In this study, a dominant caproic acid-producing strain (CPB), designated XB2, was isolated from pit mud of Baijiu using enrichment and traditional anaerobic isolation techniques. It was identified as Caproicibacterium lactatifermentans based on 16S rRNA gene sequencing and whole-genome sequencing. Comparative genomic analysis revealed that in addition to possessing a complete reverse β-oxidation (RBO) pathway, strain XB2 is the first reported Caproicibacterium strain carrying the full complement of key enzyme genes in both the fatty acid biosynthesis (FAB) pathway and the L-threonine degradation pathway for propionate synthesis. Transmission electron microscopy (TEM) revealed that strain XB2 exhibited an elliptical to fusiform morphology, differing significantly from the short-rod shape of its phylogenetic relatives. Fermentation results indicated carbon source-dependent metabolism: when glucose was used as the substrate, caproate was synthesized at a low concentration (0.23 g/L), whereas using lactate as the substrate increased butyrate production by 0.59 g/L. Under conditions with limited electron acceptors, propionate was produced at 0.21 g/L, suggesting the activation of the L-threonine pathway. This study elucidates the morphology and intraspecific metabolic diversity of C. lactatifermentans. It challenges the conventional view that CPBs rely solely on the RBO pathway for caproic acid synthesis, providing a new perspective for understanding functional redundancy and adaptability of microbial communities in pit mud of Baijiu. Furthermore, it establishes a theoretical foundation for optimizing Baijiu flavor profiles through the precise regulation of microbial metabolism.

This study aimed to explore the impact of pit mud (PM) on caproic acid biosynthesis by the microbiota in Huangshui (HS), a by-product of solid-state fermentation in Baijiu production. PM and HS from the late fermentation stage of Nongxiangxing Baijiu were used to design a PM-HS micro-fermentation system and an HS micro-fermentation system which were used to carry out four consecutive rounds of fermentation. A medium with lactic acid as the sole carbon source was used to select the core functional microorganisms degrading lactic acid. Then, 16S rRNA gene amplicon high-throughput sequencing, physicochemical analysis, redundancy analysis (RDA), and functional prediction using Picrust2 were conducted to compare the enrichment of dominant caproic acid-producing procaryotes and their potential caproic acid synthesis functions between the two fermentation systems. The results showed that the mixed fermentation system was beneficial to the enrichment of Caproiciproducens, with Caproicibacterium lactatifermentans LBM19010 being the dominant caproic acid producer. This consortium showed a strong ability to degrade lactic acid to produce caproic acid in the presence of glucose. On the other hand, the HS system was beneficial to the enrichment of the potential butyric acid-producing genus Clostridium_sensu_stricto_12, with Clostridium tyrobutyricum ATCC 25755 being the predominant strain. This strain showed a strong ability to degrade lactic acid into butyric acid in the presence of glucose. Both fermentation systems enriched strains able to degrade lactic acid into butyric acid. In the fermentation system of Nongxiangxing Baijiu, the microbial genera producing caproic acid were not the same as those degrading lactic acid. This study provides theoretical support for further research on synergistic metabolism of caproic acid-producing and butyric acid-producing microorganisms and also provides a scientific basis for the targeted enrichment of caproic acid-producing and butyric acid-producing microorganisms.

In the Chinese strong-flavor Baijiu (CSFB) fermentation ecosystem, the caproic acid-anabolism of caproic acidproducing bacteria (CPBs) is very important for improving the fermentation quality of CSFB. Therefore, it is necessary to thoroughly understand the types of CPBs and their caproic acid-anabolism characteristics. This minireview introduces readers to the diversity, phylogenetic relationship, physiological and metabolic characteristics, and caproic acid synthesis mechanism of CPBs isolated from the CSFB fermentation ecosystem as well as their synergistic metabolic relationships with other CPBs or non-CPBs. This paper provides a reference for understanding the in-situ caproic acid-anabolism pattern of CPBs from the CSFB fermentation ecosystem, and further provides a theoretical basis for the future targeted application of CPBs in CSFB fermentation and for CPBs culture engineering for the synthesis of high value-added caproic acid.