Revealing Core Microbiota Related With Fatty Acid Synthesis And Influencing Factors In Fermentation Of Soy Sauce Aroma Type Liquor

The fermentation of soy sauce aroma type liquor is a spontaneous fermentation process with multi-species interactions.Due to the unique production process,the contents of fatty acids are higher than other liquors.Fatty acids have a great contribution to the flavor and quality of soy sauce aroma type liquor.However,microbial community associated with fatty acid synthesis is unclear.It is essential to reveal core microbiota related with fatty acid synthesis and influencing factors for the improvement of quality.In this study,we analyzed the changes of fatty acids and microbial community in the fermentations of soy sauce aroma type liquor by gas chromatography and high-throughput sequencing,and revealed the microorganisms related with fatty acid synthesis.Moreover,we analyzed the abundance and transcript activity of functional genes related with fatty acid synthesis by metagenomic and metatranscriptomic analysis,constructed the pathway of fatty acid synthesis and determined the core microbiota related with fatty acid synthesis.The environmental sources of the core microbiota were investigated and the influence of environmental microbiota on fatty acid synthesis was explained.The effects of p H on fatty acid synthesis were elucidated through culturable experiments.This study has important theoretical implications for the stable improvement in flavor quality of soy sauce aroma type liquor.The main findings of this study are as follows:（1）In response to the problems that the changes of fatty acids and microbial community in fermentations of soy sauce aroma type liquor are unclear,we quantitatively analyzed the changes of fatty acids in fermentations by gas chromatography.The contents of total fatty acids,including short chain fatty acids（C2-C5）and medium chain fatty acids（C6-C10）,reached the maximum of 2.26 g·kg^-1 on the third day in stacking fermentation and 3.71 g·kg^-1 on the fifth day in liquor fermentation.Acetic acid was the highest fatty acid in fermentations,accounting for 58.20%of the total fatty acid contents.High-throughput sequencing analysis revealed that the dominant genera（average relative abundance≥1%）in the fermentations were Acetobacter,Saccharopolyspora,Acetilactobacillus,Bacillus,Kroppenstedtia,Kroppenstedtia,Byssochlamys,Thermomyces,Thermoascus,Torulaspora,Aspergillus,Pichia and Saccharomyces.Spearman’s correlation analysis revealed that 12 dominant genera and 11 fatty acids were significantly correlated（P<0.05）,suggesting that the dominant genera may be related with fatty acid synthesis in soy sauce aroma type liquor.（2）In response to the problems that core microbiota related with fatty acids synthesis and their environmental sources in fermentations of soy sauce aroma type liquor are unclear,we analyzed the abundance of functional genes encoding enzymes related with fatty acid synthesis by metagenomic analysis.The dominant genera with abundance of functional genes>400FPKM included Acetilactobacillus,Kroppenstedtia,Byssochlamys,Saccharopolyspora,Pichia and Saccharomyces.We analyzed the transcript activity of functional genes encoding enzymes related with fatty acid synthesis by metatranscriptomic analysis.The dominant genera with transcript activity of functional genes>1000 FPKM included Saccharomyces,Acetilactobacillus,Pichia,Byssochlamys and Kroppenstedtia,indicating these five species were core microbiota related with fatty acid synthesis in soy sauce aroma type liquor.We further analyzed the environmental sources of core microbiota by Source Tracker,finding that Daqu mainly provided Kroppenstedtia（34.01%）,Acetilactobacillus（3.31%）,Pichia（0.97%）Saccharomyces（0.12%）and Byssochlamys（0.10%）,and ground mainly provided Pichia（47.47%）,Saccharomyces（16.17%）,Byssochlamys（8.55%）,Acetilactobacillus（0.10%）and Kroppenstedtia（0.08%）.This provided a theoretical basis for further regulation of fatty acid synthesis.（3）In response to the problems that the regulation of core microbiota related with fatty acid synthesis was unclear,firstly,we analyzed the influence of environmental microbiota on core microbiota,and found that environmental microbiota could ultimately regulate the contents and diversity of fatty acids via changing core microbiota related with fatty acid synthesis by structural equation model.Secondly,the redundancy analysis of physical and chemical factors and core microbiota showed that acidity was an important factor affecting the core microbiota.Finally,culture experiments with metabolically active microorganism（Saccharomyces cerevisiae）showed that the yield of total fatty acids increased significantly with the decrease of p H in the range of 3.5-5.5（P<0.05）.The yields of total fatty acids,acetic acid,propionic acid,isobutyric acid,isovaleric acid and octanoic acid were the highest at p H3.5(233.34±3.63 mg·L^-1,81.55±6.34 mg·L^-1,16.73±0.11 mg·L^-1,72.21±11.81 mg·L^-1,44.37±4.72 mg·L^-1 and 18.49±2.51 mg·L^-1,respectively).This provided theoretical guidance for metabolic regulation of core microbiota related with fatty acid synthesis.