Table_1_Statistical Optimization of Novel Medium to Maximize the Yield of Exopolysaccharide From Lacticaseibacillus rhamnosus ZFM216 and Its Immunomodulatory Activity.DOCX

The traditional media used for the fermentation of Lactobacilli always contain carbohydrate polymers, which interfere with the analysis of the exopolysaccharide (EPS) produced by the bacteria. In this investigation, a novel medium formulation that could avoid such interference was successfully developed. The beef extract, yeast extract, and peptone used in this formulation were subjected to the removal of polysaccharides before use. The factors affecting the EPS production were optimized by a single factor test, Plackett–Burman design, and Box–Behnken design. The optimum formula was ascertained as: 7.5 g L–1 yeast extract, 12.5 g L–1 beef extract, 10 g L–1 peptone, 21.23 g L–1 maltose, 5.51 g L–1 yeast nitrogen base, 2 g L–1 K2HPO4, 5 g L–1 anhydrous sodium acetate, 2 g L–1 ammonium citrate, 0.58 g L–1 MgSO4⋅7H2O, 0.25 g L–1 MnSO4⋅H2O, and 1 mL L–1 Tween 80. The initial pH of the medium was 6.5. The optimized conditions for fermentation of the strain to produce EPS were as follows: seed size 1%, culture temperature 37°C, and culture time 20 h. Optimum results showed that EPS yield was 496.64 ± 3.15 mg L–1, being 76.70% higher than that of unoptimized conditions (281.07 ± 5.90 mg L–1). The EPS was mainly comprised of glucose and guluronic acid, with a weight average molecular weight of 19.9 kDa; it was also characterized by Fourier transform infrared spectroscopy and UV analysis. EPS was found to significantly enhance the phagocytic capacity, promote the NO, TNF-α, IL-1β, and IL-6 secretion, and improve mRNA expression of cytokines in RAW 264.7 macrophages, indicating its considerable immunomodulatory activity. Western bolt and immunofluorescence results demonstrated that the EPS was able to increase p65 nuclear translocation in the macrophages, indicating that EPS enhanced immunity via the NF-κB signaling pathway. EPS investigated in this work has potential as an attractive functional food supplement candidate for the hypoimmunity population.

传统用于乳杆菌（Lactobacilli）发酵的培养基通常含有碳水化合物聚合物，这类物质会干扰对该细菌所产胞外多糖（exopolysaccharide, EPS）的分析。本研究成功开发出一种可规避此类干扰的新型培养基配方。该配方中所用的牛肉浸膏、酵母浸膏与蛋白胨均在使用前脱去了多糖成分。研究通过单因素试验、普拉克特-伯曼（Plackett–Burman）设计以及Box-Behnken设计对影响EPS合成的因素进行了优化。最终确定的最优培养基配方为：酵母浸膏7.5 g·L⁻¹、牛肉浸膏12.5 g·L⁻¹、蛋白胨10 g·L⁻¹、麦芽糖21.23 g·L⁻¹、酵母氮源基础培养基（yeast nitrogen base）5.51 g·L⁻¹、磷酸氢二钾（K₂HPO₄）2 g·L⁻¹、无水乙酸钠5 g·L⁻¹、柠檬酸铵2 g·L⁻¹、七水合硫酸镁（MgSO₄·7H₂O）0.58 g·L⁻¹、一水合硫酸锰（MnSO₄·H₂O）0.25 g·L⁻¹以及吐温80（Tween 80）1 mL·L⁻¹。培养基初始pH值为6.5。该菌株产EPS的最优发酵条件为：接种量1%、培养温度37℃、培养时长20 h。优化后EPS产量可达496.64 ± 3.15 mg·L⁻¹，较未优化条件下的产量（281.07 ± 5.90 mg·L⁻¹）提升了76.70%。该EPS主要由葡萄糖与古洛糖醛酸构成，重均分子量为19.9 kDa；同时通过傅里叶变换红外光谱与紫外光谱对其进行了表征。实验发现，该EPS可显著增强RAW 264.7巨噬细胞的吞噬能力，促进一氧化氮（NO）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）以及白细胞介素-6（IL-6）的分泌，并上调细胞因子的mRNA表达水平，表明其具备可观的免疫调节活性。蛋白质免疫印迹（Western bolt）与免疫荧光实验结果显示，该EPS可促进巨噬细胞中p65蛋白的核转位，证实EPS通过核因子κB（NF-κB）信号通路增强免疫功能。本研究中所表征的EPS具备作为免疫低下人群优质功能性食品补充剂候选物的应用潜力。