【作者】 张军；

【导师】 刘光明； 林雄水；

【作者基本信息】 集美大学 ， 食品加工与安全（专业学位）， 2022， 硕士

【摘要】 刺麒麟菜（Eucheuma spinosum）含有丰富的海藻多糖,而传统方式提取制备的多糖因溶解性差、粘度高、分子量大等问题导致生物利用度差、生物活性低。益生菌发酵法被报道能显著降低多糖的分子量并提高生物活性。因此本研究利用益生菌发酵法制备发酵刺麒麟菜多糖（Fermented Eucheuma spinosum polysaccharides,F-ESP）,以低温冻融法制备的冻融刺麒麟菜多糖（Low temperature freeze-thaw Eucheuma spinosum polysaccharides,L-ESP）为对照,探究发酵对多糖化学成分、物理性质、链环结构的影响,并探究F-ESP的抗食物过敏活性及作用机理。利用清酒乳杆菌亚种（BNCC185970）和鼠李糖乳杆菌（BNCC185356）分别发酵刺麒麟菜,得到两种发酵粗多糖。利用DEAE-52纤维素柱对两种发酵粗多糖进行纯化,收集2 mol/L Na Cl洗脱部分命名为F1-ESP-3和F2-ESP-3。与L-ESP的纯化组分L-ESP-3对比发现,F1-ESP-3和F2-ESP-3的溶解性增强、表观粘度降低、分子量降低至28.30 k Da和33.58 k Da;F1-ESP-3和F2-ESP-3链环结构相似,主要由（1→3）-β-D-半乳糖和（1→4）-3,6-内醚-α-D-半乳糖糖苷键连接,均为含3,6内醚半乳糖残基的α硫酸吡喃糖。体外肥大细胞模型结果显示,L-ESP-3、F1-ESP-3和F2-ESP-3均能抑制大鼠嗜碱性粒细胞（Rat basophilic leukemia-2H3,RBL-2H3）和小鼠骨髓来源肥大细胞的脱颗粒。相较于F2-ESP-3和L-ESP-3,F1-ESP-3在200μg/m L浓度下对RBL-2H3和小鼠骨髓来源肥大细胞的脱颗粒抑制率分别高达43.33%±2.72%和40.93%±3.01%。F1-ESP-3能显著抑制RBL-2H3细胞Ca²⁺内流和细胞膜蛋白F-actin的解聚和异构,并显著减轻由肥大细胞介导的小鼠被动皮肤过敏症状,提示F1-ESP-3具有更好的体内抗过敏活性。进一步构建小鼠卵清蛋白食物过敏模型,对F1-ESP-3的体内抗食物过敏活性进行评价。结果显示,F1-ESP-3能够缓解卵清蛋白引发的小鼠食物过敏症状;显著降低小鼠血清中卵清蛋白特异性免疫球蛋白E、肥大细胞蛋白酶和组胺的水平;减少小鼠脾脏树突状细胞和肥大细胞的比例。此外,F1-ESP-3能够保护小鼠肠道屏障调节小鼠肠道拟杆菌门和厚壁菌门的丰度,上调毛螺菌属的水平,并影响核苷酸寡聚化结构域样受体、丝裂原活化蛋白激酶通路、核转录因子-k B抑制因子和抗原处理及递呈等信号通路进一步改善食物过敏小鼠肠道微环境。综上所述,通过益生菌发酵制备的刺麒麟菜多糖具有溶解性好、粘度低、分子量小等特点。F1-ESP-3能够通过稳定肥大细胞、减少脾脏肥大细胞比例、改善肠道微环境等作用发挥抗食物过敏活性。本研究结果可为海藻多糖抗过敏功能性食品的开发提供理论依据。更多还原

【Abstract】 Eucheuma spinosum is rich in seaweed polysaccharides,but the polysaccharides extracted by traditional methods have poor bioavailability and low biological activity due to poor solubility,high viscosity and large molecular weight.Probiotic fermentation has been reported to significantly reduce the molecular weight of polysaccharides and improve biological activity.Therefore,this study used probiotic fermentation method to prepare fermented Eucheuma spinosum polysaccharides（F-ESP）,Low temperature freeze-thaw Eucheuma spinosum polysaccharides（L-ESP）prepared by low temperature freeze-thaw method was used as control.Its chemical composition,physical properties and chain structure were analyzed,and its anti-food allergy activity and action mechanism are compared and analyzed.Two kinds of Eucheuma spinosum polysaccharides were obtained by fermentation of Eucheuma spinosum with the selected subspecies of Lactobacillus sakei subsp.sakei（BNCC185970）and Lactobacillus rhamnosus（BNCC185356）.After purification,the part eluted by 2 mol/L Na Cl was collected and named F1-ESP-3 and F2-ESP-3.Compared with the purified component L-ESP-3,it was found that the solubility of F1-ESP-3 and F2-ESP-3 was enhanced,the apparent viscosity was reduced,and the molecular weight was reduced to 28.30 k Da and 33.58k Da;F1-ESP-3 and F2-ESP-3 have similar chain structures.They are all contained 3,6-anhydro galactose residuesα-Pyranose sulfate,mainly composed ofβ-（1→3）-linked D-galactopyranose residues andα-（1→4）-linked 3,6-anhydro-D-galactopyranose.The results of mast cell model in vitro showed that L-ESP-3,F1-ESP-3 and F2-ESP-3 could inhibit the degranulation of rat basophilic leukemia-2H3（RBL-2H3）and bone marrow mast cells.When 200μg/m L of F1-ESP-3 is added,the degranulation inhibition rate of RBL-2H3 and bone marrow mast cells was the highest,reaching 43.33%±2.72%and 40.93%±3.01%respectively.F1-ESP-3 can significantly inhibit the Ca²⁺influx of RBL-2H3 cells and the depolymerization and isomerization of cell membrane protein F-actin.F1-ESP-3 can significantly reduce the symptoms of mast cell-mediated passive skin allergy,indicating that F1-ESP-3 may have better anti allergic activity in vivo.Further construct the mouse ovalbumin food allergy model,and evaluate the anti-food allergy activity of F1-ESP-3 in vivo.The results showed that F1-ESP-3 could alleviate the food allergy symptoms of mice caused by ovalbumin,such as hypothermia and loose stool;The levels of ovalbumin specific immunoglobulin E,mast cell protease and histamine in the serum of sensitized mice were significantly reduced;Reduce the proportion of dendritic cells and mast cells in mouse spleen.In addition,F1-ESP-3 can protect the intestinal barrier of mice and further improve the intestinal microenvironment of food allergic mice by regulating the abundance of Bacteroidetes and Firmicutes.F1-ESP-3 can further improve the intestinal microenvironment of food allergic mice by up regulating the levels of Lachnospiraceae,and affecting the signal pathways such as nod like receptor,MAPK,I kappa B and antigen processing and presentation.To sum up,Eucheuma spinosum polysaccharide prepared by probiotic fermentation has the characteristics of good solubility,low viscosity and low molecular weight.F1-ESP-3 can stabilize mast cells and exert anti-food allergy activity by regulating intestinal flora and spleen lymphocyte clustering in vivo.The results provide a theoretical basis for the development of seaweed polysaccharide anti-allergy special medical food.更多还原