Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, P.R. China 100048; Mengniu Gaoke Dairy (Beijing) Co. Ltd., Beijing, P.R. China 101100.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, P.R. China 100048.
J Dairy Sci. 2021 Mar;104(3):2693-2708. doi: 10.3168/jds.2020-19237. Epub 2021 Jan 15.
Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.
胞外多糖（EPS）产生的乳酸菌已广泛应用于乳制品中，但牛奶中主要的金属离子钙如何调节乳酸菌的 EPS 生物合成尚不清楚。本研究以益生菌植物乳杆菌 K25 为研究对象，探讨了 Ca2+对其 EPS 生物合成的影响。结果表明，在半定量培养基中添加 20mg/L 的 CaCl2 不会影响 K25 菌株的生长，但会增加 EPS 的产量，并改变聚合物的微观结构。Ca2+的存在还改变了 EPS 的单糖组成，高分子量成分减少，鼠李糖含量增加，尽管傅里叶变换红外光谱分析表明聚合物的官能团没有改变。这进一步通过 cps 基因的 mRNA 表达分析得到证实，其中 9 个基因受 Ca2+上调，包括与鼠李糖有关的 EPS 生物合成的 cps4F 和 rfbD。蛋白质组学分析表明，Ca2+上调了与碳转运和代谢、脂肪酸合成、氨基酸合成、离子转运、UMP 合成相关的大多数蛋白质。特别是，MelB、PtlIIBC、EIIABC、PtlIIC、PtlIID、Bgl、GH1、MalFGK、DhaK 和 FBPase 的表达增加为 EPS 合成提供了底物。同时，代谢组学分析显示三羧酸循环、葡萄糖代谢和丙酸代谢中的小分子代谢物发生了显著变化。其中，多半乳糖醇、木酮糖和 5-磷酸核糖等活性小分子的含量增加，有利于 EPS 的生物合成。此外，Ca2+激活了 HipB 信号通路，抑制了 ArsR、LytR/AlgR、IscR 和 RafR 等操纵子阻遏物的表达，并激活了 GntR 的表达，从而调节 EPS 合成基因。本研究为了解 L. plantarum K25 响应 Ca2+时与 EPS 生物合成相关的代谢途径的整体变化提供了依据，有利于开发其 EPS 生产潜力，应用于益生菌乳制品。
