcripts in retail cheeses by reverse transcription-quantitative PCR.

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.

New insights into sulfur metabolism in yeasts as revealed by studies of Yarrowia lipolytica.

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.

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.

Genome sequence of Corynebacterium casei UCMA 3821, isolated from a smear-ripened cheese.

Corynebacterium casei is one of the most prevalent species present on the surfaces of smear-ripened cheeses, where it contributes to the production of the desired organoleptic properties. Here, we report the draft genome sequence of Corynebacterium casei UCMA 3821 to provide insights into its physiology.

Genome sequence of Staphylococcus equorum subsp. equorum Mu2, isolated from a French smear-ripened cheese.

Staphylococcus equorum subsp. equorum is a member of the coagulase-negative staphylococcus group and is frequently isolated from fermented food products and from food-processing environments. It contributes to the formation of aroma compounds during the ripening of fermented foods, especially cheeses and sausages. Here, we report the draft genome sequence of Staphylococcus equorum subsp. equorum Mu2 to provide insights into its physiology and compare it with other Staphylococcus species.

S-methyl thioesters are produced from fatty acids and branched-chain amino acids by brevibacteria: focus on L-leucine catabolic pathway and identification of acyl-CoA intermediates.

Despite their importance as potent odors that contribute to the aroma of numerous cheeses, S-methyl thioesters formation pathways have not been fully established yet. In a first part of our work, we demonstrated that Brevibacterium antiquum and Brevibacterium aurantiacum could produce S-methyl thioesters using short-chain fatty acids or branched-chain amino acids as precursors. Then, we focused our work on L-leucine catabolism using liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry analyses coupled with tracing experiments. For the first time, several acyl-CoAs intermediates of the L-leucine to thioesters conversion pathway were identified. S-methyl thioisovalerate was produced from L-leucine, indicating that this amino acid was initially transaminated. Quite interestingly, data also showed that other S-methyl thioesters, e.g., S-methyl thioacetate or S-methyl thioisobutyrate, were produced from L-leucine. Enzymatic and tracing experiments allowed for postulating catabolic pathways leading to S-methyl thioesters biosynthesis.

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.

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.

Global regulation of the response to sulfur availability in the cheese-related bacterium Brevibacterium aurantiacum.

In this study, we combined metabolic reconstruction, growth assays, and metabolome and transcriptome analyses to obtain a global view of the sulfur metabolic network and of the response to sulfur availability in Brevibacterium aurantiacum. In agreement with the growth of B. aurantiacum in the presence of sulfate and cystine, the metabolic reconstruction showed the presence of a sulfate assimilation pathway, thiolation pathways that produce cysteine (cysE and cysK) or homocysteine (metX and metY) from sulfide, at least one gene of the transsulfuration pathway (aecD), and genes encoding three MetE-type methionine synthases. We also compared the expression profiles of B. aurantiacum ATCC 9175 during sulfur starvation or in the presence of sulfate. Under sulfur starvation, 690 genes, including 21 genes involved in sulfur metabolism and 29 genes encoding amino acids and peptide transporters, were differentially expressed. We also investigated changes in pools of sulfur-containing metabolites and in expression profiles after growth in the presence of sulfate, cystine, or methionine plus cystine. The expression of genes involved in sulfate assimilation and cysteine synthesis was repressed in the presence of cystine, whereas the expression of metX, metY, metE1, metE2, and BL613, encoding a probable cystathionine-γ-synthase, decreased in the presence of methionine. We identified three ABC transporters: two operons encoding transporters were transcribed more strongly during cysteine limitation, and one was transcribed more strongly during methionine depletion. Finally, the expression of genes encoding a methionine γ-lyase (BL929) and a methionine transporter (metPS) was induced in the presence of methionine in conjunction with a significant increase in volatile sulfur compound production.

Exploration of sulfur metabolism in the yeast Kluyveromyces lactis.

Hemiascomycetes are separated by considerable evolutionary distances and, as a consequence, the mechanisms involved in sulfur metabolism in the extensively studied yeast, Saccharomyces cerevisiae, could be different from those of other species of the phylum. This is the first time that a global view of sulfur metabolism is reported in the biotechnological yeast Kluyveromyces lactis. We used combined approaches based on transcriptome analysis, metabolome profiling, and analysis of volatile sulfur compounds (VSCs). A comparison between high and low sulfur source supplies, i.e., sulfate, methionine, or cystine, was carried out in order to identify key steps in the biosynthetic and catabolic pathways of the sulfur metabolism. We found that sulfur metabolism of K. lactis is mainly modulated by methionine. Furthermore, since sulfur assimilation is highly regulated, genes coding for numerous transporters, key enzymes involved in sulfate assimilation and the interconversion of cysteine to methionine pathways are repressed under conditions of high sulfur supply. Consequently, as highlighted by metabolomic results, intracellular pools of homocysteine and cysteine are maintained at very low concentrations, while the cystathionine pool is highly expandable. Moreover, our results suggest a new catabolic pathway for methionine to VSCs in this yeast: methionine is transaminated by the ARO8 gene product into 4-methylthio-oxobutyric acid (KMBA), which could be exported outside of the cell by the transporter encoded by PDR12 and demethiolated by a spontaneous reaction into methanethiol and its derivatives.

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.

A proteomic and transcriptomic view of amino acids catabolism in the yeast Yarrowia lipolytica.

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.

Identification of brevibacteriaceae by multilocus sequence typing and comparative genomic hybridization analyses.

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.

Growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp. during ripening in a cheese model medium.

The growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp., some common but rarely mentioned cheese bacteria, were investigated in a cheese model deacidified by Debaryomyces hansenii during the ripening process. Our results show that these bacteria had distinct growth and cheese flavour production patterns during the ripening process. P. vulgaris had the greatest capacity to produce not only the widest variety but also the highest quantities of volatile compounds with low olfactive thresholds, e.g. volatile sulphur compounds and branched-chain alcohols. Such compounds produced by P. vulgaris increased after 21 days of ripening and reached a maximum at 41 days. The three bacteria studied exhibited various degrees of caseinolytic, aminopeptidase and deaminase activities. Moreover, P. vulgaris had a greater capacity for hydrolysing casein and higher deaminase activity. Our results show that P. vulgaris, a Gram-negative bacterium naturally present on the surface of ripened cheeses, could produce high concentrations of flavour compounds from amino acid degradation during the ripening process. Its flavouring role in cheese cannot be neglected. Moreover, it could be a useful organism for producing natural flavours as dairy ingredients.

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.

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.