Characterizing the Contribution of Functional Microbiota Cultures in Pit Mud to the Metabolite Profiles of Fermented Grains.

Elevating the flavor profile of strong flavors Baijiu has always been a focal point in the industry, and pit mud (PM) serves as a crucial flavor contributor in the fermentation process of the fermented grains (FG). This study investigated the influence of wheat flour and bran (MC and FC) as PM culture enrichment media on the microbiota and metabolites of FG, aiming to inform strategies for improving strong-flavor Baijiu flavor. Results showed that adding PM cultures to FG significantly altered its properties: FC enhanced starch degradation to 51.46% and elevated reducing sugar content to 1.60%, while MC increased acidity to 2.11 mmol/10 g. PM cultures also elevated FG's ester content, with increases of 0.36 times for MC-FG60d and 1.48 times for FC-FG60d compared to controls, and ethyl hexanoate rising by 0.91 times and 1.39 times, respectively. Microbial analysis revealed that Lactobacillus constituted over 95% of the Abundant bacteria community, with Kroppenstedtia or Bacillus being predominant among Rare bacteria. Abundant fungi included Rasamsonia, Pichia, and Thermomyces, while Rare fungi consisted of Rhizopus and Malassezia. Metagenomic analysis revealed bacterial dominance, primarily consisting of Lactobacillus and Acetilactobacillus (98.80-99.40%), with metabolic function predictions highlighting genes related to metabolism, especially in MC-FG60d. Predictions from PICRUSt2 suggested control over starch, cellulose degradation, and the TCA cycle by fungal subgroups, while Abundant fungi and bacteria regulated ethanol and lactic acid production. This study highlights the importance of PM cultures in the fermentation process of FG, which is significant for brewing high-quality, strong-flavor Baijiu.

Succession of microbial community of the pit mud under the impact of Daqu of Nongxiang Baijiu.

Complex microbiomes of pit mud play significant roles in imbuing flavors and qualities of Nongxiang Baijiu during fermentation. However, pit mud microbial enrichment and succession is a long process that is also accompanied by aging. Development of high-quality artificial pit mud becomes an urgent problem. In this study, a new medium based on space (TK) Daqu was used to effectively enrich the dominant microorganisms in pit mud. The results showed that Caproiciproducens was the most preponderance in the cultures unadded Daqu, whereas Clostridium sensu stricto 12 was the most preponderance, followed by Caproiciproducens in the enrichment cultures added TK Daqu. It is worth noting that TK Daqu balanced the relative abundance of Caproiciproducens and Clostridium sensu stricto 12 in 100-year pit mud culture (S100), which was more conducive to the increase of methanogens. PICRUSt2 prediction results showed that hydrogenotrophic methanogens could promote the synthesis of caproic acid by using the product H2 as the metabolic substrate and increased significantly in the pit mud enrichment cultures with TK Daqu. The increase of lactate dehydrogenase (EC 1.1.1.27) content in S100 contributed to the degradation of lactic acid and the increase of caproic acid. Adding TK Daqu enrichment cultures is more conducive to the enrichment and metabolic balance of pit mud microorganisms.

Effects of Daqu properties on the microbial community and their metabolites in fermented grains in Baijiu fermentation system.

Daqu is the natural starter for Nong-flavor Baijiu brewing. The effects of Daqu properties on the microbial community succession and their metabolites in fermented grains (FG) during Baijiu brewing were determined. These results showed that the effect of Daqu on the bacterial communities was stronger than that of the fungal communities. Compared with the conventional Daqu (DZ), Taikong (TK), and Qianghua (QH), Daqu significantly enhanced the content of volatile metabolites (especially esters) and ethanol when they were used, respectively, for FG fermentation. In the second round of fermentation, the relative abundance of Lactobacillus decreased, the content of lactic acid decreased, and that of caproic acid increased. In particular, the abundance of Lactobacillus was also reduced by 20% in FGs of the second round when TK Daqu was used than that in the respective first round. Partial least squares structural equation model analysis also showed that physicochemical parameters and Daqu properties significantly affected FG community structure and metabolism. This study provides a theoretical basis for further study on the effect of high-quality Daqu on the quality of fresh Baijiu and lays an important theoretical foundation for the stabilization of the Baijiu fermentation system based on Daqu.

Effects of Different Daqu on Microbial Community Domestication and Metabolites in Nongxiang Baijiu Brewing Microecosystem.

The quality and yield of the fresh Baijiu mainly depend on the activity of pit mud (PM) and the quality of Daqu. However, the cultivation of PM is a long-term process, and high-quality Daqu can change the community structure of fermented grain (FG) and accelerate the evolution of PM communities. The present research aimed to investigate the four different types of Daqu on the FG-fermenting microbial community structure and metabolites and their interphase interactions with PM. These results show that Kroppenstedtia in the bacterial community of Taikong Daqu (TK) was positively correlated with ethyl caproate, which significantly increased the content of FG volatile metabolites, especially lipid components, and facilitated the accelerated evolution of Methanobacteriales and Methanosarcinales in PM. Bacillus has a high relative abundance in Qianghua Daqu (QH), which shows obvious advantages to improving the alcoholic strength of FG and contributing to increasing the abundance of Methanomicrobiales in PM. Qianghua and traditional-mixed Daqu (HH) have a similar bacterial composition to QH and a similar fungal composition to traditional Daqu (DZ), and thus also showed the advantage of increased yield, but the volatile flavor metabolites produced were not as dominant as DZ. ß-diversity analysis showed that in TK fermentation systems, FG is more likely to domesticate the structure of PM microorganisms. These results indicated that the interaction between microbial communities in Baijiu fermentation niches was significantly influenced by different Daqu. It can not only enhance the key volatiles in FG but also accelerate the evolving direction of the community in PM. Daqu fortified by functional genera or microbiota can evolve a community structure more suitable for Baijiu fermentation. The microbiota composition and interaction between the communities in both Daqu and PM significantly impacts the yield and quality of the base liquor.

Characterizing the interaction relationship of the microbial communities between Zaopei and pit mud disturbing by Daqu.

The differences of interaction between interphase microbial communities were evaluated caused by two kinds of Daqu, including conventional Daqu (CDQ) and fortified Daqu (FDQ). The community diversity, functional genera and metabolites in pit mud (PM) and Zaopei (ZP) were investigated by polyphasic detecting approaches. FDQ evolved the core microbial community fitting Baijiu brewing faster than CDQ. Compared with CPM, the abundance of Aspergillus, Hyphopichia, and Penicillium in FPM were 1.54, 14.75, and 1.68 times, while that of Lactobacillus, Bacillus, Methanobrevibacter, and Methanosaeta were 2.13, 1.85, 6.35, and 3.36 times, respectively. Furthermore, the content of key flavor components was increased in ZP using FDQ. These results suggested the interaction between interphase microbial communities in various phases of Baijiu fermentation niches was significant influenced by Daqu. It can not only enhance the key volatiles in ZP but also evolve the community to fit Baijiu fermentation by introducing functional genera to Daqu. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00975-z.

Evolving the core microbial community in pit mud based on bioturbation of fortified Daqu.

Directional stress is an effective measure to change the community structure and improve the bioactivity of pit mud (PM). In this study, the addition of fortified Daqu to artificial PM (APM) was intended to disturb the microbial community and further affect metabolites. To evaluate the effect of fortified Daqu on culturing APM, the microbial communities of APM with or without the addition of fortified Daqu were investigated by fluorescence in situ hybridization and Illumina MiSeq. The results indicated that microbes (Clostridium sp., Clostridium kluyveri, hydrogenotrophic methanogens, and acetotrophic methanogens) related to the production of key aroma compounds increased notably when fortified Daqu was added. In particular, the hydrogenotrophic and acetotrophic methanogens increased by 6.19- and 4.63-fold after 30 days of culture. Subsequently, metabolites (organic acids, volatile compounds) were also analyzed by HPLC (high-performance liquid chromatography) and HS-SPME-GC-MS (headspace solid phase microextraction - gas chromatography - mass spectrometry). The results showed that the content of butyric acid and hexanoic acid was significantly higher when fortified Daqu was added to APM. In addition, the proportion of esters and phenols was also higher than in APM without fortified Daqu. A survey of the microbial compositions of APMs with or without added fortified Daqu indicated that the microbial community evolves into a functional community favoring liquor brewing. We have developed a novel process by disturbing the community diversity.