Optimizing The Preparation Processes Of Extracting Fucoxanthin And Brown Algae Polyphenols From Laminaria Japonica

Fucoxanthin brown algae polyphenols were unique active ingredients to the brown algae,which have high values in food and pharmaceutical industries.In this study,fucoxanthin and brown algae polyphenols combined extraction,brown algae polyphenols purification and its antioxidant activities were evaluated.High-performance liquid chromatography-mass spectrometry was used to identify the main components of brown algae polyphenols.The results are as follow:Section 1:The research of fucoxanthin combined with brown algae polyphenols extraction from Kelp.In this study,the dried Kelp as raw materials,cellulose,pectinase and their compound enzyme were broken enzyme.Based on the different enzyme dosage,reaction time,pH value etal,the univariate experiment showed that compound enzymatic extraction?the extraction rate of fucoxanthin was 1.25 g/Kg,brown algae polyphenols was 1.83 g/Kg?higher than pectinase extraction?the extraction rate of fucoxanthin was 1.14 g/Kg,brown algae polyphenols was 1.70 g/Kg?and enzymatic extraction?the extraction rate of fucoxanthin was0.42 g/Kg,brown algae polyphenols was 0.84 g/Kg?.Considering the results of compound enzymatic extraction and pectinase extraction almost the same and cost issues,enzymatic joint extraction selected pectinase extraction.On the basis of results of orthogonal experiments,the best extracting processes for fucoxanthin and brown algae polyphenols as follows:the ratio of extracting solvent and material,40 L·kg^-1;enzyme dosage,20000 U·kg^-1;pH value,5.0;enzymatic temperature,60?;reaction time,90 min.In this condition,the best extracting rate of fucoxanthin was 1.24 g/Kg and brown algae polyphenols was 1.68 g/Kg.Section 2:The study of purification and ingredients identification about brown algae polyphenols.The crude brown algae polyphenols was purified followed by polarity solvent extraction,macro-porous resin colunm chromatography and silica gel column chromatography.Hexane,methylene chloride and ethyl acetate were extraction agent.The purity of different phases were dichloromethane 53 mg/g,ethyl acetate purification 47 mg/g,n-hexane purified product 29 mg/g,the aqueous phase 2.51 mg/g.Analysis of the differences of each component with full wavelength scanning method,ethyl acetate purification contains less carotenoids and chlorophyll a and other impurities,followed by methylene chloride phase and n-hexane phase.Different purified products were obtained by purifying with macro-porous resin XAD-7column chromatograph on the water phase,polyphenol content after purification was 18.77 mg/g,increase 6.5 times than 2.51 mg/g before.Silica gel column chromatography was used to further separation and purification of ethyl acetate phase.Five different components were obtained by gradient elution and stepwise collection.The purity of polyphenols in the order of F3?F4?F5?F2?F1.Among them,the part of F4 was target component and its purity was 74.25 mg/g the yield was6.00%.Two fraction were obtained by F1?F2 secondary purification,polyphenol content was68.8 mg/g and 50.08 mg/g,increased significantly compared to the previous.Used HPLC method to analysis the crude extracts,purified material hexane,methylene chloride purified product,ethyl acetate purification and macro-porous resin purification material.The result showed ethyl acetate phase has the highest purity.Two kinds of polyphenols were identified by high-performance liquid chromatography ionization mass spectrometry?HPLC-ESI-MS?,they are Fucodiphloroethol G and Dieckol.Section 3:Research the antioxidant activity of brown algae polyphenols.Contrastied the antioxidant activity of three kinds of organic phase:the polyphenol extracted by ethyl acetate has the strongest antioxidant activity,followed by methylene chloride phase and n-hexane phase.The reduction capacities of these purifications were very close.Compare these antioxidant activities of these purified productions.When the concentration of XAD-7 purification was 10 mg/mL,its ABTS scavenging rate closes to 100%and its IC₅₀0 was 3 mg/mL.While the aqueous phase concentration was 10 mg/mL,its ABTS scavenging rate was 30%.The reducing power of the XAD-7 purification was straight up far beyond the reducing ability of the aqueous phase.