Study On Hydrolysis Of Rutin/Naringin Catalyzed By Whole Cell

Flavonoids have various physiological functions,including antibacterial,anti-inflammatory,anti-allergy,anti-cancer and anti-oxidation activities,and thus widely used in food,medical and cosmetic industries.As kinds of important flavonoids,rutin,naringin and their derivatives have attracted consedarable attentions.Previous reports showed that the dehydroxylation modification of isoquercitrin and naringenin can siginicantly improve their physiological activities,.In nature,these flavonoid aglycones are few in stock,and the chemical dephosphorylation methods aremainly used for their preparation.Since the flavonoids have multiple glycosyl groups,the chemical methods or the enzyme-chemical methods required multiple protection/deprotection steps to obtain the corresponding aglycones or intermediates.The emerging enzymatic stategies can avoid the drawbacks of chemical methods,but suffered from the high cost s and low stability of enzyme prepations.Considering the above-mentioned problems,this dissertation explored the whole-cell catalyzed selective hydrolysis of rutin and naringin.The effects of microbial strain type,culture medium and reaction conditions on the catalytic properties of whole-cell catalyst and the mechanism were studied.The optimum reaction system for production of the flavonoid aglycones and their intermediates was established.Among the 14 strains of microbes tested,the whole-cells of Aspergillus niger GIM 3.25 and Pseudomonas stutzeri GIM 1.273 showed the ability to catalyze the hydrolysis of rutin.Aspergillus niger showed higher catalytic activity than Pseudomonas stutzeri,with high selectivity towardsthe production of isoquercitrin.Using Aspergillus niger Cells as the catalyst,the effects of several key factors on the reaction were investigated and the optimum conditions were confirmed as:reaction time 2 h,reaction temperature 35?,the pH value of buffer system 5.0,the shaking speed 120 r/min and the whole cell dosage 60 mg;under which the highest rutin conversion was obtained(>99%).Aspergillus niger whole-cell catalyst showed high operational stability.After arepeatedly used for10 batches,the cells remained 88% of their original activity.It was found that the culture medium composition,especially the inducer component,could significantly affect the catalytic activity of whole-cell catalysts,and its effect was varied with the strain tested.Aspergillus niger prepared by GP-2 medium containing naringin showed the highest catalytic activity,by which the conversion of rutin and selectivity were 32.2% and 99%,respectively.Pseudomonas aeruginosa showed higher activity when cultured in GP-3 medium containing carboxymethylcellulose than in other media.Naringin shown to be the best induder among eight carbon sourcesFluorescence real-time PCR showed that the expression of rutin-?-rhamnosidase gene was significantly higher than that of other carbon sources when adding flavonoids such as naringin.The optimum culture conditions for Aspergillus niger were determined as follows: naringin 3.0 g/L,yeast extract 1.5 g/L,MgSO4 ·7H2O 1.0 g/L,KH2PO4 1.0 g/L,KCl 1.5 g/L,anhydrous CaCl2 0.1 g/L,surfactant Triton X-100 0.5%(V/V);fermentation pH 5.0,fermentation temperature 35?,fermentation speed 140 r/min,fermentation time 48 h,and its optimum concentration was 3.0 g/L;under which the biomass of Aspergillus niger and the optimum rutin conversion were 1.8 g/L and 95.58%,respectively..The effects of environment-friendly ionic liquid-water mixed solvents on the preparation of naringenin by hydrolysis of naringin from whole cell of Aspergillus niger were also investigated.The results showed that the aqueous solutions containing [Bmim]BF4 and [Emim]BF4 not only improved the solubility of naringin,but also greatly improved the catalytic efficiency of Aspergillus niger whole cell in the reaction of catalyzed rutin hydrolysis.Using [Bmim]BF4 solution as the reaction solvent,the optimum reaction conditions of Aspergillus niger catalyzed hydrolysis of naringinwere: buffer solution of pH 5.5,temperature 35? and speed 140 r/min;under which the conversion of the whole-cell catalytic reaction after 24 h reached 99%,and the naringenin selectivity was 93.3%.This study is of not only high theoretical value but also good industrial application potential.The new green and safe method was developed for the synthesis of isoquercitrin and naringin with much higher activity and commercial values than the orifinal flavonoids,which also provides a new way for the safe production of other flavonoid food additives.