Screening Of Fucoidanase-producing Strains And Preparation Of Low Molecular Weight Fucoidan By Enzymatic Hydrolysis

Fucoidan is a natural marine functional resource which has a wide range of physiological activities due to its complex and variable chemical composition.However,it is difficult to be absorbed due to the large molecular weight of natural rockweed polysaccharides,so we need to find a suitable way to reduce its molecular weight.Currently,enzymatic hydrolysis has become an ideal hydrolysis method because of the advantages of mild degradation conditions,being friendly to the environment,and being easy to control the molecular weight of the products.Because most of the known fucoidanase have low enzyme activity and it is difficult to produce them on a large scale,the search for new strains that produce fucoidanase with higher enzyme activity has become an important problem to be solved.This study aimed to screen a new strain of fucoidanase-producing enzyme,prepared low molecular weight fucoidan of different molecular weights by enzymatic method,and explored the effect of enzymatic hydrolysis on the biological activity of fucoidan.This study included the following:（1）In this study,70 strains that produced fucoidanase were initially screened by using fucoidan as the sole carbon source.The strains were subjected to solid-state and liquid-state fermentation,and the fucoidanase activity was determined.It was found that strain W1207 had the highest fucoidanase activity of（0.32±0.01）U·mL^-1 under solid-state fermentation conditions.And the results of high-performance gel permeation chromatography showed that the fucoidanase from this strain could effectively reduce the molecular weight of fucoidan and produce low molecular weight fucoidan with a molecular weight size of about 2 kDa.The strain was identified by morphology and molecular biology as Aspergillus chevalier WA1207.（2）The solid-state fermentation conditions of A.chevalier WA1207 were optimized using a single-factor method.The results showed that the fungus had the highest enzyme activity of（0.41±0.02）U·mL^-1 when incubated at 10 g of bran,0.1 g of fucoidan,pH 5.0,70%(v·w^-1)water content,2.5%(v·w^-1)inoculum,and incubating at 35°C for 5 days,which was 31.43%higher than before optimization.（3）In order to further improve the yield of low molecular weight fucoidan,the enzymatic hydrolysis conditions were optimized.According to the results,the highest yield of low molecular weight fucoidan was 52.85%with the substrate concentration of 15 g·L^-1,pH 6.0,the enzymatic hydrolysis temperature of 55°C,and the enzymatic solution time of 24 h.And the sulfate content was not changed during the hydrolysis process,which means no desulfurization occurred during the hydrolysis process,which indicated that the enzyme could mildly hydrolyze fucoidan.（4）The preparation of enzyme hydrolysis products was carried out in bulk using crude enzyme solution under optimal enzyme hydrolysis conditions.Four products（F1,F2,F3 and F4）with different molecular weights were obtained from the hydrolysate after alcohol precipitation,protein removal,dialysis,ultrafiltration classification,and freeze-drying.And then their chemical compositions were analyzed.It was found that four products were mainly composed of L-fucose and galactose;The weight-average molecular weights of F1,F2,and F3are relatively small,all below 10 kDa,which are 1 250 Da,2 505 Da,and 8 227 Da,respectively,and the weight average molecular weight of F4 is 40 554 Da,which is relatively large.（5）In order to verify the effect of enzymatic hydrolysis on the activity of fucoidan,the antioxidant activity and probiotic growth capacity of the samples before and after hydrolysis were measured separately.Antioxidant results showed that all four products had better antioxidant activity than F0.Among them,the effect of molecular weight on the DPPH radical scavenging ability of the products was small,and the scavenging ability of the products did not differ much from each other.In the hydroxyl radical scavenging ability,the molecular weight was negatively correlated with the scavenging activity of the products.The probiotic growth experiments showed that the proliferation effect of F0 on three strains of Lactobacillus was not obvious and appeared to be inhibited at high concentrations,the proliferation effect of the four products was better,and the density of the bacteria was all significantly increased.