《食品工业科技》F5000论文展播：退化和正常窖泥微生物多样性的比较分析（2022年）

Objectives: During the process of brewing Baijiu (Chinese liquor), the mud pit serves as the foundation for fermentation and contains a diverse range of microbial communities, which determine the taste quality of the resulting Baijiu. A comparative analysis of the microbial community structures in normal pit mud and degraded pit mud can help to identify characteristic microorganisms associated with degraded and normal pit mud, thereby exploring the relationship between Baijiu quality and microorganisms. In this study, high-throughput sequencing technology and bioinformatics methods were applied to perform high-throughput sequencing and bioinformatics analysis of the microbial communities in normal pit mud and degraded pit mud.

Methods: Using high-throughput sequencing and bioinformatics analysis, the microbial diversity of normal pit mud and degraded pit mud were studied and compared. First, the total DNA from the pit mud samples was extracted using a soil DNA extraction kit. Subsequently, the extracted DNA was used as a template to amplify the bacterial 16S rRNA V3-V4 region using PCR. The obtained PCR products were subjected to high-throughput sequencing using the MiSeq PE300 platform; after sequencing, the obtained paired-end sequences were quality-controlled by removing low-quality sequences and adapters, and then, the QIIME platform was employed to classify the sequences into operational taxonomic units (OTUs) based on a 97% similarity threshold. The RDP (Ribosomal Database Project) database was used for taxonomic annotation at the phylum, class, and order levels to analyze the microbial community structure of the pit mud. Additionally, the α and β diversities of the pit mud microbial communities were analyzed using built-in scripts in QIIME.

Results: A total of 10 pit mud samples were collected, including 5 normal pit mud samples and 5 degraded pit mud samples. The degraded pit mud appeared silver-gray with white needle-like crystalline clumps and lacked ester aroma, while the normal pit mud was gray-brown (black) with a strong ester aroma. After MiSeq high-throughput sequencing, a total of 279982 sequences were obtained. After removing chimeras based on 97% similarity, a total of 7606 operational taxonomic units (OTUs) were identified, averaging 1339 OTUs per pit mud sample. These OTUs were classified into 26 phyla, 66 classes, 108 orders, 233 families, and 524 genera. Alpha diversity analysis indicated that the Chao1 index and Shannon index of the degraded pit mud were significantly higher than those of the normal pit mud (P<0.05). Multivariate statistical analysis revealed a significant difference in microbial community structure between normal pit mud and degraded pit mud (P<0.05). The dominant phyla in both normal and degraded pit mud included Firmicutes, Proteobacteria, and Actinobacteria, with relative abundances greater than 1%. However, the relative abundances of the phyla Spirochaetes, Synergistetes, and Chloroflexi were significantly lower in the normal pit mud than those in degraded pit mud (P<0.05). The dominant genera shared by normal and degraded pit mud included Lactobacillus and Clostridium. Statistical analysis showed that Lactobacillus, Bacillus, and Lelliottia were significantly more abundant in the normal pit mud than in degraded pit mud (P<0.05).

Conclusion: Normal pit mud was not only different from degraded pit mud in appearance, but also in microbial structure: the degraded pit mud demonstrates higher microbial richness and diversity. With the degradation of the pit mud, the dominant phyla in the microbial community shifted to include Spirochaetes, Synergistetes, and Chloroflexi, in addition to the previously dominant phyla. Furthermore, the number of dominant genera increaseed from 5 to 9 in the degraded pit mud. Additionally, we found that Lactobacillus was the absolutely dominant genus in the normal pit mud, accounting for a relative abundance as high as 67.91%. This analysis suggests that Lactobacillus may play a role in producing characteristic flavor compounds such as ethyl lactate in Baijiu and also exhibit inhibitory effects on certain miscellaneous bacteria. The microbial diversity analysis between normal and degraded pit mud confirms that bacterial diversity can serve as an objective indicator for evaluating pit mud quality which is of reference value for subsequent optimization of pit mud quality and prevention and control of pit mud degradation.