Co-production,Structural Identification And Activity Evaluation Of Polysaccharide And Polypeptide From Oyster Crassostrea Rivularis

Crassostrea rivularis is a marine bivalve mollusc of the order ostreidae in the order lamellibranchia,which is one of the four major cultured shellfish in China.There are a large number of artificial cultivation in coastal areas such as Guangdong,Guangxi,Fujian,Shandong,Zhejiang and so on.Their meat is delicious,nutritious,rich in protein and carbohydrates,because of its high zinc content,iron,calcium and other elements,known as the"sea of milk".Traditional Chinese medicine lables the oyster as town of nerves,zi Yang Yin,soft firm fights,having certain curative effect for insomnia,labor spleen,impotence,lumbago,ren and other disease.In 2017,the output of oyster industry in China reached 4.879 million tons,which is an important economic shellfish in China.However,except for direct consumption,oyster products in China are mostly directly entered into the food market through simple processing.Research on bioactive substances is the leading direction of marine research.Currently,studies on the extraction and separation of marine active ingredients at domestic and overseas is hotpoint.Relevant research reports show that oyster polysaccharides have a variety of medicinal functions.Natural marine active peptides have high stability,but the research on bioactive peptides in marine fish,shrimp and shellfish is still in the laboratory stage.The main contents and results of this study are as follows:1.We explored the best conditions of co-production of polysaccharide and polypeptide from Crassotrea rivularis by response surface methodology.Experiment was used to find out the effect of single factors which were hydrolysis time,enzymolysis proportion and solid-liquid ratio on the degree of hydrolysis,polypeptide content,glycosaminoglycan content and polysaccharide content.Then three mathematical models were set up by Design Expert 8.0.6.With three response values including polypeptide content,glycosaminoglycan content and polysaccharide content,the three-factor and three-level response surface analysis was set up.The results show that the optimal enzyme dosage are of 0.58%?Mass Fraction?Alcalase and 0.22%?Mass Fraction?Trypsase,the solid-liquid ratio is 1:3.6 and enzymolysis time is 2.0 h.Under these conditions,repeated experiments were carried out to determine the yield of rough polysaccharide in whole organ of Crassostrea rivularis was 21.29%,which has 85.15%polysaccharide and 20.02%glycosaminoglycan and the proportion of polypeptide was 0.44%.Also the yield of rough polypeptide was 31.41%,which has 84.03%polypeptide and 0.11%glycosaminoglycan and the proportion of polysaccharide was 5.68%,which was basically the same as the predicted values.2.The oyster hydrolysate was divided into three groups including 0⁸ kDa?CRRS-A?,8²00 kDa?CRRS-B?,200 kDa⁰.2?m?CRRS-C?,and the determination of components'content,molecular weight distributions?M_W?,antioxidant activities and basic structure of each group were studied.The results show that the CRRS-A was polypeptide with the content of?87.26±1.65?%,the Mw was less than 2 kDa and made up with 10 compounds.It has highest antioxidant activities(the DPPH free radical scavenging ability and hydroxyl radical scavenging ability were high,the superoxide radical scavenging ability was higher than that of antioxidant glutathione?GSH?significantly,reaching?116.67±3.22?U/g.The CRRS-B contained polysaccharide for?75.82±0.28?%,including glucuronic acid?5.06±0.60?%,methyl pentose?31.23±1.29?%,sulfate radical?23.47±0.7?%,glucosamine hydrochloride?2.29±0.04?%.Its'Mw was less than 150 kDa,assumed pyranose carbohydrate with high antioxidant activities between BHT and GSH.The CRRS-C was pyranotype glycoprotein with a molecular weight of7152 kDa and has poor antioxidant activity in vitro.Fractional ultrafiltration system can separate Crassostrea rivularis effectively and the membrane separation components of enzymatic hydrolysates from Crassostrea rivularis have high antioxidant activity in vitro.Above all can provide theoretical basis for the high value utilization of Crassostrea rivularis.3.For the purification of 8²00 kDa oyster enzymolysis,CRP-1³,after determination of the physical and chemical properties that CRP-1 total sugar content was?32.40±0.89?%and molecular mass was 124.5 kDa,composed by mannose,ribose,glucosamine,glucuronic acid,galacturonic acid,glucose,galactosamine,galactose,arabinose,which the ratio was 5.55:0.32:9.08:0.47:0.66:48.13:3.57:8.53:0.30.The total sugar content of CRP-2 was?78.50±1.90?%and the relative molecular weight was67.0 kDa.CRP-2 was composed of mannose,ribose,glucosamine,glucose,galactose and galactose.The content ratio of the six monosaccharides was 7.79:0.41:12.69:32.08:4.98:5.92.The total sugar content of CRP-3 was?73.46±2.19?%,and the relative molecular weight was 8.3 kDa,which was composed of glucosamine,galactose acid and glucose.The content ratio of the three monosaccharides was 8.91:5.71:10.15.Fourier transform infrared spectroscopy?FTIR?showed that CRP-1 was a polysaccharide compound of polyuronyl pyranoside.CRP-2 is a polyketo unsaturated polysaccharide compound;CRP-3 is a polyketo-containing carboxyl polyunsaturated polysaccharide compound.4.The antioxidative activity of hydrolysate peptide from oyster Crassostrea rivularis was investigated.Separations were performed with an 0⁸ kDa membrane,Sephadex G-25 gel filtration chromatography,and semi-prep reversed-phase liquid chromatography.Five purified peptides exhibited high radical scavenging activities in the following order:F5>F3>F1>F2>F4.Among these,the highest activities were observed for DPPH-IC₅₀=?21.75±7.08??g/mL,hydroxyl-IC₅₀=?18.75±6.49??g/mL,and Superoxide-IC₅₀=?11.00±25.42??g/mL and the reducing power was?2.54±0.17?OD at 700 nm.The amino acid sequences of the five peptides were analyzed by ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry with sequences F1?Gly-Trp-Val-Asp-Tyr?,F2?Ser-Met-Phe-Arg-Phe-Tyr?,F3?Tyr-Arg-Ile-Pro-Glu?,F4?Arg-Lys-Arg-Trp-Pro-Lys?,and F5?Tyr-Ala-Lys-Arg-Cys-Phe-Arg?,which have molecular weights of 652.76,850.07,676.82,870.14,and943.22 Da,respectively.5.Using RAW264.7 macrophage line from mouse,we demonstrated that CRRS-A and CRP-1³ from oyster Crassostrea rivularis have immunoregulatory activity to macrophages,which can enhance its phagocytosis to neutral red and FITC-labeled Escherichia coli,induce the secretion of NO?TNF-??IL-6?IL-1?and lead mRNA expression with genes of iNOS?TNF-??IL-6?IL-1?.And the ability of the pure polysaccharide from Crassostrea rivularis is that CRP-3>CRP-1>CRP-2.