Chen Liang, Gu Qing, Zhou Tao
Key Laboratory for Food Microbial Technology of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China.
Front Nutr. 2022 Jun 2;9:924495. doi: 10.3389/fnut.2022.924495. eCollection 2022.
The traditional media used for the fermentation of 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 yeast extract, 12.5 g L beef extract, 10 g L peptone, 21.23 g L maltose, 5.51 g L yeast nitrogen base, 2 g L KHPO, 5 g L anhydrous sodium acetate, 2 g L ammonium citrate, 0.58 g L MgSO⋅7HO, 0.25 g L MnSO⋅HO, and 1 mL L 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, being 76.70% higher than that of unoptimized conditions (281.07 ± 5.90 mg L). 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.
用于发酵的传统培养基总是含有碳水化合物聚合物，这会干扰细菌产生的胞外多糖（EPS）的分析。在本研究中，成功开发了一种可以避免此类干扰的新型培养基配方。该配方中使用的牛肉浸出物、酵母浸出物和蛋白胨在使用前进行了多糖去除处理。通过单因素试验、Plackett-Burman设计和Box-Behnken设计对影响EPS产生的因素进行了优化。确定的最佳配方为：7.5 g/L酵母浸出物、12.5 g/L牛肉浸出物、10 g/L蛋白胨、21.23 g/L麦芽糖、5.51 g/L酵母氮源、2 g/L KH₂PO₄、5 g/L无水乙酸钠、2 g/L柠檬酸铵、0.58 g/L MgSO₄·7H₂O、0.25 g/L MnSO₄·H₂O和1 mL/L吐温80。培养基的初始pH值为6.5。该菌株发酵生产EPS的优化条件如下：接种量1%、培养温度37℃和培养时间20 h。最佳结果表明，EPS产量为496.64±3.15 mg/L，比未优化条件下（281.07±5.90 mg/L）高出76.70%。EPS主要由葡萄糖和古洛糖醛酸组成，重均分子量为19.9 kDa；还通过傅里叶变换红外光谱和紫外分析对其进行了表征。发现EPS能显著增强RAW 264.7巨噬细胞的吞噬能力，促进NO、TNF-α、IL-1β和IL-6的分泌，并改善细胞因子的mRNA表达，表明其具有相当强的免疫调节活性。蛋白质免疫印迹和免疫荧光结果表明，EPS能够增加巨噬细胞中p65的核转位，表明EPS通过NF-κB信号通路增强免疫力。本研究中研究的EPS有潜力成为免疫力低下人群有吸引力的功能性食品补充剂候选物。
