Studies On Extraction, Purification And Antibacterial Properties Of Protamine From Squid

Protamine is a kind of polyphosphate cationic peptide which is existed in the testis ofa variety of animals. Generally speaking, This protein is combined into DNA of nuclearand has important applications in the fields of medicine and food. In this paper, theextraction processing of protamine was optimized from the by-products of Todarodespacificus Steenstrup—testis. Then, the purification of protamine was explored withmodern separation and purification technology. At last, the antibacterial properties andthe application in foods of this protein were studied preliminarily. The details are asfollows:1. Single factor and orthogonal array design experiments of squid protamineextraction conditions were studied. The results showed that the yield of squid protaminewas3.5%in the optimal condition of3times volume of0.3M sulfuric acidconcentration to extract protamine, repeating for2times, and3times volume of coldethanol. The squid protamine contained93%protein which amino acids accounted for81.70%and was rich in all kinds of amino acids in which basic amino acids accountedfor26.2%, accounting for1/3of the measured amino acids total. The three basic aminoacids(arginine, lysineand, histidine) accounted for9.61%,14.84%,1.75%respectively.2. Crude squid protamine extracted from squid testis was successfully separated andpurified by Sephadex G-50gel chromatography and CM Sepharose FF ion exchangechromatography. And then they were verificated with the help of specific argininereaction—Sakaguchi reaction and Tricine-SDS-PAGE. The results showed that thesecond peak of CM Sepharose FF ion exchange chromatography was the target proteinpeak, which protein contained four kinds of components, the molecular weight ofwhich were9.0kDa、11.0kDa、13.5kDa、21.0kDa respectively, in which the twocomponents with the molecular weight11.0kDa and13.5kDa accounted for the most.The four kinds of the target protein were also validated by the technology ofRP-HPLC.3. The antibacterial properties and the application test in food of this protaminewere studied. The results showed that the squid protamine had an obvious effect onthe Staphylococcus aureus and Salmonella sp. in antimicrobial aspects but had noinhibitory effect on the Rhizopus nigricans and Saccharomyces cerivisiae. This protein had a good effect on the antibacterial properties in neutral or alkalineconditions. High temperature treatment would diminish the antibacterial activity ofthis protein, so it could only be used in the low temperature pasteurization of food orfresh food preservation. Metal ions would affect its antibacterial activities. Within acertain concentration range, the antibacterial activity of this protamine had adownward trend with the rise of ion concentration and under the same conditions, thehigh-cost mental ions of its antibacterial activities were greater than the low-costmental ions. The study also showed that this protamine could be digested in intestinalby pepsin and trypsin enzyme and so on and lost its antibacterial properties which didnot threaten the growth of normal human intestinal microflora. In the experiments ofapplication in foods, this protein could extend the shelf life and freshness of fish andslow down its deterioration under the same conditions. What’s more, it also played apreservative effect according to inhibit the growth of microorganisms in oyster.