Study On The Preparation And Physiological Activity Of Alginate Degradation Products

The development of marine resources and blue economy have become strategic choices for sustainable development in the world.And the deep processing of seaweed polysaccharides is of great significance for environmental protection and high value utilization of polysaccharide resources,so it has received wide attention.However,the biological activity of alginate is limited due to its large molecular weight,high viscosity and easy gelation,so the targeted preparation of active degradation products of alginate is one of the important means to improve its biological activity.In this paper,a scientific and accurate quantitative analysis method of alginate oligosaccharide was established using alginate as raw material.And the technological conditions for the efficient preparation of alginate oligosaccharide by the coupling of hydrothermal method and enzymatic method were optimized.On this basis,alginate active enzymatic products with different molecular weight ranges were prepared by degrading alginate with alginate lyase,and the effects of different molecular weights on its in vitro antioxidant activity and in vitro immune enhancing activity were investigated.The main research findings are as follows:A quantitative analysis method of alginate oligosaccharide based on ethanol fractionation precipitation-carbazole-sulfuric acid chromatography was established.And a preparation method that based on ethanol fractionation of alginate active enzymatic products with different molecular weight ranges was determined.On this basis,with the yield of alginate oligosaccharide as the evaluation index,the process conditions of alginate degradation by hydrothermal method,enzymatic method and hydrothermal and enzymatic coupling treatment were optimized through single factor experiments.Finally,the optimal conditions for the preparation of alginate oligosaccharide from alginate by hydrothermal and enzymatic coupling degradation were as follows:after 30 min of hydrothermal treatment at 100℃,15 U/g of alginate lyase was added,and the enzymatic hydrolysis time was 24 h.Under these conditions,the yield of alginate oligosaccharide was 34.02%;Compared to direct hydrothermal treatment,the yield increased by3.17 times,while compared to enzymatic treatment,the yield increased by 1.21 times.Therefore,the introduction of hydrothermal pretreatment in the preparation process of alginate oligosaccharide can significantly improve the degradation intensity of alginate.And explored the changes in the yield of each component of alginate degradation products with different molecular weights during the enzymatic hydrolysis preparation process,which has been rarely reported in literature.The effects of the molecular weights of the alginate enzymatic products on their in vitro antioxidant activities were investigated based on the results obtained by graded alcohol precipitation with heavy average molecular weights of 13.4 k Da,5.73 k Da and 3.85 k Da,respectively.Based on this,the effect of molecular weight of alginate enzymatic products on their in vitro antioxidant activity was investigated.Firstly,the ability of alginate enzymatic products to scavenge reactive oxygen species（DPPH·radicals,hydroxyl radicals,superoxide anions）in vitro was investigated through chemical methods.It was found that alginate enzymatic products with different molecular weights exhibited in vitro free radical scavenging activity.Secondly,using the H₂O₂induced macrophage RAW 264.7 oxidative damage model,the protective ability of alginate enzymatic products with different degrees of polymerization against cellular oxidative damage was measured,and the main pathways by which they exert antioxidant activity in the cell model were identified.The results showed that the molecular weight of alginate enzymatic products was closely related to its antioxidant activity in vitro,and the antioxidant activity of alginate oligosaccharide was the best.In an in vitro model of macrophage RAW 264.7,the immune enhancing activity of different molecular weight alginate enzymatic products was evaluated.And it was found that different molecular weight alginate enzymatic products could promote the secretion of NO,tumor necrosis factor-α（TNF-α）,interleukin 6（IL-6）and mouse toll-like receptor 4（TLR4）in RAW 264.7 cells.The molecular weight of 5.73 k Da fractions were significantly more capable of promoting cytokine secretion than other degradation fraction.The alginate enzymatic product with a molecular weight of 5.73 k Da was used as the raw material for component separation using DEAE Sepharose Fast Flow column chromatography,and further investigated the immune enhancing activity of the separated component.The unsaturated sugar components were qualitatively analyzed using elemental analysis.On this basis,the TLR4 blocker TAK-242 was introduced,and its effect on the immune enhancing activity of alginate enzymatic products was measured to determine the immune stimulation pathway through which alginate enzymatic product induce cascade reactions by inducing macrophage TLR4 secretion.In conclusion,alginate active degradation product is a bio-non-toxic in vitro antioxidant and alginate oligosaccharides have very significant in vitro antioxidant activity.Meanwhile,alginate active degradation products can promote the secretion of cytokines by macrophages RAW 264.7in vitro,which can be used as a health food or an auxiliary treatment of diseases.The results of the study provide a theoretical basis for the high value utilization of alginate.