Preparation,Structural Characterization And Anti-Fatigue Activity Of Glycoprotein From By-Products Of Hairtail

In the process of hairtail processing,by-products such as fish head,viscera and bone are produced,accounting for about 50% of raw materials.These by-products contain rich protein content and high nutritional value.Glycoprotein is a kind of binding protein formed by the covalent bond between the sugar chain and the polypeptide chain.It has the functional properties of anti-oxidation,anti-fatigue,hypoglycemic,cholesterol-lowering and so on.It has broad application prospect in the development of functional food.In this study,hairtail by-products glycoprotein was extracted by ultrasonic-assisted salt extraction.Response surface design was adopted to optimize the extraction conditions of glycoprotein,and the composition and antioxidant activity of glycoprotein were determined.On this basis,animal experiments and metabolomics were used to evaluate the anti-fatigue efficacy of glycoprotein from by-products of hairtail.The membrane separation technology was used to separate and purify glycoprotein components and to study their physical and chemical properties and structures,in order to provide theoretical basis for the development and application of hairtail by-products glycoprotein in functional food excipients.The main research contents and results are as follows:(1)Optimization of preparation process of hairtail by-products glycoproteinThe results showed that the contents of moisture,ash,fat,total sugar and protein in by-products of hairtail were 75.16±0.34%,2.86±0.12%,6.86±0.07%,0.82±0.03% and 14.12±0.02%,respectively.The content of crude protein in hairtail by-products glycoprotein was high,it is a good source of protein.The hairtail by-products glycoprotein was prepared by ultrasonic-assisted salt extraction method,and the effects of different factors(Na Cl concentration,extraction time,extraction temperature,solid-liquid ratio,ultrasonic power and ultrasonic time)on the yield of glycoprotein were investigated.On the basis of single factor experiment,a response surface design with four factors and three levels was carried out.Through response surface optimization,the optimal preparation conditions of hairtail by-products glycoprotein processing were obtained as follows: Na Cl concentration was 0.4 mol/L,extraction time was 1.5 h,extraction temperature was 78℃,solid-liquid ratio was 1:10(g/m L),ultrasonic power was 525 W,ultrasonic time was 21 min.According to the above optimized preparation conditions,the yield of hairtail by-products glycoprotein was 9.20±0.2 mg/g.(2)Composition and antioxidant activity of hairtail by-products glycoprotein in vitroGlycoprotein from by-products of hairtail was extracted by optimized preparation technology,and its composition and antioxidant activity in vitro were determined.The protein content of hairtail by-products glycoprotein was 64.32±3.05% by coomath bright blue method and 9.63±0.13% by phenol-sulfuric acid method.Galactose,glucose,fructose and sucrose were detected by ion chromatograph,and the molar ratio was 4.04: 6.48: 4.22: 14.50.The results of amino acid composition showed that there were 17 kinds of amino acids in the hairtail by-products glycoprotein,and the content of essential amino acids accounted for 55.28% of the total amino acids.The free radical scavenging ability of hairtail by-products glycoprotein in vitro showed that ABTS radical scavenging rate,DPPH radical scavenging rate,superoxide anion radical scavenging rate,hydroxyl radical scavenging rate and total antioxidant capacity were 89.29±1.89%,64.77±0.56%,77.40±0.31%,43.15±0.90%,2.72±0.04 umol/m L respectively.The hairtail by-products glycoprotein contains rich amino acids and has good antioxidant ability in vitro test.(3)In vivo anti-fatigue activity of hairtail by-products glycoproteinBased on the studies on the components and antioxidant activities of hairtail by-products glycoprotein,the antifatigue activities of hairtail by-products glycoprotein were further verified by animal experiments in vitro.Shuttle test results showed that the number of active escape of mice in different dose groups was significantly higher than that in blank group after glycoprotein intervention.The open field test showed that the proportion of mice in the central region in different dose groups was higher than that in blank group.The results of weight-bearing swimming test showed that the exhaustive swimming time of mice after glycoprotein intervention was higher than that of mice in blank group,and the swimming time of medium and high dose groups was higher than that of positive control group.Compared with blank control group,SOD,CAT and GSH-Px activities were increased and MDA levels were decreased after glycoprotein intervention.The hairtail by-products glycoprotein can increase the content of liver glycogen and reduce the content of lactic acid and blood urea nitrogen.The results of liquid chromatography-tandem mass spectrometry(LC-MS)showed that the serum samples of mice treated by different groups were effectively separated,indicating that their blood metabolites were significantly different.Hairtail by-products glycoprotein can improve the learning and memory ability,activity and exercise endurance of mice,and play an anti-fatigue role by slowing down metabolism and increasing glycogen reserve.(4)Physicochemical properties and structural characterization of hairtail by-products glycoproteinThe target glycoprotein GHB-1 was isolated by membrane separation technology.The electrophoresis results showed a single band,indicating that the protein reached electrophoretic purity.The protein and sugar content of GHB-1 were determined.The results showed that GHB-1 was a protein-based glycoprotein,and the protein and sugar content accounted for 92.75±2.14% of the total.The results of thermal stability analysis show that the thermal denaturation temperature of GHB-1 is 89.4℃,compared with the non-glycosylated protein,the thermal stability is better.Infrared spectroscopy and circular dichrography showed that there were characteristic absorption peaks of protein and polysaccharide molecules in GHB-1,and the glycosides were mainly pyran type.The secondary structure of the protein was mainly random coil and α-helix structure.The O-glycopeptide bond was found in GHB-1by β-elimination reaction and hydrogen magnetic resonance spectroscopy,and the structure of glycopeptide residues was mainly β-type pyranose.Hairtail by-products glycoprotein of GHB-1 is a β-pyranoid glycoprotein with α-helix and random coil.