Study On The Preparation Of Pinctada Martensi Immunomodulating Peptide And Its Immunity Function

Immunomodulating peptide can enhance the immunity of organism, immune cell activity, and strengthen resistance to disease-carrying germs, which exhibited many benefits as a low molecular weight, easily absorbed by the human body, weak immunogenicity, and so on. It was reported that the bioactive peptides generated by enzymatic hydrolysis methods usually came with biological stability issue in vivo, which was that it would only exhibit low biological activity due to the possible biodegradation in digestive tract through oral intake. To address the above issue, simulated in vitro digestion enzymolysis method was adopted to prepare Pinctada martensi immunomodulating peptide, and ultrafiltration, gel chromatography, ion exchange chromatography et al. were used for its separation and purification, while the productivity of oligopeptide and in vitro assays of cellular immune activity were selected as screening indexes. Meanwhile, the immune mechanism of bioactive peptides was also studied. Through these ways, Pinctada martensi immunomodulating peptide was obtained, which could provide a theoretical basis and technical support for the high value utilization of the material and industrialization development of immunomodulating peptide health-care product. The search results of this study were as follows:(1) Pinctada martensii was used as the raw material, its oligopeptide was obtained by simulating the conditions of the gastrointestinal tract digestive system. After hydrolyzed with pepsin, the hydrolysis process was optimized under the combined action of trypsin and chymotrypsin. According to the analysis of artificial neural networks, the outcome demonstrated that enzyme-to-substrate level of 4500U/g(trypsin/chymotrypsin ratio of 6:1), substrate-to-liquid ratio of 1:2 and reaction time of 3.5h were the optimal conditions to generate oligopeptide(200u-2000 u Mw). Under these conditions, a relatively high yield of pinctada martensii oligopeptide was obtained, which would lay a foundation for its further exploration and utilization.(2) Using ultrafiltration technology to separate Pinctada martensii hydrolysates, the basic composition and molecular weight distribution of separated fractions were studied, proven that ultrafiltration could give satisfactory separation results in the treatment of Pinctada martensii hydrolysates. Then the composition and content of amino acid of different ultrafiltration components were discussed, showed that there might be strong bioactive sequences among the ultrafiltration fractions.(3) Using in vitro assays of cellular immune activity as screening indexes, two immune activity peptides coded SP1 and SP2 were separated and purified by using Sephadex gel chromatography, ion exchange chromatography and Superdex Peptide chromatography. The sequences of these peptide were identified by ESI MS/MS as follows. The sequence of immunomodulating peptide SP1 was Ala-Arg /Pro-Met with molecular mass of 245.0 u, and the sequence of immunomodulating peptide SP2 was Val-Arg with molecular mass of 274.0 u. After multi-step separation and purification, immunomodulating peptide SP1 and SP2 were obtained, which showed excellent immunomodulating abilities in vitro, while peptide SP2 had a stronger effect than other separated factions.(4) The effects of Pinctada martensi immunomodulating oligopeptide on immunomodulatory activities in mice were investigated by oral administration. Results indicated that the oligopeptide could significantly enhanced spleen lymphocyte transformation(p<0.05), humoral immunity level(p<0.05), as well as T cellular immune response ability(p<0.01) in mice, while showed limited effects on its nonspecific immunity.