Milk microbiota has a great influence on the safety and quality of dairy products. However, few studies have investigated the variations of bacterial composition in raw milk. In this study, raw milk samples were collected in 12 successive months, and their bacterial compositions were determined by 16 S rRNA gene sequencing. The highest diversity of bacterial composition was detected in June, while the lowest was in December. Firmicutes, Proteobacteria and Actinobacteria were the most abundant phyla and exhibited a counter-balanced relationship. Pseudomonas, Lactococcus and Acinetobacter were the most prevalent genera (>1%), and a tiny core microbiota (Acinetobacter and Pseudomonas) was observed. Temperature and humidity were the determining factors for most variation in bacterial compositions at both the phylum and genus levels. Higher abundances of Pseudomonas, Propionibacterium and Flavobacterium were correlated with low temperature. Furthermore, Pseudomonas/Propionibacterium and Lactobacillus/Bifidobacterium were two pairs of genera that had synergistic effects. Associations between the microbiota and milk quality parameters were analyzed. The abundances of Propionibacterium and Pseudoalteromonas were negatively correlated to total bacterial count, which meant that they helped to maintain milk quality, while a series of environmental microorganisms contributed to the spoilage of raw milk.

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Lacto-N-neotetraose (LNnT), one of the oligosaccharides in human milk, has many beneficial effects on infant health. In a recent work, we have constructed a recombinant Bacillus subtilis strain for the production of LNnT. Here, we further improved LNnT production with a xylose-induced clustered regularly interspaced short palindromic repeats interference system. In particular, the expressions of pf kA and pyk genes in the Embden−Meyerhof−Parnas pathway module, zwf gene in the pentose phosphate pathway module, and mnaA gene in the teichoic acid synthesis module were downregulated. The LNnT titer was increased from 1.32 to 1.55 g/L. Furthermore, to improve the conversion efficiency of lacto-N-triose II to LNnT, we knocked out tuaD gene in branch pathway and improved the expression of lgtB gene, resulting in the further increase of LNnT titer to 2.01 g/L. Finally, the addition time and amount of inducer xylose were optimized, and LNnT titer reached 2.30 g/L in shake flask and 5.41 g/L in 3 L bioreactor.

Background Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation. Results In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy. Conclusions In general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

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