Bioconversion Of Shrimp Processing Waste With Medicinal Fungi And ACE Inhibitory Activity Of Its Product

Shrimp processing industry has developed rapidly and production of shrimp processing wastes has increased over the last two decades because of the steady increasing of the global shrimp production.How to recycle the shrimp processing wastes fully to prevent the environment of nearshore coastal from pollution,become the problem to be solved.Biotechnology has become increasingly concerned for its’ recycling the shrimp processing wastes with high added value and without secondary pollution.The shrimp processing wastes was bioconverted by edible/midicinal fungi through submerged fermentation in this study.And then,antihypertensive activity of the water extracts from converted products was determined through testing the inhibitory activity of the water extracts on Angiotensin I-Converting Enzyme(ACE).Then,the conditions of bioconversion were optimized through Plackett-Burman(PB)experimental design,Path of Steepest Ascent and Response Surface Methodology(RSM)sequently.And then,the ACE inhibitor(ACEI)was isolated and purified by ethanol precipitation,macroporous resin adsorption,chloroform extraction,silica gel thin-layer chromatography and gel column chromatography in turn,and then identified with LC-MS/MS and GC/MS.Finally,the active components were synthesized for verifiing their ACE inhibitory activity.Molecular docking simulations were carried out with ACE and the dipeptide inhibitors to explore mechanics and structure-activity of their ACE inhibitory property.The main results are as follow:The chromatographic conditions for determining the content of hippuric acid(Hip)with RP-HPLC were established to evaluate the ACE inhibitory activity.The mobile phase was the water solution contained 30% of acetonitrile and 0.2% of acetic acid.The flow rate was 1.0 mL/min.The detection wavelength was 228 nm.The tempreture of the column was 30℃.These chromatographic conditions had wide linear range(2-200 ng),high precise and repestibility,the lowest limit of the detection by which was 5 ng.Hip in reaction system can be isolated and detected well without extraction in these chromatographic conditions.Inaddition,the reaction system of 50 μL was established,in which the terminal concentration of ACE and HHL were 50 mUand 4.61 mmol/L,respectively.The time for samples reaction with ACE in the ACE inhibitory assay was confirmed as 5 min.Boletus edulis,Suillus bovinus,Xerocomus badius,Cantharellus cibarius and Sarcodon aspratus were selected as the fungi for converting experiment of shrimp processing wastes from 18 kinds of fungi because of their relatively higher ACE inhibitory activity and mycelia biomass.Results of the converting experiment indicated that the shrimp processing wastes was deodorized after the bioconversion.In addition,water extacts from converted products of Xerocomus badius had the highest ACE inhibitory activity(IC50= 0.40 ± 0.00 mg/mL)and relatively higher yields(8.63 ± 0.19 g/L).IC50 value and yields of water extacts from converted products of Cantharellus cibarius were determined as 0.84 ± 0.01 mg/mL and 10.72 ± 0.34 g/L,which were relatively higher.So,Xerocomus badius and Cantharellus cibarius were selected as the fungi for optimization of bioconversion conditions.Results of PB experimental design of bioconversion implied that the solid to liquid ratio,bran powder content and concentration of glacial acetic acid were the significant factors effect the mycelium biomass of Xerocomus badius,the percentage effect of them was 92.559%;the solid to liquid ratio and concentration of glacial acetic acid were the significant factors effect the ACE inhibitory activity of water extracts from mycelium of Xerocomus badius,the percentage effect of them was 89.309%.As to Cantharellus cibarius,the solid to liquid ratio and concentration of glacial acetic acid were the significant factors effect the mycelium biomass and ACE inhibitory activity of water extracts from mycelium,the percentage effect of them was 90.22% and 93.33% repectively.Effect of solid-liquid ratio on the response values were positive,but negative effects of acetic acid content was.Results of RSM indicated that the optimized bioconversion condition of Xerocomus badius was established as 12.421% of solid to liquid ratio,7.964% of bran powder content and 1.117% of concentration of glacial acetic acid,when the mycelium biomass and the ACE inhibitory activity of water extracts from mycelium were both kept in mind.Under this model,the mycelium biomass was determined as 30.24 ± 0.21 g/L,enhanced by 38.210%;ACE inhibitory activity of water extracts from mycelium(IC50= 0.21 ± 0.02 mg/mL)was decreased significantly(P<0.05).In addition,the optimized bioconversion model of Cantharellus cibarius was established as 11.29% of solid to liquid ratio and 1.25% of concentration of glacial acetic acid,when the mycelium biomass and the ACE inhibitory activity of water extracts from mycelium were both kept in mind.Under this model,the mycelium biomass reached 27.011 g/L,was improved by 17.633%;ACE inhibitory activity(IC50= 0.39 ± 0.07 mg/mL)of water extracts from mycelium was decreased significantly(P<0.05).After being optimized,both of the mycelium biomass and ACE inhibitory activity of water extracts from mycelium of Xerocomus badius were higher than that of Cantharellus cibarius.So,the ACEI from Xerocomus badius was isolated and identified in this study.The ACEIs isolated from the converted products were identified by LC-MS/MS and GC/MS.Some fractions(IC50=0.11 ± 0.00 mg/mL)was identified as npropyl-2-hydroxy-1-oxohexahydro-1H-azepine or botanedioic acid,hydrozy-dibutyl ester,hexadecanoic acid,2-methylpropyl ester or hexadecanoic acid,butyl ester.Other fractions of them was proved to be peptides(IC50=0.14 ± 0.00 mg/mL),molecular weight of them were all lower than 500,components has the top 2 of abundance were Cys-Cys and Cys-Arg.The synthesized Cys-Cys(IC50=4.37 ± 0.07 μmol/L)has significantly(P<0.001)higher ACE inhibitory activity than Cys-Phe(IC50=13.82 ± 0.09 μmol/L),which has already been reported,whereas ACE inhibitory activity of Cys-Arg(IC50=475.95 ± 0.11 μmol/L)was significantly lower than that of Cys-Phe(P<0.001).Differences in ACE inhibitor potency among the three dipeptides could be explained by results of molecular docking.Cys-Cys ineracted with ACE through hydrogen bonds at Ala354(O),Ala356(HN),Trg523(OH),a bump at His387(NE2)and a crucial coordination between its carboxyl oxygen and Zn(II)(2.088 ?).Cys-Phe also formatted 3 hydrogen bonds and a coordination between its carboxyl oxygen and Zn(II)(2.146 ?).The absence of the bump at His387(NE2),especially the shorter crucial coordination between the carboxyl oxygen and Zn(II),increased the IC50 value by 216.25%.Cys-Arg formatted more hydrogen bonds and stronger bump with ACE at its active site.But the absence of the crucial coordination between its carboxyl oxygen and Zn(II)induced a 108.91 fold higher IC50 value of Cys-Arg compared to Cys-Cys,and 34.44 fold higher IC50 value compared to Cys-Phe.Findings in this study highlighted the importance of coordination between carboxyl oxygen and Zn(II)and its distance in defining peptide ACE inhibitor potency.Through bioconversion shrimp processing waste by Xerocomus badius,the novel ACE inhibitory dipeptides,Cys-Cys and Cys-Arg,with high activity could be isolated from the bioconverted products,which have the potential for being natural,safe and portent antihypertensive drugs.Functional mechanics were explored by using simulation of molecular docking.The results have provided the basis for development of new and efficient natural anti hypertensive drugs and sufficient resources of shrimp processing waste.