Isolation And Identification Of Intracellular Peptides From Lactobacillus Helveticus And Bioactive Activity In Vitro

This research established a method to isolate,purify and identify intracellular peptides from Lactobacillus helveticus.More than intracellular 20 peptides based on the milk proteins are identified by centrifugation,ultrasonic-broking,ultrafiltration,solid phase extraction,simulated gastrointestinal digestion and HPLC-MS-Biolynx identification,and 12 peptides has been identified as novel bioactive peptides unreported before.This complete and effective method can be used for identifying intracellular peptide in other matrices.Total results were listed as follows:(1)In order to make whole experiment more feasible,three key experimental points are researched deeply during the experiment,that are MTT assay viable count of Lactobacillus helveticus,determination of centrifugal conditions and detailed study on identification of intracellular bioactive peptides.(2)Basing on the method which combine with computer programs,Biolyxn software and HPLC-ESI-TOF-MS/MS,20 peptides from milk proteins are identified,including 12 unreported bioactive peptides.There are RLHSMKQGI?WNIPMGLIV?YELLCLNN?GLPQEVLNE?PIHNSL?DELQDKIH?GTQYTD?SAEP?FGYSGAFKCL?NQFYQKF?YVTA?YNGVFQE.(3)Anti-digestive and antioxidant activity of intracellular peptides were measured by free radical scavenging method(DPPH)and total antioxidant capacity assay(ABTS).Peptides GTQYTD?SAEP and VYVE have the best stability,and WNIPMGLIV?YELLCLNN?GLPQEVLNE?PIHNSL?FGYSGAFKCL,YNGVFQE are better,other peptides RLHSMKQGI,DELQDKIH are poorer.Peptide NQFYQKF is the poorest one in stability.We found that intracellular peptides WNIPMGLIV,YELLCLNN,GTQYTD,SAEP and FGYSGAFKCL have abilty of free radical scavenging,because their values of IC₅₀ were lower than 10 mg/ml.Besides,intracellular peptides WNIPMGLIV?YELLCLNN?GTQYTD?FGYSGAFKCL?YVTA?YNGVFQE showed high total antioxidant capacity,which total antioxidant value are more than 0.5.Peptides WNIPMGLIV?YELLCLNN and FGYSGAFKCL are approximately equal to capacity of Vitamin E in antioxidant capacity.