乳酸菌发酵及添加益生元对大豆蛋白调控肠道屏障和诱导口服耐受的影响

Effects of lactic acid bacteria fermentation and prebiotic supplementation on soy protein's regulation of intestinal barrier and induction of oral tolerance

摘要

[目的]本文旨在研究乳酸菌发酵及益生元添加对大豆蛋白影响肠道屏障稳态及调控免疫耐受的作用。[方法]采用乳酸菌发酵大豆分离蛋白(SPI)制备发酵大豆蛋白(FSPI)，分别添加 5 种益生元:低聚果糖(FOS)、低聚木糖(XOS)、水苏糖(SOS)、低聚半乳糖(GOS)和低聚异麦芽糖(IMO)，共发酵制备益生元组样品。通过体内动物试验比较不同样品对肠道屏障稳态的维持作用以及口服免疫耐受的诱导作用。试验分为空白对照组(PBS-)、致敏组(PBS+) 和 7 个耐受处理组(SPI、FSPI、F-FOS、F-XOS、F-SOS、F-GOS 和 F-IMO)。通过测定组胺、小鼠肥大细胞蛋白酶-1(mMCP-1)以及辅助型 T 细胞 Th1、Th2、Th17 型细胞因子评价小鼠的免疫反应性;通过测定肠道组织学、紧密连接(TJ)蛋白表达水平评价小鼠肠道屏障完整性;通过测定调节性 T 细胞(T_reg)、树突状细胞表面标志物(CD103、CD80 和 CD86)以及白细胞介素 10(IL-10)、转化生长因子 β(TGF-β)和视黄酸(RA)等免疫耐受相关因子的表达水平评价不同样品的耐受诱导作用。[结果] 与 PBS+组和 SPI 组相比，FSPI 与益生元处理均可显著下调组胺、mMCP-1、Th2 和 Th17 型细胞因子的表达水平(P<0.05)，从而降低小鼠的免疫反应性;益生元处理可显著上调 Th1 型细胞因子的表达(P<0.05)，有效恢复大豆蛋白引发的 Th1/ Th2 向 Th2 方向偏移的失衡现象;FSPI 与益生元处理组的肠绒毛损伤程度均减轻，肠绒毛高度和隐窝深度显著增加(P<0.05)，有效缓解了食物过敏导致的肠道绒毛损伤现象，但 FSPI 处理上调 TJ 蛋白和诱导免疫耐受的作用较弱;F-XOS 处理可显著上调 Claudin1、ZO-1 和 Occludin mRNA 的表达水平(P<0.05)。此外，F-FOS 和 F-XOS 可通过上调耐受细胞因子(IL-10、TGF-β 和 RA)和 CD103 的表达，下调 CD80 和 CD86 的表达，促进 T_reg细胞的生成，从而诱导口服耐受。[结论]FSPI 可降低小鼠对大豆蛋白的免疫反应性，维持小鼠肠道屏障完整性;添加益生元共发酵物后可进一步诱导口服耐受，F-FOS 和 F-XOS 的效果最佳。

Abstract

[Objectives] This study aimed to investigate the effects of lactic acid bacteria (LAB) fermentation and prebiotic addition on soy protein in maintaining intestinal barrier homeostasis and regulating immune tolerance. [Methods] Fermented soy protein (FSPI) was prepared by fermenting soy protein isolate (SPI) with LAB. Additionally, five different prebiotics, including fructooligosaccharides (FOS), xylooligosaccharides (XOS), stachyose (SOS), galactooligosaccharides (GOS), and isomaltooligosaccharides (IMO), were co-fermented with FSPI to create prebiotic-treated samples. In vivo animal experiment was conducted to compare the effects of different samples on intestinal barrier homeostasis and the induction of oral immune tolerance. The experiment included a negative control group (PBS-), an allergen-sensitized group (PBS+), and seven tolerance treatment groups, namely SPI, FSPI, F-FOS, F-XOS, F-SOS, F-GOS, and F-IMO groups. Immune responsiveness in mice was evaluated based on histamine level, mouse mast cell protease-1 (mMCP-1), and the expression of cytokines associated with T-helper Th1, Th2 and Th17 cells. The intestinal barrier integrity was assessed through histological analysis and the expression of tight junction (TJ) proteins. The induction of immune tolerance was comprehensively evaluated by measuring regulatory T cells (T_reg), dendritic cells surface markers (CD103, CD80, and CD86), and immune tolerance-related factors, including interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and retinoic acid (RA). [Results] Compared to the PBS+ and SPI groups, FSPI and prebiotic treatments significantly reduced the expression levels of histamine, mMCP-1, Th2 and Th17 cytokines (P < 0.05), thereby reducing immune reactivity in mice. The prebiotic treatments significantly upregulated Th1 cytokines (P<0.05), effectively restoring the Th1/ Th2 imbalance caused by soy protein allergy, which tends to shift towards the Th2 response. The FSPI and prebiotic-treated groups reduced intestinal villus damage, with significant increase in villus height and crypt depth (P<0.05), mitigating food allergy-induced intestinal villus injury. However, the FSPI group showed weaker effects in upregulating TJ proteins and inducing immune tolerance compared to prebiotic treatments. Notably, F-XOS significantly upregulated the mRNA expressions of Claudin-1, ZO-1, and Occludin (P<0.05). Furthermore, F-FOS and F-XOS promoted oral tolerance induction by increasing the expressions of tolerance-related cytokines (IL-10, TGF-β, and RA) and CD103, while downregulating CD80 and CD86, thereby facilitating the differentiation of T_reg cells. [Conclusions] FSPI could reduce the immune reactivity of mice to soy protein and maintain intestinal barrier integrity. The addition of prebiotics during co-fermentation further enhanced oral tolerance induction, with F-FOS and F-XOS demonstrating the most significant effects.

关键词

大豆蛋白 / 乳酸菌发酵 / 益生元 / 肠道屏障 / 口服耐受

Key words

soy protein / lactic acid bacteria fermentation / prebiotic / intestinal barrier / oral tolerance

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基金资助

国家自然科学基金项目(32372440)

江苏省农业科技自主创新项目(CX(24)3121)