Directionally controlling flavor compound profile based on the structure of synthetic microbial community in Chinese liquor fermentation.

Most traditional fermented foods are produced by spontaneous fermentation. It is difficult to produce traditional fermented foods with desired flavor compound profile. In this study, using Chinese liquor fermentation as a case, we aimed to directionally control flavor compound profile in food fermentation. Twenty key flavor compounds were identified in 80 Chinese liquor fermentations. Six microbial strains, identified as high producers of these key flavor compounds, were used to generate the minimal synthetic microbial community. A mathematical model was established to link the structure of the minimal synthetic microbial community and the profile of these key flavor compounds. This model could generate the optimal structure of synthetic microbial community to produce flavor compounds with desired profile. This work provided a strategy to realize the directional control of flavor compound profile via controlling the structure of the synthetic microbial community in Chinese liquor fermentation.

Metagenomics reveals the habitat specificity of biosynthetic potential of secondary metabolites in global food fermentations.

BACKGROUND: Fermented foods are considered to be beneficial for human health. Secondary metabolites determined by biosynthetic gene clusters (BGCs) are precious bioactive compounds with various biological activities. However, the diversity and distribution of the biosynthetic potential of secondary metabolites in global food fermentations remain largely unknown. In this study, we performed a large-scale and comprehensive investigation for the BGCs in global food fermentations by metagenomics analysis. RESULTS: We recovered 653 bacterial metagenome-assembled genomes (MAGs) from 367 metagenomic sequencing datasets covering 15 general food fermentation types worldwide. In total, 2334 secondary metabolite BGCs, including 1003 novel BGCs, were identified in these MAGs. Bacillaceae, Streptococcaceae, Streptomycetaceae, Brevibacteriaceae and Lactobacillaceae contained high abundances of novel BGCs (≥ 60 novel BGCs). Among 2334 BGCs, 1655 were habitat-specific, originating from habitat-specific species (80.54%) and habitat-specific genotypes within multi-habitat species (19.46%) in different food fermentation types. Biological activity analysis suggested that 183 BGC-producing secondary metabolites exhibited high probabilities of antibacterial activity (> 80%). These 183 BGCs were distributed across all 15 food fermentation types, and cheese fermentation contained the most BGC number. CONCLUSIONS: This study demonstrates that food fermentation systems are an untapped reservoir of BGCs and bioactive secondary metabolites, and it provides novel insights into the potential human health benefits of fermented foods. Video Abstract.

Metabolic cooperation between conspecific genotypic groups contributes to bacterial fitness.

Microbial interactions are important for the survival of species and the stability of the microbial ecosystem. Although bacteria have diverse conspecific genotypes in the natural microbial ecosystem, little is known about whether wild-type strains within species would interact with each other and how the intraspecific interaction influences the growth of the species. In this work, using Lactobacillus acetotolerans, a dominant species with diverse conspecific genotypes in natural food fermentation ecosystems as a case, we studied the interactions between different genotypic groups of this species. In interspecific and intraspecific pairwise cocultures, the growth of L. acetotolerans decreased, but the increase of the phylogenetic similarity would reduce this negative effect, indicating a potential intraspecific interaction of this species. Meanwhile, the strain classification method affected the analysis of intraspecific interactions, which can be efficiently demonstrated using 99.5% average nucleotide identity (ANI) as the strain-level classification method. Using this ANI classification method, we revealed the population fitness significantly increased in cocultures of different genotypic groups. Facilitation involving 11 amino acids was identified between different ANI genotypic groups, which was beneficial for increasing population fitness. This work revealed that wild-type conspecific strains could interact with each other via cooperative metabolic changes and benefit each other to increase fitness. It shed new light on the survival and stability of species in natural microbial ecosystems.

Regulation of auxotrophic lactobacilli growth by amino acid cross-feeding interaction.

Microbial interaction plays an important role in the assembly of microbial communities in spontaneous food fermentation. Amino acids are important for the microbial interaction. However, the effect of amino acids on the microbial interaction is still unclear in food fermentation. Here, we studied the cross-feeding of amino acids between Saccharomyces cerevisiae and two lactobacilli species (Limosilactobacillus panis and Lentilactobacillis buchneri) from Chinese liquor fermentation. The inoculation of S. cerevisiae and concentrations of amino acids significantly influenced the growth of two lactobacilli species (P < 0.05). Li. panis and Le. buchneri were respectively auxotrophic of 11 and 4 amino acids through single omission technique experiment. S. cerevisiae would promote the growth of Li. panis and Le. buchneri through excreting their essential amino acids, including valine and isoleucine. Herein, we used response surface methodology to establish a predictive model for the growth of lactobacilli species by the regulating factors, the initial concentrations of valine and isoleucine, and inoculum size of S. cerevisiae. Based on the optimized levels of the three factors, the biomass of Li. panis increased from 6.5 ± 0.3 to 7.6 ± 0.1 log10 cells/mL, and the biomass of Le. buchneri increased from 7.2 ± 0.2 to 8.4 ± 0.3 log10 cells/mL. This work revealed the interaction pattern between S. cerevisiae and the two lactobacilli species, it would facilitate the regulation of microbial growth in Chinses liquor fermentation.

Gradient Internal Standard Method for Absolute Quantification of Microbial Amplicon Sequencing Data.

High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R 2 average = 0.998; P < 0.001) and accurate (P internals versus microscopy > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition.IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.

Construction of a synthetic microbial community for the biosynthesis of volatile sulfur compound by multi-module division of labor.

3-(Methylthio)-1-propanol, reminiscent of cauliflower and cooked vegetable aroma, is an important sulfur compound in Baijiu. It is important to develop a method to increase 3-(methylthio)-1-propanol content to improve flavor quality of products. In this study, a synthetic microbial community was employed to enhance the content of 3-(methylthio)-1-propanol by multi-module division of labor approach. Firstly, the synthetic pathway of 3-(methylthio)-1-propanol was reconstructed and classified into three modules. Later, the hyper producers in each module were isolated and negative interaction between the members was relieved. Finally, a synthetic microbial community was constructed using three species containing one hyper producer from each module. Furthermore, the transcription characteristics of the species in each module were validated by metatranscriptomic analysis. The constructed synthetic microbial community can be used to biosynthesize 3-(methylthio)-1-propanol for Baijiu. This work provided a novel and workable strategy to design synthetic microbial community to enhance the flavor feature of other fermented foods.

Chinese Liquor Fermentation: Identification of Key Flavor-Producing Lactobacillus spp. by Quantitative Profiling with Indigenous Internal Standards.

Identifying the functional microbes in spontaneous food fermentation is important for improving food quality. To identify the key flavor producers in Chinese liquor fermentation, we propose a novel quantitative microbiome profiling method that uses indigenous internal standards to normalize high-throughput amplicon sequencing results. We screened Lactobacillus acetotolerans and Lactobacillus jinshani as indigenous internal standards based on their high distribution frequencies and relative abundances. After determining the absolute abundance of indigenous internal standards using quantitative PCR with species-specific primers, the liquor-fermented bacterial community and its dynamics were better characterized by internal standards normalization. Based on quantitative microbiome profiling, we identified that Lactobacillus was a key flavor producer correlated with eight flavor compounds. Metatranscriptomic analysis indicated that Lactobacillus was active in transcribing genes involving the biosynthesis of flavor compounds and their precursors. This work has developed a novel and extensible absolute quantification method for microbiota that will alleviate concerns in the statistical analyses based on relative microbiome profiling, and shed insights into the function of Lactobacillus in food fermentation. It can potentially be applied to other microbial ecology studies.IMPORTANCE In this study, we developed a novel strategy using indigenous internal standards to normalize the high-throughput amplicon sequencing results. We chose two Lactobacillus species as indigenous internal standards and characterized the absolute abundance of the bacterial community. Further, we identified Lactobacillus as the key flavor producer using quantitative microbiome profiling combined with multivariate statistics and metatranscriptomic analysis. This work developed a novel strategy for absolute quantitative abundance analysis of microbiota and expanded our understanding of the role of Lactobacillus in food fermentation.

Zygosaccharomyces bailii Is a Potential Producer of Various Flavor Compounds in Chinese Maotai-Flavor Liquor Fermentation.

Zygosaccharomyces bailii is a common yeast in various food fermentations. Understanding the metabolic properties and genetic mechanisms of Z. bailii is important for its industrial applications. Fermentation characteristics of Z. bailii MT15 from Chinese Maotai-flavor liquor fermentation were studied. Z. bailii MT15 produced various flavor compounds, including 19 alcohols, six acids, three esters, three ketones, and two aldehydes. Moreover, production of acids and aldehydes were increased by 110 and 41%, respectively, at 37°C (the maximum temperature in liquor fermentation) compared with that at 30°C, indicating its excellent flavor productivity. Z. bailii MT15 is a diploid with genome size of 20.19 Mb. Comparative transcriptome analysis revealed that 12 genes related to amino acid transport were significantly up-regulated (2.41- to 5.11-fold) at 37°C. Moreover, genes ARO8, ARO9, and ALDH4 involved in amino acid metabolism also showed higher expression levels (>1.71-fold) at 37°C. Increased substrate supply and a vigorous metabolism might be beneficial for the increased production of acids and aldehydes at 37°C. This work revealed the potential contribution of Z. bailii to various flavor compounds in food fermentation, and produced insights into the metabolic mechanisms of Z. bailii in flavor production.