摘要Interactions between proteins/peptides and polyphenols have been widely investigated to improve their properties and functions.Previous studies have shown that covalent and non-covalent products exhibit different structural and bioactivity characteristics.The pentapeptide PAYCS(PS)was reported to be neuroprotective in amnesia mice,and catechin(CA)conjugation could improve its activity.However,different mechanisms on memory deficits alleviation between covalent and non-covalent conjugates were little investigated.In this study,the structure of PS-CA(PS and CA covalent conjugates)and PS+CA(PS and CA non-covalent complex)was characterized by using HPLC-MS/MS.Single peak for newly formed compound was identified in PS-CA and modification of tyrosine(Tyr)and cystine(Cys)was observed.In the APP/PS1 transgenic mice model experiments,behavioral tests showed that PS-CA and PS+CA could both exhibit better memory enhancing effects than that of PS.The 16S rRNA results mainly showed the alteration of gut microbiota,especially the changes in the Bacteroidota/Firmicutes ratio and significant changes in the abundance of Verrucomicrobiota.Additionally,the treatments of PS+CA and PS-CA could primarily regulate the contents of certain short chain fatty acids(SCFAs)and brain neurotransmitters,respectively.The quantitative polymerase chain reaction(qPCR)results indicated that the cholinergic system,neuronal apoptosis,and partial antioxidant pathways could be also regulated by both the treatment of PS-CA and PS+CA.Correlation analysis confirmed the relationship between the abundance of Verrucomicrobiota and other factors.The serum peptidomes results revealed that PAY contained peptides were mostly identified in PS+CA group.Especially,PS+CA showed better effects on regulating certain SCFAs and 5-hydroxytryptamine(5-HT)related neurotransmitters while PS-CA exhibited better alleviation effects on behavioral tests and neuroprotection.All these results suggested that covalent and noncovalent modification of PS with CA showed different neuroprotective effects.