Study On The ACE Inhibition And Antioxidative Activities Of Royal Jelly By Enzymatic Modification

In this paper, after the moderate enzymatic modification by protease, the fresh royal jelly were separated by sephsdex G-25. And then the ACE inhibition activity and antioxidant activity of hydrolysate and fractions separated by gel chromatography were determined by different methods In order to explore the relations between amino acid composition and sequence and physiological activities, the fraction with highest activity were analysed by amino acid composition analysis and MALDI-TOF-MS analysis which would provide certain experimental basis and theoretical direction for royal jelly productions after the enzymatic modification.The main research contents and experimental results were as follows:(1) The selection of protease for enzymatic modification of royal jelly:With ACE inhibition activity as index.compared five kinds of protease hydrolysate with fresh royal jelly when the mass concentration of royal jelly was0.05g/mL (wet weight),and the experimental results indicated that the fresh royal jelly hardly expressed ACE inhibition activity, while the enzymatic modification products had prominent ACE inhibition activity,especially the enzymatic modification product of Amano protease P expressed the strongest ACE inhibition activity, reaching to71.47%. So, the Amano protease P was chosen for the next enzymatic modification of royal jelly and then the best modification condition was determined.(2) The Optimization of technological conditions for enzymatic modification:the single factor experiments were conducted with hydrolysis time, mass concentration of royal jelly and enzyme addition as single factors respectively. On the basis of single factor levels, the optimum enzymatic modification conditions of Amano protease P were obtained by Box-Behnken quadratic rotating orthogonal test followed by Response Surface Analysis:hydrolysis time:196min; mass concentration of royal jelly:0.0653g/mL (wet weight); enzyme addition:1503u/g.(3) The separation conditions Optimization of enzymatic modification Products by gel chromatography:The royal jelly was modified by Amano protease P in the optimum enzymatic modification conditions, and then the hydrolysate were separated by sephadex G-25, the optimal elution conditions were:eluent:lmol/L acetic acid; flow rate:0.8mL/min;sample amount:1.5mL; Three peaks were isolated followed by collection, concentration and freeze-drying and then named GFC-1, GFC-2and GFC-3.(4) The comparison of ACE inhibition activity for royal jelly, hydrolyzate and three fractions separated by Gel chromatography separation: the fresh royal jelly hardly expressed ACE inhibition activity at all, but after enzymatic modification, the ACE inhibition activity of hydrolysate enhanced signally(p<0.01). In the concentration of4.5mg/mL (dry weight), the ACE inhibition ratio of fresh royal jelly was only5.45±1.67%, while the ACE inhibition ratio of royal jelly modified by Amano protease P increased greatly to48.19±1.98%, and the GFC-3had the highest ACE inhibition activity.reached to72.75±2.12%. There are obvious difference between hydrolysate and the three fractions separated by Gel chromatography in ACE inhibition activities, the IC50of GFC-3which expressed highest ACE inhibition activity was3.193mg/mL(5) The comparison of antioxidant activities for royal jelly, hydrolyzate and three fractions separated by Gel chromatography separation:①The inhibition ratio of linoleic acid peroxide by thiobarbituric acid reactive substances method (%):In the concentration of3.8mg/mL, the inhibition ratio of linoleic acid peroxide of fresh royal jelly was12.69±0.28%while the inhibition ratio of linoleic acid peroxide of hydrolysates and the three fractions increased greatly(P<0.01) and the inhibition ratio of hydrolyzate was64.7±1.16%, and the GFC-3had the highest inhibition ratio, reaching to74.73±2.25%.The IC50of GFC-3which had the highest inhibition ratio (P<0.01) in different concentrations was3.382mg/mL.②The clearance rate of superoxide anion by pyrogallol method(%):In the concentration of2.4mg/mL, the clearance rate of superoxide anion of fresh royal jelly was only11.38±1.07%while the clearance rate of superoxide anion of hydrolyzate and three fractions increased greatly(P<0.01); the clearance rate of hydrolyzate was63.07±0.7%, and the GFC-3had the highest inhibition ratio, reaching to72.38±0.68%. The IC50of GFC-3which expressed highest clearance rate (P<0.01) in different concentrations was1.626mg/mL.③The clearance rate of hydroxy radical by Fenton method(%):In the concentration of5.6mg/mL, the clearance rate of hydroxy radical of fresh royal jelly was only11.16±1.57%while the clearance rate of hydrolyzate and three fractions increased greatly(P<0.01); the clearance rate of hydrolyzate was63.04±1.14%and the GFC-3had the highest inhibition ratio, reaching to70.16±1.44%.The IC50of GFC-3which expressed highest clearance rate(P<0.01) in different concentrations was4mg/mL.④The comparison of reducing capacity to Fe3+:the Fe3+reducing capacity of fresh royal jelly, enzymatic modification product and the three fractions separated by Gel chromatography separation were compared and the experimental results indicated that In the concentration of9mg/mL, the ability value of restoring Fe+to Fe2+for fresh royal jelly A7oonm was0.127±0.008while the ability value of restoring Fe3+to Fe2+for hydrolysate and three fractions increased greatly (P<0.01); the ability value of restoring Fe3+to Fe2+for hydrolysate A700nm was0.63±0.009and the A700nm of GFC-3which had the strongest reducing capacity was0.74±0.006. The GFC-3expressed highest Fe3+reducing capacity(P<0.01) in different concentrations. Comparing the reducing capacity of hydrolysate and the three fractions separated by Gel chromatography with A700nm-0.5as evaluation index, the concentration of GFC-3was7.126mg/mL.(6) The amino acid composition analysis and MALDI-TOF-MS analysis representation of GFC-3:in all,17kinds of amino acid were observed from GFC-3which expressed the strongest activities by the amino acid composition analysis, and the content of Asp, Glu, Lys, Val, Ser were more than other amino acid. The structure of GFC-3was analysed by MALDI-TOF-MS. According to the primary mass spectrum analysis of GFC-3, the molecular weight of GFC-3were all under1400Da. There were8peaks between800Da-1400Da.The parent ions with the stronger peak values were analysed by secondary mass spectrum analysis, The amino acid sequence of parent ion with molecular weight814was Gln-Cys-Ser-Trp-Ser-Met while the amino acid sequence of parent ion with molecular weight1311was Asp-Cys-Cys-Gly-Tyr-Thr-Thr-Cys-Asp.