The present study here establishes a complete and effective method for isolating, purifying and identifying

extracellular and intracellular peptides, and also describes the characters and bioactivities of

peptides from fermented casein with Lactobacillus helveticus. Intracellular peptides are much larger in

quantity and more complex in composition than extracellular peptides, between which the correlation

reveals proteolytic and metabolic mechanisms.In addition, totally 241 different peptide sequences

were identified by Nano LC–MS/MS from casein (212) and Lactobacillus helveticus proteins (29). These

casein-derived peptides mostly originated from -casein, followed by S1-casein, -casein, and S2-

casein, and came from extracell (69) and intracell (143), in which common peptides have a total of

27. Forty-four of the identified peptides were previously described as bioactive, including angiotensinconverting

enzyme (ACE)-inhibitory, antioxidant, immunomodulating, antimicrobial, DPP-IV inhibitory,

antiamnesic and anticancer effects and so on. Thirteen peptides with the potential of some biological

activities are obtained, which were described in previous studies. A total of 47 novel peptides of 5 to 26

amino acids that were not disclosed were obtained. The new sources of natural bioactive peptides may

have the very high application value as potential new peptide drugs for treatment human diseases. The

product peptide DELQDKIHPF found in both extracell and intracell was quantitatively analyzed using the

MRM mode of UPLC-U3Q, 23.1 and 9.76 ng/mL, respectively. The quantitative analysis of the potential

bioactive peptide may also advance the production of peptide products in the future.