母乳中活性多肽测定及对坏死性小肠结肠炎的预防作用

Determination of active polypeptides in breast milk and its preventive effect on necrotizing enterocolitis

目的对母乳中活性多肽进行生物学分析，寻找具有抑菌活性的多肽，探讨母乳中活性多肽对坏死性小肠结肠炎的防治作用。方法采集6例早产儿的身体健康母亲产后2～5d的母乳5mL／次，共6次，混匀，运用超滤截留法分离早产儿产妇乳汁中的活性多肽，串联质谱法对分离的活性多肽进行分析，生物信息学软件筛选出具有生物学功能的多肽，再应用蛋白功能聚类在线分析软件，根据其前体蛋白的生物学功能预测出与感染有关的多肽。选用坏死性小肠结肠炎常见致病菌大肠杆菌菌株一大肠杆菌标准菌株（ATCC25922）悬液，用头孢哌酮舒巴坦钠溶液，进行纸片法药敏试验和杀菌动力学试验，对乳汁中的活性多肽的抑菌效果进行验证。结果母乳中含有的4311条多肽被成功鉴定，其中1370条肽为非差异多肽，具有生物学活性的多肽188条，其中有11条肽与感染相关。母乳中内含肽复合物药敏纸片、杀菌动力学试验对大肠杆菌均有抑菌活性。结论母乳中活性肽复合物具有抑菌活性，是母乳预防坏死性小肠结肠炎的重要环节。期待11条与感染相关的肽中蕴含具有体内外生物活性，对坏死性小肠结肠炎有预防作用的抑菌肽。

Objective To analyze the biological activity of bioactive peptides in human breast milk and to find the polypeptides so as to investigate the preventive and therapeutic effects of breast milk - derived bioactive peptides on neonata necrotizing enterocolitis（NEC）. Methods Six mothers who gave birth to preterm neonates were enrolled in this study and 5 mL of their breast milk secreted within 2 - 5 postnatal days were collected for 6 times and blended sub- sequently. Bioaetive peptides from maternal milk of the preterm infants were separated by uhrafihration and analyzed by using tandem mass spectrometry. Polypeptides possibly with biological function were screened out by using bioinformatics software and the protein function cluster online analysis software was used to predict the polypeptides associated with infection according to the biological function of their precursor proteins. The ATCC25922, an Escherichia coli strain commonly associated with infection in NEC and drug solution （sulbactam/cefoperazone） were used to conduct the drug susceptibility testing and bactericidal kinetics testing, so as to verify the antibacterial effects of bioactive peptides in the breast milk. Results Four thousand three hundred and eleven peptides contained in breast milk were identified successfully,of which 1 370 were non- differential peptides, and 188 peptides possibly with biological activity and 11 peptides were associated with infection. The peptide compound in the breast milk had antimicrobial activity and bacteri- cidal power against Escherichia coli. Conclusions The active peptide compounds in the breast milk have antimicrobial activity,which play an important role in the prevention of NEC. Finding the true antimicrobial peptides with in vivo and in vitro biological activity by using antimicrobial spectrum test is expected.