Optimizing The Processes Of Extracting And Purifying Fucoxanthin And Preparation Of Lowweight Fucoidans From Laminaria Japonica

This dissertation is mainly consisted of four sections described as follows:Section 1:Fucoxanthin,widely and largely existing in brown algae,is one of carotenoid which has a variety of biological activities.In this work,the dried Laminaria japonica was selected as the experimental material for the high content of fucoxanthin.Based on the different extracting rate of fucoxanthin of the different polar organic solvents,the best extracting solvent was optimized.Subsequently,the single factor and orthogonal experiments were used to optimize the best extraction parameters in the processes of extracting fucoxanthin.Then the crude fucoxanthin extracts was purified twice times by silica gel column chromatography and its purity was detected by high-performance liquid chromatography?HPLC?.The results showed that methanol is the most suitable organic solvent for extracting fucoxanthin among the seven kinds of selected organic solvents;on the basis of the results of orthogonal experiment,the best extracting processes for fucoxanthin as follows:the extraction solvent,methanol;extracting temperature,60?;the ratio of material and extracting solvent,1:40?g/mL?;extracting time,2times and each time 1 h.In this condition,the best extracting rate of fucoxanthin was 0.375 mg/g.After purified,the purity of fucoxanthin was about 86%.Section 2:In order to obtain fuco-oligosaccharides which possessed centralized molecular weight and remarkable bioactivity,enzymic degradation method,due to it's mild reaction conditions and no destructive effect on the structure of fucoidan,was adopted in this work.So,the fucoidan lyases,derived from hepatopancreas of abalone?Haliotis diversicolor supertexta?,were isolated and partially purified to hydrolyze fucoidans which were derived from Laminaria japonica.According to the electric properties of protein,organic solvent method,ammonium sulfate precipitation method and isoelectric precipitation method were employed to isolate and purify the fucoidan lyases and their purification effects were compared.Firstly,for the isoelectric precipitation,the enzymic activity of precipitates at pH 3.0 to 9.0 were measured with the viscometric method aiming to shrink the isoelectric range of fucoidan lyases.The results showed that the precipitate at pH 5.0 condition exhibited observably?p<0.05?higher enzymic activity than the other ones.It was indicated that the isoelectric point of fucoidan lyases ranged from pH4.0 to 6.0.Then,the enzyme activity of precipitates which were obtained by the methods of isoelectric precipitation at pH 4.5,5.0 and 5.5,ammonium sulfate precipitation and organic solvent,were measured with the DNS method.Finally,The results indicated that the crude fucoidan lyases had the highest enzymic activity at pH 4.5 and 5.0,and the enzyme activity recovery rate of the fucoidan lyases were 44.4%and 42.5%,respectively.Meanwhile,the content of sulfate groups of the hydrolysed products?fuco-oligosaccharides?at pH 4.0 to 6.0were measured.The results showed that there were no significantly?P>0.05?decrease of sulfate groups for the fuco-oligosaccharides compared with that of the original fucoidans,which was implied that no sulfatase existed in the crude fucoidan layses at pH 4.5 and 5.0.Due to the better enzymic activity of the crude fucoidan layses precipitated at pH 4.5 and 5.0,theirs enzymology characteristics were discussed in further.The results displayed that the optimum hydrolysis reaction conditions were 2%?g/mL?of substrate concentration for 60 min at 38?.Then,the crude fucoidan layses were further isolated and purified with ammonium sulfate precipitation and gel filtration chromatography.The results showed that when the saturation of ammonium sulfate reached 30%and 40%,the crude fucoidanase and?-L-fucosidase were obtained,respectively.In the end,the result indicated that the enzyme activity of fucoidanase was 0.43 U and?-L-fucosidase was 0.27 U after gel filtation,and the enzyme activity recovery rate of the two fucoidan lyases was both 16%,which manifested that the enzymic activity of fucoidan lyases had a loss in the process of isolation and purification.The experimental results showed that two kinds of fucoidan lyases could effectively degrade fucoidans whose degration rates were8%-15%and the sulfate groups of fucoidans were not obviously destroyed.Consequently,low molecular weight oligosaccharides?Fuco-Oligosaccharides?were produced and the relative binding rate of sulfate groups maintained over 85%.Section 3:In order to research the effect of the methods of H₂O₂ degradation in steps or single step on the production of the low molecular weight fucoidan and its retention rate of SO₄^2-.The study used the H₂O₂ mass fraction of 1.2%in singal step and of 0.6%in two steps to degrade fucoidan,finding the best technical parameters of the production of the low molecular weight fucoidan and its retention rate of SO₄^2-;at the same time,researching the varation in viscosity and the H₂O₂ residual amount of fucoidan solution under different times.Results showed that when the production of low molecular weight fucoidan reached a stable state under the condition of 60?water bath,the H₂O₂ mass fraction of 1.2%in single step generated the more yields of the low molecular weight fucoidan and higher retention rate of SO₄^2-than the method which the H₂O₂ mass fraction of 0.6%used in two times in the whole degradation process?P>0.05?.Section 4:Fucoidan lyase was extracted from hepatopancreas of abalone?Haliotis diversicolor supertexta?and acted as enzyme source in the experiment.We adopted three kinds of physicochemical methods,such as hydrochloric acid,hydrogen peroxide and ultrasonic aiding hydrogen peroxide,to pretreat fucoidan?derived from the Laminaria japonica?in a way while the pretreated fucoidan acted as substrate for enzyme degradation.Those methods were used to increase the chance of fucoidan lyase closing to the glycosidic bonds to improve the rate of degradation.Meanwhile,the final goal of experiment is to find out the technology parameters of enzymolysis pretreated substrate by physicochemistry combined with enzyme methods,and the enzymatic hydrolysates had been highest degraded in the process of reaction and owned the best retention rate of SO₄^2-at the same time.The results of experiment showed that:the 0.5%concentration of hydrogen peroxide oxidized to degrade fucoidan in water bath?65??for 2 h firstly and the fucoidan lyase worked on pretreated substrate for 1 h in 38?water bath environment condition.After the datas were deal with by Excel software,the relative viscosity of reaction sugar solution fell to 81.3%and the content of SO₄^2-of substrate kept well.When we made a comparision among the results of experiment,we found Physicochemical combining with enzyme technology methods had higher degradation rate and better relative retention of SO₄^2-of substrate than a single method?physical chemistry method or enzyme degradation method?.The combining technology provided a theoretical basis and innovative ideas for the large-scale preparation of low molecular weight fucoidan.