Unraveling multifunction of low-temperature Daqu in simultaneous saccharification and fermentation of Chinese light aroma type liquor.

Chinese liquor is produced by a representative simultaneous saccharification and fermentation process. Daqu, as a starter of Chinese liquor fermentation, affects both saccharification and fermentation. However, it is still unclear how Daqu contributed to the simultaneous saccharification and fermentation process. Here, using Chinese light aroma type liquor as a case, we identified low-temperature Daqu-originated enzymes and microorganisms that contributed to the simultaneous saccharification and fermentation using metaproteomic analysis combined with amplicon sequencing analysis. α-Amylase and glucoamylase accounted for 95 % of total saccharifying enzymes and were identified as key saccharifying enzymes. Lichtheimia was the key producer of these two enzymes (> 90 %) in low-temperature Daqu. Daqu contributed 90 % α-amylase and 99 % glucoamylase to the initial liquor fermentation. These two enzymes decreased by 35 % and 49 % until day 15 in liquor fermentation. In addition, Daqu contributed key microbial genera (91 % Saccharomyces, 6.5 % Companilactobacillus) and key enzymes (37 % alcohol dehydrogenase, 40 % lactic acid dehydrogenase, 56 % aldehyde dehydrogenase) related with formations of ethanol, lactic acid and flavour compounds to the initial liquor fermentation. The average relative abundances of these fermentation-related key microorganisms and enzymes increased by 2.78 times and 1.29 times till day 15 in liquor fermentation, respectively. It indicated that Daqu provided saccharifying enzymes for starch hydrolysis, and provided both enzymes and microorganisms associated with formations of ethanol, lactic acid and flavour compounds for liquor fermentation. This work illustrated the multifunction of low-temperature Daqu in the simultaneous saccharification and fermentation of Chinese light aroma type liquor. It would facilitate improving liquor fermentation by producing high-quality Daqu.

Molecular Organization and Functional Analysis of a Novel Plasmid-Borne cps Gene Cluster from Lactiplantibacillus plantarum YC41.

Capsular polysaccharide (CPS) can tightly attach to bacterial surfaces and plays a critical role in protecting microorganisms from environmental stresses. However, the molecular and functional properties of some plasmid-borne cps gene clusters are poorly understood. In this study, comparative genomics of the draft genomes of 21 Lactiplantibacillus plantarum strains revealed that the specific gene cluster for CPS biosynthesis was observed only in the 8 strains with a ropy phenotype. Furthermore, the complete genomes showed that the specific gene cluster cpsYC41 was located on the novel plasmid pYC41 in L. plantarum YC41. In silico analysis confirmed that the cpsYC41 gene cluster contained the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene. The insertional inactivation of the rmlA and cpsC genes abolished the ropy phenotype and reduced the CPS yields by 93.79% and 96.62%, respectively, in L. plantarum YC41 mutants. These results revealed that the cpsYC41 gene cluster was responsible for CPS biosynthesis. Moreover, the survival rates of the YC41-rmlA- and YC41-cpsC- mutants under acid, NaCl, and H2O2 stresses were decreased by 56.47 to 93.67% compared to that of the control strain. Furthermore, the specific cps gene cluster was also confirmed to play a vital role in CPS biosynthesis in L. plantarum MC2, PG1, and YD2. These findings enhance our understanding of the genetic organization and gene functions of plasmid-borne cps gene clusters in L. plantarum. IMPORTANCE Capsular polysaccharide is well known to protect bacteria against various environmental stresses. The gene cluster for CPS biosynthesis is typically organized in the chromosome in bacteria. It is worth noting that complete genome sequencing showed that a novel plasmid pYC41-borne cpsYC41 gene cluster was identified in L. plantarum YC41. The cpsYC41 gene cluster included the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the wzx gene, which was verified by the significantly decreased CPS yield and the absent ropy phenotype in the corresponding mutants. The cpsYC41 gene cluster plays an important role in bacterial survival under environmental stress, and the mutants had decreased fitness under stress conditions. The vital role of this specific cps gene cluster in CPS biosynthesis was also confirmed in other CPS-producing L. plantarum strains. These results advanced a better understanding of the molecular mechanisms of plasmid-borne cps gene clusters and the protective functionality of CPS.

Raw Material Regulates Flavor Formation via Driving Microbiota in Chinese Liquor Fermentation.

Raw material is important for flavors in fermented foods. Here, the effect of hulless barley on the microbiota in Chinese liquor was studied using two main cultivars (heilaoya and dulihuang). Six genera (Lactobacillus, Saccharomyces, Komagataella, Aspergillus, Pichia, and Weissella) were identified as flavor producers. Komagataella, mainly correlated with esters, dominated in heilaoya, and Pichia, mainly correlated with carbonyls, dominated in dulihuang. The Mantel test indicated reducing sugar drove the succession of microbiota (heilaoya: P = 0.001; dulihuang: P = 0.006). Especially, glucose (P = 0.0226) and fructose (P = 0.0168) presented the most significant correlations with Pichia and Komagataella, respectively. The simulative fermentation confirmed Komagataella phaffii QK2 grew better in heilaoya with more fructose, whereas Pichia fermentans PF grew better in dulihuang with more glucose. This work highlighted the effect of raw material on microbiota, which would be beneficial for regulating the quality of fermented foods.