Preparation, Purification And Characteristics Study On ACEIP From Aquatic By-product Protein By Biological Method

This paper mainly focused on the preparation of ACEIP (Angiotensin 1 ConvertingEnzyme Inhibitory Peptide) based on aquatic product or by-product protein bybiological methods including controlled hydrolysis, multiple enzymatic andfermentation. After the optimization of the biological process conditions, thefermentation kinetic model, separation and purification, amino acid sequence andcharacteristics of ACEIP were systematically studied.First, to screen the optimal material from aquatic product protein includingmarine fish protein and by-products, freshwater fish protein and mussels protein,basic components were analyzed of 15 aquatic protein, ribbonfish (Trichiurushaumela) backbone was screened as the most optimal material for the preparationof ACEIP according to ACE half-inhibitory concentration of hydrolysates byacid protease and alcalase.Hydrolysis effects of acid protease, alcalase, pepsin, trypsin, flavory protease,papain, neutral protease and elastease on ribbonfish backbone were evaluated byACEIC₅₀ and anti-oxidant activity (reducing power, OD₇₀₀). Acid protease wasselected out the best enzyme. The best conditions of controlled hydrolysis gainedby using response surface methodology (RSM) were: pH 2.67, temperature 45℃,time 7 h, substrate to water ratio 1: 4.38 (m/v, g/mL), enzyme to substrate ratio1/150 g/g protein. ACEIP obtained under these conditions had the properties ofACEIC₅₀ 1.05 mg/mL and reducing power 0.638.After that, multiple enzyme hydrolysis of ribbonfish backbone was studiedusing orthogonal test and the optimal two step enzymes hydrolysis conditionsevaluated by ACEIC₅₀ and anti-oxidant activity (reducing power, OD₇₀₀) were:hydrolyzed at 37℃, pH 4.5, substrate to water ratio 1:4.5 (m/v, g/mL) , enzyme tosubstrate ratio 1/100 g/g protein for 3 h by elastease; then hydrolyzed at 37℃, pH2.0, enzyme to substrate 1/300 g/g protein for 2 h by pepsin. The ACEIC₅₀ reached0.88 mg/mL under these conditions.The controlled hydrolysis was limited by kinds of enzyme and the enzymeprices as concerning to the industry producing. This is the first time of the study toresearch preparation of ACEIP by biological method of fermentation. We screenedthe suitable bacteria from different kinds of bacteria and fungi stored in our lab, and got a strain of Bacillus lichenformis EL314CF10 with best result. Afteroptimization using RSM, the optimum conditions of fermentation were obtained toproduce ACEIP from ribbonfish backbone: sucrose content 4.42%, ribbonfishbackbone content 4.39 g/25mL, K₂HPO₄ 0.1%, MgSO₄ 0.01%, pH 7.0 (aftersterilization), temperature 37℃, rotate speed 200 rpm, medium volume 25 mL/250mL flask, cultivation period of seed 18 h, inocula volume 4%, cultivation peroid28 h. The ACEIC₅₀ of the fermentation suspension was 0.78 mg/mL, and thereducing power was 0.812, nearly the reducing power of 2 mg/mL Vc.To combine the experiment in lab and industry producing, we studied theexpand fermentation in 5 L bioreactor. Based on analysis of characteristics ofcultivation course and kinetic data, the kinetic models were established usingLogistic equation and Luedeking-Piret equation:Cell growth model:Change of 1 /ACEIC₅₀ model:Sucrose consumption model:All the models fitted the experiment data very well, the F test of the threemodels showed p＜0.01, indicated that the models were practical.The mixture of bio-active peptides were separated by new separation techniquesincluding ultra filtration, gel chromatography and RP-HPLC. The peptides withmost bio-activity were mainly fraction with molecular weight lower than 5000 Da.The results showed 14 peaks after separation by gel chromatography. Peptideswith ACE inhibitory activity had molecular weight lower than 1000 Da generally, yet anti-oxidant peptides had bigger molecular weight ranging from 500 Da to3000 Da.Further separation steps were carried out of the most ACEI active peak from thefirst separation step. A fraction above 95% purity was gained. The molecularweight of this fraction was 317.25 determined by LC-MS, and the amino acidsequence was LW, ACEIC₅₀ was 5.6μM.To further confirm the function of ACEIP from ribbonfish backbone, animalexperiments were carried out including single administration and long termadministration of SHR (Spontaneously Hypertensive Rat) and mice swimmingexperiment. The results showed that ACEIP from ribbonfish backbone had theanti-hypertensive function, the low and high dose treatments reduced the bloodpressure of 8.8 and 20 mmHg of SHR in the single administration experiment,respectively; and 15 and 20 mmHg of SHR in the long term administrationexperiment. Oral administration of the ACEIP from ribbonfish backboneprolonged the swimming time of mice, indicated that it could enhance theendurance of mice.