Screening Of Fucoindanase-producing Bacteria, Fucoindanase Characerization, And Establishment Of The Hydrolyzate Antioxidant Activity Predication System

Kelp is a kind of important economic alga in China, and it is popular for its highnutritional value and various biological activities. At present, the output of kelp inChina ranks first in the world. However, the kelp industry is unable to grow vigorousdue to the current low-level processing and low utilization rate, which is just30%.Therefore, in order to realize development of kelp industry, an important way is torealize value-added utilization through further research on biological activities of kelp.This paper conducts research on the HDFuc in kelp, and its degrading products.Because of the great advantages of lower molecular weight HDFuc in activityapplication and structure study, it is essential to degrade HDFuc and to obtain lowermolecular weight components. Enzymic hydrolysis is the best way for fucoidandegrading, but it cannot be achieved due to the lack of Fucoidan degrading enzyme（FUCenzyme） in the market.In this thesis, the first step of research is to screen FUCenzyme-producing microbe.Using HDFuc as the sole carbon source, a FUCenzyme-producing bacterium namedRC2is obtained from Laminaria japonica in the sea area of Rongcheng. It isidentified to be a new member of Flavobacteriacea by its physiology andbiochemistry characteristic and its16s rDNA analysis. FUCenzyme, a kind ofendoenzyme, can be produced stably by RC2. According to tests of HPSEC and TLC,RC2is confirmed to produce FUCenzyme and generate lower molecular weighthydrolyzate.To increase the FUCenzyme-producing ability of RC2, fermentation medium andenzyme-producing conditions are optimized. The optimal medium is0.2%Fucoidanand0.4%beef extract, prepared with filtered seawater. FUCenzyme activity reachesto178U/mL after72h-cultivating by RC2under the optimal fermentation conditions: 25℃, natural pH,150rpm of shaking speed,20%of the liquid loading size in250mLflask,10%of inoculation size. On this base, the expanding fermentation test in5Lfermentation tank is done and110530U enzyme activity in total is obtained after96h-cultivating by RC2under the conditions:25℃,200rpm of stirring speed and1.0L/min of air flow.Thereafter, the FUCenzyme is purified and its characteristics are analyzed. CrudeFUCenzyme is separated by ammonium sulfate precipitation and QFF ion exchange,and then a single component with enzymic activity is obtained. The single componentis proved to be a purified FUCenzyme by the evidence of a single band in SDS-PAGE.Its molecular weight is41.0KDa according to standard molecular weight marker. Inthe whole process of purification, FUCenzyme is purified up to11.8times and therecovery of enzymic activity is11.3%. Enzymic property research shows that theoptimal conditions are50℃, pH8.0. The FUCenzyme, stable only at20℃and30℃,is with poor thermal stability. But it is with good stability when stored at4℃. It isstable at pH8.0, then at pH7.0. And the enzyme can show its activity only in thesituation with NaCl, and the optimal NaCl concertration is0.4mol/L or0.6mol/L.Zn²⁺, K⁺and Ca²⁺have function of activation to the enzyme, and among them, thefunction of Zn²⁺is greatest. While, Cu²⁺, Hg²⁺and Ag+have function of inhibition,and among them, the function of Cu²⁺is greatest. In addition, NaF and EDTA-2Nainhibit the FUCenzyme strongly. Change of reducing sugars content is monitoredduring the catalyzing process, and the results show that the content of reducing sugarsincreases markedly in the early12hours, and is with little change after12hours. It isproved that the rate of catalyzing declines clearly after12hours.For the first time, a visual antioxidant activity prediction system is established,which realizes the prediction of antioxidant activity of the hydrolyzate catalyzed byFUCenzyme. Three BP network models are established to predict respectively thescavenging ratio of DPPH˙,˙OH and O2-. The three models are verified to be suitablefor use, with the maximum error lower than10%. On the basis of BP netwo rk models,a visual antioxidant activity prediction system is established under the platform ofMatlab, making the visual antioxidant activity prediction of hydrolyzate comes true. Further, GA is used for seeking optimal value of BP network models. The resultsshow that when adding FUCenzyme22.0U to1mg HDFuc and hydrolyzing at25.2℃for6.4hours, the hydrolyzate is generated with highest antioxidant activity （DPPH˙scavenging ratio39.85±1.00%,˙OH scavenging ratio82.08±5.74%, O2-scavengingratio27.67±3.45%, reducing power0.2348±0.0044, iron-chelation activity35.64±3.01%）.Then the in vivo antioxidant study of HDFuc and enzymic hydrolyzate is done.Hydrolyzate is obtained when catalyzing HDFuc under the optimal conditionsmentioned above. It shows significant antioxidant activity in mice of HDFuc and itshydrolyzate when using D-galactose induced subacute aging model. Furthermore, thehydrolyzate is better than HDFuc in the aspect of protecting the mice antioxidantenzyme activity.In conclusion, a FUCenzyme producing bacterium is successfully obtained and thepurification of the FUCenzyme is conducted. Lower molecular weight enzymatichydrolyzate is obtained taking advantage of the enzyme, and a visual antioxidantactivity prediction system of hydrolyzate is established for the first time. Using GAfor optimizing, highest activity enzymatic hydrolyzate is got. And the enzymatichydrolyzate is proved to be with good antioxidant activity by animal experiments. Theachievement in this thesis lays a foundation for development and application ofHDFuc, which will promote the high-value utilization of kelp and development ofkelp industry.