| P. undulata is a good source of protein with low fat. The yield of P. undulataincreased year by year. However, the products are mainly marketing fresh and lacking deepprocessing technology, which caused low efficiency of resource utilization and loweconomic benefit. Currently, adopting controlled enzymolysis technology preparingbioactive peptide is becoming a hot research. Therefore, P. undulata was used as rawmaterial in this paper, controlled enzymolysis technology, ultrafiltration, Sephadex G-25gel chromatography, HPLC et al. used to optimize enzyme hydrolysis, separate and purifyactive peptide in order to make full use of protein resources from P. undulata. The resultsof the research are as follows:(1) Extraction and characteristic of P. undulata protein were investigated tounderstand the composition and the properties of raw material. Results indicated that theproportion of each protein among total protein was myofibril protein> sarcoplasmicprotein> stromal protein; SDS-PAGE electrophoresis showed that myosinogen stripsdistributed widely between29.0kDa and200kDa; Myofibril protein strips mostly rangedfrom66.4kDa to200kDa and29.0kDa to38.0kDa; Stromal protein had obvious bandingin190kDa,97kDa,44kDa and29kDa.All of the three protein fractions contained higher contents of essential amino acidsand the amino acids which are associated with immune activity and antioxidant activity,including alkaline amino acid, hydrophobic amino acid and branched-chain amino acid;and the denaturation temperatures of sarcoplasmic protein, myofibrillar protein and stromalprotein were53.4℃,47.23℃and55.73℃, respectively.(2) The degree of hydrolysis and DPPH free radical scavenging rate were used asindex to screen the optimal protease. Results indicated that the DPPH radical scavengingactivity of the enzymatic hydrolysate prepared by Alkaline protease was significantlyhigher than other three proteases (p <0.05), with DPPH free radical clearance rate of44.22%; Hydrolysis parameters, including temperature, time and the ratio of alkalineprotease to P. undulata meat (E/S), were optimised by single factor analysis and responsesurface methodology design to optimize enzymatic hydrolysis conditions. The optimumconditions were obtained as follows: temperature,50℃; time,3h; E/S,4,000U/g.(3) There was a certain correlation between DPPH radical scavenging activity and immunomodulatory with a positive correlation coefficient of0.84. The optimum conditionof ethanol precipitation was90%ethanol solution with three times volum. Under thisconditions, the decreasing rate of polysaccharide was (89.21±2.63)%，and the recovery ofpeptides was (90.08±2.25)%; The effect of <5kDa fraction on peritoneal macrophagefunction was investigated. Compared with the blank control group, ultrafiltration treatment(The concentration is0.6mg/mL.) and <5kDa fraction (The concentration is0.6mg/mL.)can promote secretion ability of IL-6and NO of peritoneal macrophage significantly withthe concentration of0.6mg/mL. However, there was no significant effect on secretionability of NO. Compared with the fractions without ultrafiltration treatment,<5kDafraction has the stronger induced ability in peritoneal macrophage function, such asenhancing the phagocytic ability of peritoneal macrophage, promoting secretion ability ofIL-6and NO of peritoneal macrophage. This showed that the ultrafiltration can enrichactive peptide. Similar phenomena were observed in amino acid composition of thefraction without ultrafiltration treatment and <5kDa fraction. But the contents of Val, Metand Phe of <5kDa fraction were significantly higher than the fraction withoutultrafiltration treatment and the3kinds of amino acids were related to immune activity,which can also explain that compared with the fractions without ultrafiltration treatment;<5kDa fraction has the stronger induced ability in peritoneal macrophage function.(4) Four fractions were obtained by gel filtration on a Sephadex G-25column.Compared with the blank group, P2(The molecular weight ranged from757Da to2856Da)and P3(The molecular weight ranged from634Da to1229Da) fractions couldsignificantly enhance lymphocyte proliferation ability at a concentration of0.5mg/mL;Peptide spectrum analysis revealed that the composition the four fractions were notcomplicated, which indicated that the ultrafiltration fractions were separated effectively.The four fractions could enhance peritoneal macrophage function. Moreover, P2(Themolecular weight ranged from757Da to2856Da) and P3(The molecular weight rangedfrom634Da to1229Da) fractions could significantly enhance peritoneal macrophagefunction with high and low concentration (p <0.05). However, the effect of the fourfractions on NO secreting ability of peritoneal macrophage was worse than the phagocyticability and IL-6secreting ability of peritoneal macrophage. |
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