【作者】 李婷；

【导师】 奚春宇；

【作者基本信息】 吉林大学 ， 食品科学与工程， 2023， 硕士

【摘要】 溃疡性结肠炎(Ulcerative colitis,UC)正严重威胁着青少年儿童的健康,利用碱性蛋白酶酶解蛋清制备的蛋清肽(Egg white peptides,EWPs)具有多种功能活性,如抗氧化、抗炎和调节肠道紊乱等,且已被证明可以预防成年小鼠的溃疡性结肠炎,但对幼鼠溃疡性结肠炎的调控作用尚不明确。本文以EWPs为主要研究对象,首先评估了不同批次间EWPs的批次稳定性,然后构建幼鼠结肠炎模型探究EWPs对幼鼠溃疡性结肠炎的调控效果,并通过16s rRNA测序技术和肠道菌群功能预测分析EWPs预防幼鼠溃疡性结肠炎的作用及其机理。全文的主要研究内容及结果如下:(1)以蛋清为原料,通过碱性蛋白酶酶解制备6个批次的EWPs(150～1000 Da),并对其氨基酸组成、肽序列、体外抗氧化活性进行表征及分子对接的研究。结果表明:6个批次的200 mg/m L的EWPs溶液中游离氨基酸的含量处于138.39±2.24～162.12±2.20μg/m L之间,酶解产物主要是肽。EWPs具有良好的抗氧化活性,批次间具有一定的稳定性。确定出6个批次共28条肽段,包括在批次间重复出现的四条肽段:QMDDFE、WDDDPTD、DEPDPL和FKDEDTQ。(2)将实验小鼠分成空白组(CK)、模型组(M)、蛋清处理组(EW)和EWPs低剂量处理组(EWPs L),EWPs中剂量处理组(EWPs M)和EWPs高剂量处理组(EWPs H),通过预先给予不同剂量蛋清肽干预,然后构建溃疡性结肠炎模型,探究EWPs对溃疡性结肠炎的预防效果。结果表明:EWPs处理可以提高实验小鼠血清氨基酸含量、体重和结肠长度。与M组相比,EWPs处理显著降低了实验小鼠血清中促炎细胞因子肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、IL-8和IL-1β的含量,提高了结肠组织中谷胱甘肽过氧化物酶(GSHPx)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性并降低了髓过氧化物酶(MPO)的浓度。同时改善了结肠炎小鼠结肠组织通透性增加和病理学损伤。综上所述,EWPs可通过调节炎症因子、氧化应激、肠道通透性以及紧密连接蛋白表达等,达到调控溃疡性结肠炎的作用效果。(3)基于16s r RNA和靶向代谢组学技术,分析CK、M、EW、EWPs L、EWPs M和EWPs H组中实验幼鼠的盲肠代谢物,对肠道菌群和肠道菌群代谢产物短链脂肪酸(Short chain fatty acids,SCFAs)进行研究。通过考察各组间的差异菌群和代谢产物,并将差异菌群、代谢产物和炎症因子进行相关性分析,探究EWPs调控幼鼠溃疡性结肠炎的作用机制。结果表明:EWPs的干预可以上调有益菌的丰度、下调有害菌的丰度,调节肠道紊乱,恢复SCFAs水平,起到调控幼鼠溃疡性结肠炎的作用。更多还原

【Abstract】 Ulcerative colitis(UC)is posing a serious threat to the health of adolescents and children.Egg white peptides(EWPs)produced by enzymatic hydrolysis of egg white with alcalase have various functional activities,such as antioxidant,anti-inflammatory,and regulating intestinal flora disorders.It has been proven to prevent UC in adult mice,but the regulatory role of EWPs on ulcerative colitis in young mice is unclear.This study takes EWPs as the main research object,first evaluating the batch stability of EWPs between different batches,then constructing a young mice UC model to explore the regulatory effect of EWPs on young mice,and analyzing the role and mechanism of EWPs in preventing UC in young mice through 16 s rRNA sequencing technology and functional prediction of intestinal flora.The main research content and results of the full text are as follows:(1)Six batches of EWPs(150～1000 Da)were prepared from egg white by enzymatic hydrolysis with alcalase.The amino acid composition,peptide sequence,in vitro antioxidant activity,and molecular docking were studied.The results showed that the content of free amino acids in six batches of 200 mg/m L EWPs solution ranged from 138.39 ± 2.24 to 162.12 ± 2.20 μg/m L,the enzymatic hydrolysis product is mainly peptides.EWPs have good antioxidant activity and have certain stability between batches.A total of 28 peptide sequences were identified from 6 batches,including four peptide sequences that repeatedly appeared between batches: QMDDFE,WDDDPTD,DEPDPL,and FKDEDTQ.(2)The experimental mice were divided into control group(CK),model group(M),egg white treatment group(EW),low dose EWPs treatment group(EWPs L),medium dose EWPs treatment group(EWPs M),and high dose EWPs treatment group(EWPs H).Different doses of EWPs were given in advance to intervene,and then a model of UC was constructed to study the preventive effect of EWPs on UC.The results showed that EWPs treatment could increase the serum amino acid content,body weight,and colon length of experimental mice.Compared with the M group,EWPs treatment significantly reduced the inflammatory factor tumor necrosis factor(TNF-α)in the serum of experimental mice,Interleukin-6(IL-6),IL-8,and IL-1β.The activity of glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD)in colon tissue was increased,and the concentration of myeloperoxidase(MPO)was decreased.At the same time,it improved the permeability increase and pathological damage of colon tissue in mice with UC.In summary,EWPs can achieve the effect of regulating ulcerative colitis by regulating inflammatory factors,oxidative stress,intestinal permeability,and tight junction protein expression.(3)Based on 16 s r RNA and targeted metabolomics techniques,the cecal metabolites of experimental young mice in CK,M,EW,EWPs L,EWPs M,and EWPs H groups were analyzed,and the intestinal flora and its metabolites,short chain fatty acids(SCFAs),were studied.To investigate the mechanism of EWPs in regulating UC in young mice,we investigated the differential flora and metabolites among groups,and analyzed the correlation between the differential flora,metabolites,and inflammatory factors.The results showed that the intervention of EWPs could upregulate the abundance of beneficial bacteria,downregulate the abundance of harmful bacteria,regulate intestinal flora disorder,restore the level of SCFAs,and play a role in regulating ulcerative colitis in young mice.更多还原