Study On Enzymatic Conversion Of Naringin And Bioactivities Of The Product

Naringin can be enzymolysed by naringin.Firstly,it is hydrolysed by?-L-rhamnosidase into rhamnose and prunin.Prunin,with a one-third bitterness ratio to naringin can be further hydrolysed by the?-D-glucosidase into glucose and tasteless naringenin simultaneously.It is well known that naringin exhibits various biological activities such as anti-ulcer,anti-inflammatory,anti-oxidant and anti-bacterial activities.However,the applications of naringin in food,drug and cosmetic industry are limited by the poor solubility.So in order to improve its biological value,enzymolysis of free and immobilized naringin by naringinase was studied,and then the antimicrobial activities and the radical scavenging activities in vitro of them were evaluated preliminarily.The main results were listed as follows:?1?The results of dynamic conversion process analyzed by DNS assay were concordant with those by HPLC method,so the dynamic conversion process of naringin can be analyzed by DNS assay.And the optimal conditions of variables for naringin bioconversion were determined as temperature 50oC,pH5.0,enzyme dosage 8 U/mL,substrate concentration 0.2 g/100 mL.Under the optimal condition,the conversion rate of naringin reached 85.1%after 150 min,then subsequently increased little with the reaction on approximately.The double reciprocal plot of Lineweaver-Burk fit equation is y=0.0094x+22.736,with K_m=413.44?g/mL,V_max=0.022mg/?min·mL?.The change trends of yields of reducing sugars were similar among each batch feeding under continous batch feeding.However,the reaction time to reach balance delayed with the feeding times increasing,and conversion rates of naringin were identical when the reaction run 150 min after each feeding.The final product,obtained through separation and purification,was naringenin identified by ultraviolet spectrometry,IR spectroscopy and HPLC method,and it was 92.1%pure.?2?The weak polar poly alkyl ester macroporous adsorption resin XAD-7HP,as the most appropriate support,was selected to immobilize naringin.The optimal conditions of variables for naringin bioconversion were determined as temperature 50oC,pH5.0,enzyme dosage 8 U/mL,substrate concentration 0.2 g/100 mL,vibration rate 80 times/min.Under the optimal condition,the conversion rate of naringin reached 93.5%after 6 h,subsequently increased little with the reaction on approximately.But the best The double reciprocal plot of Lineweaver-Burk fit equation is y=0.0008x+0.8627,with K_m=927.32?g/mL,V_max=0.023 mg/?min·mL?.Under the optimum condition,the changes of enzymolysis of naringin were identical before and after the extend experimentation.Conversion rate of naringin reached 89.8%after the reaction run 6 h in the enlarging experiment,and the enzymolysis of naringin slowed down when the naringin loadings increased to 5 folds of that of the former run,and the conversion rate of naringin reached 90.2%after 10 h.Compared to continous batch feeding,the rate of enzymolysis of naringin of one-time feeding was much faster,and which was more suitable for industrial production.The final product,obtained through separation and purification,was naringenin identified by ultraviolet spectrometry,IR spectroscopy and HPLC method,and it was 95.8%pure.?3?The agar disk diffusion procedure was used to assess the antimicrobial activities of naringin and naringenin against three kinds of bacterial strains and a type of fungus,while three complementary in vitro essays:the radical scavenging activities of naringin and naringenin against DPPH radical and hydroxyl radical,and metal chelating ability were used to evaluate the antioxidant activities of naringin and naringenin.The antimicrobial experiments showed that the antimicrobial activities of naringin and naringenin were greatly different at the same concentration,and naringenin showed a relatively better antimicrobial activity than naringin,while naringnin failed to inhibit the growth of all of the tested microorganisms.Moreover,naringenin showed a better antimicrobial activity for Gram-positive bacteria than that for Gram-negative bacteria.The results of in vitro essays against the radical scavenging activities of naringin and naringenin showed that the IC₅₀ of naringin,naringenin and BHT against DPPH radical was7.044 mg/mL,1.277 mg/mL and 0.035 mg/mL,respectively,while the IC₅₀ of naringin,naringenin and BHT against hydroxyl radical was 1.499 mg/mL,1.288 mg/mL and1.081 mg/mL.In addition,the reducing capacities of them were as follows in order:BHT>naringenin>naringin.The work expounded the dynamic conversion processes of free and immobilized naringin towards naringenin by naringinase and showed the differences between them.In addition,the antimicrobial in vitro essays and antioxidant experiments showed naringenin had better bio-activities than naringin,which provided a theoretical basis for them to be food preservatives and antioxidants.