Study On Producing Agarase Strain Screening And Biological Enzyme Preparation Of Agaro Oligosaccharides System Solutions

There are a large number of oligosaccharides with various biological activities existed in the seaweeds.The oligosaccharides are widely used in the field of medicines,dietary supplements,food additives and cosmetics,due to the functional characteristics,such as antioxidant,anticancer,antiviral,anti-inflammatory,immune enhancement,moisturizing and whitening.As one of the main raw materials for the production of alginate oligosaccharides,the agar has the considerable value in exploitation and application.Traditionally agar oligosaccharides are prepared with acid hydrolysis method,which is always accompanied with severe hydrolysis reaction,uncontrolled experimental conditions,and the loss of biological activities of polysaccharides.Therefore,biological enzyme hydrolysis method is introduced and shows the potential application in the agar oligosaccharides production.Algae are the main food for abalones,and previous studies show that there are a variety of microorganisms for agar degradation in the digestive tract of abalone.Based on that,the thesis was aimed to obtain the high yield of agarase-producing strains from the intestinal tract of abalone,and to do the molecular biological identification of the strains;then the fermentation performance of the strains was systematically investigated to determine the optimal fermentation conditions,and to obtain the crude enzyme liquid containing agarase with high activity;in addition,the crude enzyme was used as the biocatalyst in the process of agar degradation to optimize the degradation parameters;the agar oligosaccharide components were identified and analyzed with thin-layer chromatography(TLC)and high performance liquid chromatography(HPLC),and the antioxidant ability of agar oligosaccharides was studied.The main research and results are summarized as follows:(1)Eighteen agarase-producing strains were successfully separated from the intestinal tract of haliotis diversicolor purchased from Fuzhou market.One strain containing high yield agarase,as the target strain,was selected out from the 18 strains using agar as the sole-carbon source.After analysis of the 16S rDNA sequence of the target strain and the NCBI and the ribosomal database comparison,it is verified that the target strain belongs to the genus Vibrio bat(Vampirovibrio sp.),and is named as Vampirovibrio sp.fjfst-2013001.(2)The single factor test together with orthogonal design was applied to optimize the fermentation conditions for Vampirovibrio agar sp.fjfst-2013001.The optimal medium components were agar 0.25%(w/v),salinity 2.5%(w/v),yeast 0.15%(w/v)and tryptone 0.6%(w/v).The best fermentation conditions were:initial pH8.0,8%of inoculation quantity,25 ? of culture temperature,100r/min of shaking speed and 48h for fermentation time.Under the optimal conditions,the enzyme activity of the agarase in the crude enzyme liquid was 4.17U/ml,which was enhanced by 241.04%compared to that without fermentation optimization(1.73U/ml).(3)The separation,purification and the enzyme properties of the agarase were investigated.The fermentation liquid was concentrated using the salting out method.Then the concentrated crude enzyme was treated with ion exchange chromatography using DEAE-Sepharose Fast flow column and with Sephcryl S-100 chromatography.The final enzyme liquid was purified by about 29.5 times,and the recovery was 39.53%.After the electrophoresis identification of the protein,a single strip was obtained.The technological conditions for agar degradation were optimized using the agarase as the biocatalyst:45? for enzymolysis temperature,pH8 for the enzyme solution,0.25%of agar concentration,15mins for enzymolysis.According to the test results from large molecule polysaccharide hydrolysis reaction the crude enzyme does not show any hydrolysis ability for chitosan,carrageenan,xanthan gum,sodium alginate,soluble starch,sucrose and Isomaltooligosaccharide,and it is also indicated that the crude enzyme liquid has a good specificity.As demonstrated by the tolerance ability test of metal ions,the crude enzyme has the tolerance for certain metal ions except for copper ion,aluminum ion,iron ion and surfactant SDS which could destroy the enzyme activity.(4)The agar enzymatic hydrolysate was analyzed and identified using TLC and HPLC.The results show that Neoagarohexaose is the only product from the crude enzyme degradation of agar.And then the antioxidant test was applied to Neoagarohexaose to study for the scavenging effect on hydroxyl radicals and superoxide anions,as well as the ability of removing DPPH and total antioxidant capacity.The result shows that the Neoagarohexaose possesses certain antioxidant ability in vitro.