Effects Of Ejiao Peptide-iron Chelate On Intestinal Inflammation In Mice With Iron Deficiency Anemia And Its Microencapsulation

Iron deficiency anemia（IDA）is one of the nutritional diseases with the highest incidence in the world,which can directly lead to the decline of immune function.With excess iron intake,there is a risk of iron overload leading to multiple organ damage and increased intestinal inflammation.At present,the research on iron supplements focuses more on exploring the blood-replenishing activity,but there is a lack of corresponding attention on its effect on intestinal inflammation.Ejiao peptide-iron chelate（EPI）is the blood-replenishing active ingredient in Ejiao,which has a good blood-replenishing effect.In this study,EPI was used as the research object to evaluate the improvement effect of EPI on inflammation in IDA mice through non-targeted metabolomics,inflammatory factors and histological analysis,combined with intestinal iron concentration,gut microbiota sequencing and non-targeted metabolomics to explore the mechanism of EPI improving intestinal inflammation,and further guide the optimization of EPI according to this mechanism.First,by establishing an IDA mouse model,the improvement effect of EPI on inflammation in iron deficiency anemia mice was evaluated from the tissue and molecular levels.EPI blood tonic activity evaluation results showed that EPI could significantly improve the blood indicators RBC,HGB,HCT,MCV and MCH in mice（P<0.05）,and significantly reduce the organ index（P<0.05）,and had a good effect of iron supplementation,and shows a dose-dependent effect.Plasma non-targeted metabolomic analysis showed that at high dose levels,Fe SO₄ supplementation caused metabolic disorders,and the levels of inflammation-related metabolites arachidonic acid and thromboxane B2 increased,while EPI could effectively restore the metabolic disorders in IDA mice and improve the iron homeostasis.EPI can regulate the levels of serum immunoglobulins,blood cells,inflammatory factors（IL-6,IL-10,TNF-α,CRP）and their gene expressions to restore them to near normal levels.H&E staining analysis and real-time quantitative PCR showed that EPI could effectively improve the colonic mucosal barrier damage and inflammatory cell infiltration caused by IDA,and significantly increase the expression of tight junction proteins Occludin,Claudin and ZO-1（P<0.05）.This indicates that EPI has a protective effect on the colonic mucosal barrier.Among the different dose groups,the middle dose had the best improvement effect on various indicators of inflammation and metabolism;the improvement effect of EPI was better than that of Fe SO₄ at the same dose.Secondly,through the analysis of colonic iron concentration and oxidative stress level,combined with high-throughput sequencing of gut microbiota 16S r DNA and fecal non-targeted metabolome analysis,to explore the mechanism of EPI improving intestinal inflammation.The results showed that,compared with Fe SO₄,EPI significantly decreased iron concentration and oxidative stress level in the intestine（P<0.05）due to its higher relative bioavailability（123.5%）.The results of colon microbiota analysis showed that supplementation of EPI had better recovery effect on species diversity,and the middle does showed the best improvement;at high dose,compared with Fe SO₄,EPI could reduce the abundance of harmful bacteria（eg Helicobacter、Erysipelatoclostridium）in the gut microbiota,and increase the abundance of beneficial bacteria（eg.Bifidobacterium）to improve intestinal inflammation levels.The results of fecal metabolome analysis showed that EPI had a better effect on the metabolism of the body,and could effectively regulate the recovery of the levels of 257 differential metabolites caused by IDA,mainly involved in the regulation of histidine metabolism,β-alanine metabolism and other pathways.The correlation analysis between the results of KEGG metabolism prediction and the results of plasma and feces metabolomics showed that the effect of EPI in improving intestinal inflammation was related to the regulation of fatty acid metabolism pathways.Finally,according to the mechanism of EPI improving intestinal inflammation,single factor experiment and orthogonal experiment were used to modify EPI by microencapsulation,in order to prolong the release time of EPI and reduce intestinal iron concentration.The effects of the wall-to-core ratio,the ratio of soybean protein isolate（SPI）to pectin,and the stirring time on the embedding rate were investigated by single-factor experiments,and the most suitable wall-to-core ratio was 2:1,and the ratio of SPI to pectin was 5:5,the stirring time was40 min.The primary and secondary relationship of the influence of each factor on the encapsulation rate of EPI microcapsule core material was further investigated by orthogonal experiments,and the optimal parameter combination was obtained:the wall-to-core ratio was3:1,the ratio of SPI to pectin was 6:4,and the stirring time was 30 min,the embedding rate was 86.69%.Then,the physicochemical properties of the EPI microcapsules prepared under the optimal conditions were characterized.The results showed that the particle size distribution of the EPI microcapsules was narrower,with an average particle size of 119.50μm;the water absorption was significantly reduced to 25.64%±0.97%.Microencapsulation significantly reduced the Fe²⁺content released by EPI during simulated digestion in the gastrointestinal tract（P<0.05）,and significantly improved the chelation rate of EPI at 20-80°C（P<0.05）,indicating its improved digestive stability and thermostability.EPI microcapsules can reduce the residual iron content in feces and delay the peak time of intestinal iron content by 2 h.