Preparation Of Polysaccharides From Two Kinds Of Seaweed By Fermentation And Enzymatic Hydrolysis And Their Moisturizing And Antioxidant Activities

Copper algae and asparagus are important economic seaweeds which widely distribute and yield highly in China.These two kinds of seaweeds are rich in nutrients and active substances.Because of the characteristics of the seaweed polysaccharide,including excellent moisture absorption,moisture retention and oxidation resistance,it has a good application prospect in the cosmetics industry.However,there are still some issues,such as high cost and low efficiency of polysaccharide extraction.Studies have shown that the biological activity of seaweed polysaccharide is closely related to its molecular weight.The key to the industrial application of seaweed polysaccharide is to find a preparation method suitable for industrial production of seaweed oligosaccharides and establish the structure-activity relationship between molecular weight and activity of polysaccharide.Based on the screening of enzyme-producing strains,this paper used microbial fermentation coupled enzymatic hydrolysis to prepare oligosaccharides from copper algae and Gracilaria lemaneiformis.In order to provide reference for efficient,environmentally friendly and low-cost preparation of oligosaccharides from seaweed and for its application in cosmetics industry,here further investigated the relationship between the molecular weight of the two trehalose,moisture absorption and antioxidant activity.The main results of this paper are as follows:(1)The process of preparing two trehalose by hot water extraction coupled with chemical degradation.In this paper,seaweed polysaccharides were prepared by hot water extraction,alcohol precipitation and H₂O₂-ultrasonic combined reduction method using Gracilaria lemaneiformis and copper algae as raw materials respectively.The results showed that after soaking in hot water at 100?for 2 h,the yield of asparagus polysaccharide was12.13%and that of copper algae polysaccharide was 4.98%.After H ₂ O ₂-ultrasound combined degradation for 3 h,ultrafiltration was carried out to obtain polysaccharide components with average molecular weights of>10 kDa,3?10 kDa and<3 kDa.The yields of each component of Gracilaria lemaneiformis polysaccharide were 75.48%,5.27%and15.36%respectively.The yields of each component of copper algae polysaccharide were69.8%,6.32%and 20.38%respectively.(2)The process of preparing two kinds of seaweed polysaccharides by fermentation and enzymatic hydrolysis.This part of the process is divided into two types,the first is direct fermentation.The direct fermentation method is based on the detection of polysaccharide lyase activity,selecting strains with high enzyme activity for direct fermentation,and using the extracellular enzymes secreted by the strains themselves to achieve polysaccharide degradation.The second process is the method of first enzymolysis and then coupling strain fermentation enzymolysis.0.3%cellulase and 1.0%pectinase were used to pretreat the raw materials before fermentation.The results of the direct fermentation method showed that Gracilaria lemaneiformis was fermented by two strains of Pseudomonas(Pseudoalteromonas sp.70006)and Aspergillus niger(As3350),and the yields of polysaccharides with average molecular weights of>10 kDa,3?10 kDa and<3 kDa were 48.82%,16.38%and 29.63%,respectively.Aspergillus niger was fermented for 72 h,and the yields of three polysaccharides with average molecular weight were 49.7%,16.05%and 28.23%respectively.Using copper algae as substrate,direct fermentation,after 48 h fermentation with Pseudomonas,the fermentation broth was separated by membrane to obtain three polysaccharide components with average molecular weight>10 kDa,3?10 kDa and<3 kDa in yields of 78.33%,6.83%and 11.03%,respectively.Aspergillus niger was fermented for 96 h,and the yields of three copper algae polysaccharide components with average molecular weight were 73.5%,11.34%and 11.34%respectively.The results of the fermentation process of the coupled strain before enzymatic hydrolysis showed that the yields of Gracilaria lemaneiformis polysaccharide with average molecular weight>10 kDa,3?10 kDa and<3 kDa were 60.93%,10.77%and 24.38%respectively.After the hydrolysis of the two enzymes for 2.5 h and then fermentation by Aspergillus niger for 72 h,the yields of the three Gracilaria lemaneiformis polysaccharide were 56.64%,14.55%and 24.54%.Using copper algae as substrate,the yields of polysaccharide components with average molecular weights>10 kDa,3?10 kDa and<3kDa were 71.27%,10.95%and 12.38%,respectively.Enzymatic hydrolysis was 2.5 h coupled with Aspergillus niger fermentation for 72 h,and the yields of three polysaccharide components with different average molecular weights were 75.71%,8.29%and 8.12%,respectively.The analysis of the above results showed that under the same conditions,the effect of preparing low molecular weight Gracilaria lemaneiformis polysaccharide by enzymatic fermentation of two kinds of bacteria was better than that of copper algae polysaccharide,and the proportion of oligosaccharides below 3 kDa was close to 30%.The above results were also higher than the yield of chemical degradation method,but the fermentation process of first enzymolysis and then coupling strain had no advantage over the direct fermentation process.(3)Study on moisture absorption and moisturizing activity of seaweed polysaccharides with different molecular weights.Two kinds of seaweed polysaccharides with average molecular weight ranges of>10 kDa,3?10 kDa and<3 kDa were used respectively.Glycerol and hyaluronic acid were used as controls to investigate the moisture absorption and moisturizing activities of polysaccharides with different molecular weights.The moisture absorption results showed that the two polysaccharides had the best moisture absorption effect in the components with an average molecular weight of less than 3 kDa,and the moisture absorption rate of Gracilaria lemaneiformis polysaccharide reached the maximum 52.66%,which was the 57.57%of hyaluronic acid 70.2%and glycerol,and was higher 47.3%and 24.58%than the two components with an average molecular weight of>10 kDa and 3?10 kDa(P<0.05).The moisture absorption rate of the polysaccharide component of the copper algae was the most 79.66%,reaching the 87.08%of glycerol,which was higher than that of hyaluronic acid 6.2%,and has higher 120.05%and 39.43%than the two components with an average molecular weight of>10 kDa and 3?10 KDa(P<0.05).The moisturizing resulted showed that the components with average molecular weight<3 kDa of Gracilaria lemaneiformis polysaccharide had the highest moisturizing rate,which was 88.09%,reaching the 97.59%of hyaluronic acid moisturizing rate and the 96.33%of glycerin moisturizing rate,higher 3.6%and 3.72%than the two components with average molecular weight>10 kDa and 3?10 kDa(P<0.05).The moisture retention rate of the components with molecular weight>10 kDa of copper algae polysaccharide was the largest,which is 90.59%,and there was no significant difference with hyaluronic acid,glycerin and the other two components(P>0.05).(4)Study on antioxidant activity of seaweed polysaccharides with different molecular weights.Two kinds of seaweed polysaccharides with molecular weight ranges of>10 kDa,3?10 kDa and<3 kDa were used respectively.The antioxidant activities of the two kinds of trehalose in vitro were investigated by detecting the scavenging rate of DPPH,·OH,O₂^-and their total antioxidant activities.The results showed that the clearance rate and total antioxidant activity of DPPH,·OH and O₂^-of the two seaweed polysaccharides with different components increased with the increase of concentration,but different components showed different antioxidant effects.At the maximum addition concentration of 2.5 mg/m L,Gracilaria lemaneiformis polysaccharide with molecular weight range of 3?10 kDa has the best removal effect on DPPH and O₂^-,with the maximum removal rates of 53.89%and41.47%respectively,reaching the 55.58%and 41.76%of Vc control group respectively.<3kDa molecular weight range of asparagus polysaccharide to·OH clearance rate of the maximum 61.92%,the 55.58%of the control group.The DPPH clearance rate of the components with average molecular weight>10 kDa of copper algae polysaccharide was the 98.42%at the concentration of 2.5 mg/m L,which was 0.15%higher than that of the control group(P<0.05).The components with<3 kDa had the best clearance effect on·OH and O₂^-,with clearance rates of 69.3%and 77.78%respectively,which were the 70.43%and 78.34%of the control group.The total antioxidant effect of copper algae polysaccharide in the molecular weight range of 3?10 kDa was the best.