Preparation,Modulation Of Gut Microbiota And Anti-Inflammatory Activity Of In Vitro Glycosylated Bovine Immunoglobulin G And Fc Fragments

Immunoglobulin G(Ig G)is one of the principal biological active antibodies in bovine colostrum and one of the blood’s main antibodies.It is mainly responsible for identifying,neutralizing,and eliminating pathogens and toxic or harmful antigens.However,sialylated Ig G has been confirmed to show anti-inflammatory activity,and it could play a good role in relieving symptoms in rheumatoid arthritis and thrombocytopenic purpura(ITP)models.Besides,sialylated Ig G also has a specific function of regulating intestinal microbes.Our previous research has achieved a layer-by-layer self-assembly microcapsule with protein and polyphenol to protect Ig G from digestion.This study provides the possibility for the work of studying the effect of b Ig Gs on intestinal microbes.To lay scientific foundation for the nutritional value evaluation and product development of bovine immunoglobulin G(b Ig G),the methods of digesting bovine N-glycolylneuraminic acid(Neu5Gc)from b Ig G and ligating with human N-acetylneuraminic acid(Neu5Ac)were explored to realize the transformation from non-human-source sialic acids to human-source sialic acids of b Ig G.Then,the preparation conditions of the crystallizable fragment(Fc)were investigated.In addition,we also studied its biological activities: stimulating inflammation in the RAW264.7 cell by means of LPS to study the anti-inflammatory activity of the Ig G as mentioned earlier and its Fc fragment,and the anaerobic fermentation model of the intestinal flora of healthy volunteers is used to study the influence of the in vitro glycosylated Ig G and its Fc fragment on the structure of the intestinal flora.The main research contents of this thesis are as follows:1.In vitro glycosylation of bovine immunoglobulin G and preparation of Fc fragmentIn this section,we used the in vitro glycosylation method to prepare sialylated b Ig G.First,the Neu5 Gc residue of b Ig G was enzymatically digested with optimized neuraminidase hydrolysis conditions to avoid this non-human sialic acid form’s potential hazards.Then Two glycosyltransferases,B4GALT1 and ST6GAL1,were used to transfer galactose and Neu5 Ac to b Ig G.After optimizing and selecting the conditions for in vitro glycosylation of b Ig G by papain digestion,the Fc fragment of in vitro glycosylated b Ig G was prepared.We can obtain glycosylated b Ig G Fc fragment under this condition: 10 mg/m L b Ig G,0.05 mg papain/mg b Ig G,10 m M cysteine activator,2 m M EDTA solution,p H 7.0,enzymatically digestion of b Ig G for 3 h.The yield of b Ig G was 71.7%,and the yield of the Fc fragment was 20.8%.Analysis by UHPLC-MS showed that the sialic acid forms are Neu5 Ac.This research helps us to develop and utilize animal-derived food humanized products and the evaluation of the nutritional value of animal-derived products.2.The effect of in vitro glycosylated Ig G and its Fc fragment on the intestinal flora during in vitro anaerobic fermentationIn this section’s study,we firstly prepared the NG and SG samples,and secondly used an in vitro anaerobic fermentation model to study the effect of b Ig Gs on intestinal microbes.The results show that b Ig Gs can maintain the diversity of microorganisms to a certain extent.Compared with the BLK group,the GG,SG,GFc and NG groups can all achieve higher relative abundances of Megamonas and Lachnospiraceae＿unclassified genera,and significantly reduce the relative abundance of Bacteroides,Parasutterella and Dialister genera.GG and GFc can significantly reduce Bilophila and Escherichia-Shigella,while the relative abundance of Parabacteroides of SG is the lowest in the b Ig Gs samples;SG sample can significantly increase the total acids content.3.Anti-inflammatory activity of glycosylated Ig G and its Fc fragment in vitroIn this section of the thesis,the anti-inflammatory activity of sialylated b Ig Gs was studied using LPS to stimulate the RAW 264.7 macrophage inflammation model.The results showed that SG,GG and GFc samples have a specific inhibitory effect on the release of NO,but SG has a limited inhibitory effect on the m RNA expression of inflammatory factors and inflammation-related genes.GG and GFc can reduce the production of IL-6 and TNF-α,the reason can be the transcriptional inhibition of related genes,and they can inhibit the m RNA expression of COX-2 and i NOS to a certain extent.GG can also significantly inhibit IL-1βm RNA expression.Therefore,overall,the anti-inflammatory activity of GG is the most important.