In Vitro Antioxidant Activities Of Squid(Todarodes Pacificus)Viscera Autolysates And Purification And Application Of Squid Viscera Proteases

Squid viscera is one of the by-products produced in the manufacturing process of squid, belongs to the inedible part, it nearly accounts for 15% of the wet weight, is the by-product of the process of most during the squid processing. Squid viscera in large-scale enterprises is usually made into squid dissolving pulp and then on sale, while small enterprises would like to bury it or discard it casually. The handling is serious waste of squid visceral this precious resource, and is unfavorable to the local ecological environment, does not conform to the sustainable development strategy in our country. In order to solve this problem, this dissertation mainly studied:(1) the squid visceral autolysates' nutritional characteristics and in vitro antioxidant activities, purification and identification of antioxidant peptide peptides in SVAs;(2) isoelectric solubilization/precipitation(ISP) was used to extract squid visceral proteases, enzyme type and molecular weight was studied;(3) the physical and chemical properties of the squid visceral proteases, including enzyme optimum temperature and the optimum p H, enzyme to temperature, p H and salinity tolerance;(4) using squid visceral proteases to hydrolysis yellow carp(Se Pti Pinna taty) protein, the antioxidant activity in vitro of the yellow carp hydrolysates was studied.According to the nutritional evaluation of SVAs, its protein content and soluble peptides content is high, amino acid composition is reasonable, the first limiting amino acid is cysteine + methionine, the other essential amino acids were higher than the values of the FAO/WHO recommended. Its DPPH free radical, hydroxyl radical scavenging ability and reducing power were higher than that of L- carnosine under the same concentration. The results implied the SVAs has the potential to develop into functional additive. By Sephadex G25 gel chromatography technology and RP – HPLC, four components were harvested, and SVAs 3 exhibited the best antioxidant activities in vitro; by ESI/MS/MS technology, six peptides SVAs3-?(LLAPPER), SVAs3-?(FPGLADR), SVAs3- ?(WVAPLK), SVAs3- ?(FFNPVH), SVAs3- ?(FNVVLK), SVAs3- ?(LELPLK) were identified from SVAs3. Six peptides showed antioxidant activities more or less, and SVAs3-? and SVAs3-? had the best DPPH radical-scavenging ability, SVAs3-? and SVAs3-? showed the best hydroxyl radical-scavenging ability, the reducing power of SVAs3-? was much better than the other five peptides in 5 mg/m L(7.02 m M)(p < 0.05).In this paper, the method of ISP was used to extract the proteases from squid viscera. Studies showed that the best condition to obtain proteases from squid viscera by ISP was p H 3.00. After ultrafiltration, enzyme activity of the proteases from squid viscera reached 1.06 U/m L, specific activity reached 8.83 U/mg. Compared with the crude enzyme, the proteases purified was about 3.96 times, protein recovery was 54.55%. SDS-PAGE showed that the purified enzymes contained at least two kinds of proteins, their molecular weight were 45 k Da and 28.06 k Da, respectively, and zymography indicated that it contained at least one kind of protease, with molecular weight of 45 k Da. UPLC-ESI-MS/MS showed that the protease was cathepsin D [Todarodes Pacificus], with molecular weight of 42.748 k Da.The physical and chemical properties of squid visceral protease studies showed that the squid visceral protease of optimum temperature is 60 ?, the best p H is 3.00, the enzyme with temperature range of 30-50 ? and p H with range of 1.00-5.00 was much stable. In addition, the enzyme in Na Cl concentration of 8%, maintained its 80% of the initial enzyme activity after 60 min incubation. Application test showed that yellow carp hydrolysate in the degree of hydrolysis of 8.63% had the best DPPH radical-scavenging ability, and the hydrolysates under the degree of hydrolysis of 15% had the best hydroxyl radical-scavenging ability and reducing power, were 83.69% and 0.78 respectively. Therefore, squid visceral proteases have application value, can be taken as a potential way to make full use of squid viscera.