| Immunoglobulin G(IgG),as a kind of glycoprotein,is produced by the adaptive immune system and can be commonly found in the serum,tissue fluid,and breast milk of animals.However,while being exposed to pepsin in gastric juice of adult,the intact IgG would be divided into pFc’ and F(ab’)2 fragments,which can only bind antigen without other immunological functions.So oral exogenous IgG cannot fully exert its physiological effects in adults.Since the non-natural substances are highly cytotoxic during the Layer-by-Layer self-assembly(LbL)process,natural products such as polyphenols and proteins can protect the biological activity of IgG.Studies have shown that polyphenols can be used as crosslinking agents since polyhydric phenol structures of polyphenols have the unique physical and chemical capability of binding to proteins.In addition,as a protein that may withstand proteolytic degradation of gastric digestive enzymes,bovine serum albumin(BSA)presents another characteristic of being cleaved by intestinal enzymes.Therefore,the LbL multilayers microcapsule can be prepared based on BSA,(-)-Epigallocatechin gallate(EGCG),tannic acid(TA)and caffeoylquinic acid(CQA)for protecting IgG in stomach digestion and releasing in small intestinal tract.Though studies have demonstrated that sialylation of Nlinked glycan could directly convert IgG from an inflammatory antibody to an antiinflammatory mediator,the effect of sialylated IgG as a natural food additive on the intestinal flora is still ignoring.The dietary components containing sialic acid have an essential effect on the intestinal flora.Also,it can be inferred that sialylated IgG can promote the Bifidobacterium based on the analysis of carbohydrate-active enzymes related genes.Moreover,research has shown that probiotics,including Bifidobacterium,can relieve the symptoms of colitis.The first aim of this study is to develop a delivery system based on BSA and EGCG/3,4-diCQA/TA LbL multilayers microcapsules for protecting IgG in the stomach digestion and releasing it in small intestinal tract.The second aim is to investigate the effects of sialylated IgG on gut microbiota structure with anaerobic fermentation models of healthy humans and patients with colitis intestinal microflora in vitro and elucidate the mechanism of sialylated IgG as food additive effect on gut microbiota structure.The main research contents and results are presented as follows:1.Preparation of IgG-(BSA-EGCG/3,4-diCQA/TA)n microcapsule and its characterization in gastrointestinal tractThe LbL self-assembly microcapsule was prepared with BSA,EGCG,3,4-diCQA,and TA to protect IgG in the stomach and release it in the small intestine in this chapter.The results showed that three kinds of polyphenols were loaded in the EGCG,3,4-diCQA,and TA sequence to prepare BSA-polyphenols LbL membrane.The composition of IgG-(BSAEGCG/3,4-diCQA/TA)n microcapsule and its stability and releasing ability in gastrointestinal tract were evaluated.Also,by binding these three kinds of polyphenols to BSA,the thermal denaturation temperature and ordered secondary structure of the BSA-polyphenols microcapsules were increased,and the time of scavenging activity on ABTS free radicals was significantly prolonged.These findings suggest that the(BSA-EGCG/3,4-diCQA/TA)n microcapsule can protect IgG in food processing and stomach digestion and release it in the small intestinal tract for bioactive delivery.2.Effects of sialylated IgG on gut microbiotaIn this study,the effect of glucose,sialylated IgG,and non-sialylated IgG as only carbon source on gut microbiota was investigated in vitro fermentation.The results showed that sialylated IgG significantly promoted the proliferation of Bifidobacterium,while nonsialylated IgG resulted in a significant increase in the number of Lactobacillus.At the same time,the Firmicutes/Bacteroidetes value in group of sialylated IgG is closer to that of the healthy intestinal flora of feces than others,which means that sialylated IgG can maintain the original state of the intestinal flora from the perspective of energy intake.The effects of sialylated IgG and non-sialylated IgG on intestinal flora pH and SCFAs are significantly different.The content of acetic acid and butyric acid were significantly increased in the group with sialylated IgG,while non-sialylated IgG significantly increased the content of lactic acid.3.Mechanism of sialylated IgG in promoting the proliferation of Bifidobaterium in vitroIn this chapter,three strains named B.bifidum CCX 19061,B.breve CCX 19041,and B.longum subsp.infantis CCX 19042 were isolated.Furthermore,B.breve CCX 19041 and B.longum subsp.infantis CCX 19042 showed co-cultivation growth properties with B.bifidum CCX 19061 in a sialylated IgG-based medium,which was also supported by changes of free monosaccharides and N-glycan structure.These findings suggest the increase of Bifidobacterium in vitro fermentation is attributed to the commensal relationship of three bifidobacterial species through utilizing sugars released from sialylated IgG.4.Effect of DiCQAs on gut microbiota in patients with colitisIn this section,the effects on gut microbiota of sialylated IgG and non-sialylated IgG in vitro with samples from colitis patients were investigated.The results showed that the effects of each group on intestinal flora were significantly different.Compared with the intestinal flora of healthy volunteers,sialylated IgG can not only significantly promote the relative abundance of Bifidobacterium,but also improve the diversity of intestinal flora,the value of Firmicutes/Bacteroidetes,pH and SCFAs in the intestinal flora of patients with colitis.Although non-sialylated IgG has improved the intestinal flora of patients with colitis,it is not as effective as sialylated IgG.Therefore,sialylated IgG can be used as a prebiotic to regulate gut microbiota imbalance,inhibit the growth of non-beneficial bacteria,and then relieve and improve the symptoms of colon inflammation in patients with colitis. |
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