Optimization Of Enzymatic Hydrolysis Process Of Kelp And Extraction And Detection Of Enzymatic Hydrolysis Products

With the development of marine science,the algae and their derivatives algae polysaccharides in the ocean are increasingly concerned by the scientific community because of their strong biological activity,development value and application prospects.However,due to its easy-to-coagulate characteristics,it is limited in application.And its degradation product algae oligosaccharide has a small molecular weight,which is easy for passive plants to use.Make up for the shortcomings in the application of algae polysaccharide.Therefore,this paper mainly studies the process optimization of the preparation of algae oligosaccharides by enzymatic hydrolysis,and the qualitative and quantitative detection of algae oligosaccharides in the enzymolysis solution and plant hormones in enzymolysis solution and extraction of algal oligosaccharides in enzymolysis solution.(1)Preparation for the crude enzyme solution of alginate lyase and optimization of enzymatic hydrolysis conditionsAlginate lyase enzyme activity test:respectively select marine algae biolysis 75%and 100%,biolysis 100%placed for 1d,biolysis 100%placed for 2d,biolysis 100%placed for 3d to extract crude enzyme solution as added 10%to kelp enrichment medium and cultivate for 4 days.Daily detection of enzyme activity and observation of kelp lysis was performed.The results show that when the algae are cracked at 75%,the enzyme activity is higher and the algae can be fully liquefied.Therefore,through the above experiments,it can be determined that the alginate lyase should be extracted when the algae decompose 75%,and the activity is better at this time.Optimization of fermentation conditions:experiment through the alginate lyase in different kelp lysis medium,shock time,kelp concentration and so on for 4 directions,comprehensive analysis obtained alginate lyase in medium with inorganic salt ions such as ammonium sulfate,sodium chloride,the alginate lyase activity is relatively high and algal oligosaccharide production is also the highest.The optimal concentration enzymatic hydrolysis of kelp in the medium is 30%,the temperature is 42°C,and keep the shaking with 160 rpm.(2)Separation and extraction of algal oligosaccharides from kelp fermentation brothCentrifuge the kelp fermentation broth at 5000rpm for 10min and take the supernatant,first add the Coomassie brilliant blue solution,the color of the supernatant of the fermentation broth becomes blue.It is prove that the supernatant of the fermentation broth contains a certain amount of protein,then add ethanol,proteinase K,lead acetate and other substances to precipitate the protein in the kelp fermentation broth and the colorimetric method was used to detect the protein and oligosaccharide content in the supernatant at 280nm and 235nm respectively.The protein removal rate of proteinase K reached 82.83%,the lead acetate solution is only 35.59%.The results show that proteinase K precipitate the protein effectively,but this method has certain drawbacks,for instance,it will cause some oligosaccharides to be lost.(3)Establishment of detection method of algae oligosaccharides in kelp fermentation brothThe six sugars of glucose,sucrose,fructooligosaccharides,dextran,sodium alginate,and algae oligosaccharides were tested for their OD value at 490 nm using the sulfuric acid-phenol method.Through data processing,it was found that the OD value of 0.3995 after sodium alginate hydrolysis was significantly greater than 0.315 before hydrolysis.So select sodium alginate as the standard product of the standard curve,then exploring the detection system,hydrolysis time and temperature,and ice bath time through single factor variables,determine the final standard curve:y=348.13x(R~2=0.9975).Thin layer chromatography(TLC)method:through the analysis of the moisture content and pH value of the fermentation broth,the experiment found that the band is clearer when the pH=10 after concentration 25 times,and there is a certain migration distance and the band is at the lower position of the disaccharide,speculate that there are more trisaccharides in the fermentation broth.(4)Determination of phytohormone in kelp fermentation brothCentrifuge the kelp fermentation broth at 5000rpm for 10min and take the supernatant,qualitative and quantitative detection of three phytohormone(auxin,cytokinin,abscisic acid)in fermentation broth by high performance liquid mass spectrometry and chromatography.Among them IAA0.60ng/ml;TZR 0.00024ng/ml;C-zeatin 0.024ng/ml;IP 0.0040ng/ml;IPR 0.12ng/ml;ABA 0.027ng/ml.The kelp fermentation broth contains the three plant hormones detected above and the kelp fermentation broth can be used as a growth stimulant on crops...