Process Optimization Of Extraction And Purification Of Astaxanthin Produced By Lactobacillus Plantarum CHU-R

Lactobacillus plantarum CHU-R has the characteristics of high astaxanthin content,easy culture and short production cycle,so it is expected to become a strain of astaxanthin industrial production.However,the extraction process of astaxanthin has so many problems such as low extraction efficiency,large amount of organic reagent,the stability of astaxanthin is not high and easy residue and so on.Therefore,it is important to study how to extract astaxanthin from this strain,and to futher isolate and purify the crude astaxanthin extract.Through the research,the optimization and comparison of different astaxanthin extraction and purification methods were performed,and the following experimental results were obtained:(1)Organic reagent methodThe single-factor test and orthogonal array design experiment were employed to optimize the organic reagent method.The results showed that when the dosage of dimethyl sulfoxide was 1: 4(Bacterial: DMSO,w/v),the dosage of acetone was 1: 6(Bacteria: acetone,w/v),the water bath temperature was 60°C for 90 min,the extraction efficiency was the best.Under the optimal conditions the astaxanthin recovery was 5.63 mg/g dry cell.(2)Grinding assisted enzyme methodUsing grinding method and lysozyme to work together,optimized by single-factor test and orthogonal arry design,the optimal conditions were as follow: reaction temperature at 38°C,water bath for 24 h,lysozyme concentration 4 mg/L,astaxanthin amount can be reached 2.49 mg/g dry cell.(3)Acid-heat methodThe single-factor test and orthogonal arry design of acid-heat method showed that when the boiling water bath treatment time was 5 min,the hydrochloric acid concentration was 3.5 mol/L,the treatment times of boiling water bath and ice bath were 6 times,the ratio of solid to liquid(w/v)was1: 7.Under this conditions,the extraction amount of astaxanthin per gram of dried bacteria can be up to 5.8 mg/g.(4)Cryogenic ball millingUsing cryogenic ball milling to broken cell wall and then extract astaxanthin with organic reagent.After optimization of the process,the best conditions were as follow:milling time 6 h,bacterial bead ratio 3: 5(w/w),and the dosage of ethanol was 5: 2(w/v),speed 450 r/min,under the optimal conditions,the extraction amount of astaxanthin per gram of dried bacteria can be obtained to 4.25 mg/g.(5)Stability comparisonThe effects of organic reagent method,acid heat treatment,low temperature ball milling and grinding-assisted enzyme method on the astaxanthin extraction and stability under the optimal conditions were compared.The results showed that the extraction efficiency of acid heat was slightly higher than that of organic reagent method and cryogenic ball milling method.The grinding-assisted enzymatic method had the lowest extraction efficiency,but due to its mild reaction conditions,the astaxanthin extracted had the best photothermal stability.Each extraction method had certain advantages and disadvantages,and different extraction methods can be selected according to the specific application direction of the astaxanthin product.(6)Saponification process optimizationBased on the single factor test,the saponification conditions of the astaxanthin ester of Lactobacillus plantarum were further optimized through orthogonal design.We found that the maximum saponification rate of astaxanthin in CHU-R lactobacillus could reach 87.31% under the conditions of NaOH-ethanol concentration 1mol/L,saponification time 75 min,and saponification temperature 25°C.(7)PurificationTLC and HPLC were used to isolate and detect astaxanthin extract from Lactobacillus plantarum.Using silica gel thin-layer plates with n-hexane: acetone=7: 3 as developing solvent,and the development time of 8 min,the crude astaxanthin can be well separated.The results of TLC and HPLC showed that after saponification,the content of free astaxanthin increased and the content of astaxanthin decreased.The saponified Lactobacillus plantarum crude astaxanthin sample was further purified by 80-100 m silica gel column chromatography.The purified sample was detected by TLC.The results showed that there was only one point with the sameRf value as the astaxanthin standard.The peak area determined by HPLC was calculated according to the area normalization method,and the purity of free astaxanthin was 89.21%.