发酵天山雪莲粗多糖的菌株筛选及护肤功效研究

Screening of strains for producing Saussurea involucrata polysaccharide with skincare efficacy

摘要:

【目的】利用微生物发酵植物可以提高多糖的产量，并且能够将原有的植物多糖转化成活性更高的新型发酵多糖，本研究围绕天山雪莲的粗多糖，基于发酵后的活菌数、多糖产量和护肤功效进行发酵菌种筛选，旨在获得适宜发酵天山雪莲粗多糖的优良菌株。【方法】利用不同菌株发酵天山雪莲粗多糖，通过平板菌落计数法测定活菌数，采用蒽酮比色法测定发酵液的多糖含量；采用细胞屏障损伤和抗炎模型，利用噻唑蓝(MTT)法检测细胞活率，利用格里斯法检测NO含量，评价发酵多糖在细胞模型中的护肤功效；利用特应性皮炎小鼠模型，分析发酵多糖对皮肤组织表观及经皮失水率、皮肤组织病理及表皮厚度变化和皮肤组织屏障蛋白-丝聚合蛋白的影响，评价发酵多糖在动物模型中的功效。【结果】不同菌株发酵天山雪莲粗多糖后的活菌数和多糖产量差异较大，其中枯草芽孢杆菌(Bacillus subtilis) CCFM1162和165-M1、干酪乳杆菌(Lactobacillus casei) CCFM1073、罗伊氏乳杆菌(L. reuteri) CCFM8631、清酒乳杆菌(L. sakei) GD17-9的活菌数较高，均不低于2.0×10⁸ CFU/mL；而酿酒酵母(Saccharomyces cerevisiae) HN7-A5、干酪乳杆菌CCFM1073、罗伊氏乳杆菌CCFM8631、清酒乳杆菌GD17-9发酵天山雪莲粗多糖的多糖产量较高，达到1.37 g/L以上。综合多糖产量和活菌数，选取干酪乳杆菌CCFM1073、罗伊氏乳杆菌CCFM8631、清酒乳杆菌GD17-9、酿酒酵母HN7-A5的发酵多糖进行细胞护肤功效评价，结果显示所选菌株的发酵多糖对十二烷基硫酸钠(sodium dodecyl sulfate, SDS)诱导的HaCaT细胞存活率影响不大，而清酒乳杆菌GD17-9、罗伊氏乳杆菌CCFM8631、干酪乳杆菌CCFM1073、酿酒酵母HN7-A5的发酵多糖对降低脂多糖(lipopolysaccharide, LPS)诱导的RAW264.7细胞NO含量效果较好，其中清酒乳杆菌GD17-9、罗伊氏乳杆菌CCFM8631的发酵多糖分别降低了81%和71%。结合活菌数、多糖产量、细胞护肤功效评价，选取清酒乳杆菌GD17-9和罗伊氏乳杆菌CCFM8631发酵的天山雪莲粗多糖进行动物护肤功效验证，发现罗伊氏乳杆菌CCFM8631发酵天山雪莲粗多糖功效更好。【结论】综合活菌数、多糖产量以及细胞和动物模型的护肤功效，罗伊氏乳杆菌CCFM8631菌株是发酵天山雪莲粗多糖的最适宜菌株。

Abstract:

[Objective] The microbial fermentation of plant can increase the yield of polysaccharide and transform the original plant polysaccharide into a new fermentative polysaccharide with higher activity. According to the viable count, yield of Saussurea involucrata polysaccharide, and skincare efficacy, we conducted strain screening, aiming to obtain a strain suitable for the production of S. involucrata polysaccharide. [Methods] Different strains were used to produce S. involucrata polysaccharide. The viable count was determined by plate colony counting method, and the content of polysaccharide in the fermentation broth was determined by anthrone colorimetry. The cell models of barrier damage and inflammation were used to evaluate the skincare efficacy of the polysaccharide in cells, with the cell viability measured by MTT method and NO content measured by Griess method. The mouse model of atopic dermatitis was used to evaluate the efficacy of the polysaccharide in animals in terms of skin appearance, trans-epidermal water loss, skin pathology, changes in epidermal thickness, and the barrier protein filaggrin of skin tissue. [Results] There were significant differences in the viable count and yield of crude polysaccharide after fermentation by different strains. The viable counts of Bacillus subtilis CCFM1162, B. subtilis 165-M1, Lactobacillus casei CCFM1073, L. reuteri CCFM8631, and L. sakei GD17-9 were no less than 2.0×10⁸ CFU/mL and higher than those of the other strains. However, Saccharomyces cerevisiae HN7-A5, L. casei CCFM1073, L. reuteri CCFM8631, and L. sakei GD17-9 produced more than 1.37 g/L polysaccharide. On the basis of the polysaccharide yield and the viable count, the skincare efficacy of crude polysaccharide from S. involucrata fermented by L. casei CCFM1073, L. reuteri CCFM8631, L. sakei GD17-9, and S. cerevisiae HN7-A5 was evaluated with cell models. The results showed that the polysaccharides produced by the selected strains had little effect on the survival rate of HaCaT cells induced by sodium lauryl sulfate, while the polysaccharides decreased the NO content in the RAW264.7 cells exposed to lipopolysaccharide (LPS). The polysaccharides produced by L. sakei GD17-9 and L. reuteri CCFM8631 had better effect, reducing the NO content by 81% and 71%, respectively. According to the viable count, polysaccharide yield, and the skincare efficacy in cell models, we selected the crude polysaccharides from S. involucrata fermented by L. sakei GD17-9 and L. reuteri CCFM8631 to validate the skincare efficacy in the animal model. The results showed that the crude polysaccharides from S. involucrata fermented by L. reuteri CCFM8631 had better skincare efficacy. [Conclusion] L. reuteri CCFM8631 was the most suitable strain for the production of crude polysaccharide from S. involucrata, considering the viable count, the polysaccharide yield, and the skincare efficacy in cell and animal models.