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      Bio-Heat Is a Key Environmental Driver Shaping the Microbial Community of Medium-Temperature Daqu

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          ABSTRACT

          “Daqu” is a saccharifying and fermenting agent commonly used in the traditional solid-state fermentation industry (e.g., baijiu and vinegar). The patterns of microbial community succession and flavor formation are highly similar among batches, yet the mechanisms promoting temporal succession in the Daqu microbial ecology remain unclear. Here, we first correlated temporal profiles of microbial community succession with environmental variables (temperature, moisture, and titratable acidity) in medium temperature Daqu (MT-Daqu) throughout fermentation. Temperature dynamics significantly correlated ( P < 0.05) with the quick succession of MT-Daqu microbiota in the first 12 d of fermentation, while the community structure was relatively stable after 12 d. Then, we explored the effect of temperature on the MT-Daqu community assembly. In the first 4 d of fermentation, the rapid propagation of most bacterial taxa and several fungal taxa, including Candida , Wickerhamomyces , and unclassified Dipodascaceae and Saccharomycetales species, significantly increased MT-Daqu temperature to 55°C. Subsequently, sustained bio-heat generated by microbial metabolism (53 to 56°C) within MT-Daqu inhibited the growth of most microbes from day 4 to day 12, while thermotolerant taxa, including Bacillus , unclassified Streptophyta , Weissella , Thermoactinomyces , Thermoascus , and Thermomyces survived or kept on growing. Furthermore, temperature as a major driving force on the shaping of MT-Daqu microbiota was validated. Lowering the fermentation temperature by placing the MT-Daqu in a 37°C incubator resulted in decreased relative abundances of thermotolerant taxa, including Bacillus , Thermoactinomyces , and Thermoascus , in the MT-Daqu microbiota. This study revealed that bio-heat functioned as a primary endogenous driver promoting the formation of functional MT-Daqu microbiota.

          IMPORTANCE Humans have mastered the Daqu preparation technique of cultivating functional microbiota on starchy grains over thousands of years, and it is well known that the metabolic activity of these microbes is key to the flavor production of Chinese baijiu. The pattern of microbial community succession and flavor formation remains highly similar between batches, yet mechanistic insight into these patterns and into microbial population fidelity to specific environmental conditions remains unclear. Our study revealed that bio-heat was generated within Daqu bricks in the first 4 d of fermentation, concomitant with rapid microbial propagation and metabolism. The sustained bio-heat may then function as a major endogenous driving force promoting the formation of the MT-Daqu microbiota from day 4 to day 12. The bio-heat-driven growth of thermotolerant microorganisms might contribute to the formation of flavor metabolites. This study provides useful information for the temperature-based modulation of microbiota function during the fermentation of Daqu.

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          Most cited references28

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          Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays.

          Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure.
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            Developments in the use of Bacillus species for industrial production.

            Bacillus species continue to be dominant bacterial workhorses in microbial fermentations. Bacillus subtilis (natto) is the key microbial participant in the ongoing production of the soya-based traditional natto fermentation, and some Bacillus species are on the Food and Drug Administration's GRAS (generally regarded as safe) list. The capacity of selected Bacillus strains to produce and secrete large quantities (20-25 g/L) of extracellular enzymes has placed them among the most important industrial enzyme producers. The ability of different species to ferment in the acid, neutral, and alkaline pH ranges, combined with the presence of thermophiles in the genus, has lead to the development of a variety of new commercial enzyme products with the desired temperature, pH activity, and stability properties to address a variety of specific applications. Classical mutation and (or) selection techniques, together with advanced cloning and protein engineering strategies, have been exploited to develop these products. Efforts to produce and secrete high yields of foreign recombinant proteins in Bacillus hosts initially appeared to be hampered by the degradation of the products by the host proteases. Recent studies have revealed that the slow folding of heterologous proteins at the membrane-cell wall interface of Gram-positive bacteria renders them vulnerable to attack by wall-associated proteases. In addition, the presence of thiol-disulphide oxidoreductases in B. subtilis may be beneficial in the secretion of disulphide-bond-containing proteins. Such developments from our understanding of the complex protein translocation machinery of Gram-positive bacteria should allow the resolution of current secretion challenges and make Bacillus species preeminent hosts for heterologous protein production. Bacillus strains have also been developed and engineered as industrial producers of nucleotides, the vitamin riboflavin, the flavor agent ribose, and the supplement poly-gamma-glutamic acid. With the recent characterization of the genome of B. subtilis 168 and of some related strains, Bacillus species are poised to become the preferred hosts for the production of many new and improved products as we move through the genomic and proteomic era.
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              Metagenomic analysis of kimchi, a traditional Korean fermented food.

              Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

                Author and article information

                Journal
                Applied and Environmental Microbiology
                Appl. Environ. Microbiol.
                American Society for Microbiology
                0099-2240
                1098-5336
                November 16 2017
                December 01 2017
                December 01 2017
                September 29 2017
                : 83
                : 23
                Article
                10.1128/AEM.01550-17
                5691423
                28970223
                c2a5ee46-c41d-48f9-a0c8-bd503c2a56f6
                © 2017
                History

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