Fermentation And Preparation Of Chlorogenic Acid Hydrolase And Application In Sunflower Seed By Aqueous Extraction Process

Sunflower seed protein is a high-quality protein resource of high nutritional value.Browning of sunflower seed protein caused by chlorogenic acid（CGA）oxidation limited its usage in food and beverage to great extent.The totally removal of CGA from sunflower seeds has not been achieved by any physicochemical dephenolization method.CGA hydrolase is a kind of esterase that catalyzes the hydrolysis of CGA,and also has the esterification and transesterification activity.It has great advantage in the preparation of caffeic acid and caffeic acid phenethyl ester.But now there are very few research reports about it.By screening and mutagenesis,strains of producing CGA hydrolase were obtained,and the CGA hydrolase was isolated and purified.It is used in the preparation of sunflower seed protein by aqueous extraction processing.The effects of enzymatic hydrolysis of CGA on sunflower seed protein were studied in this paper.The main results were as following:Firstly,Using CGA as the single source of carbon source,we screened strains producing CGA hydrolase from the honeysuckle planting soil,in which the activity of SD-14 is the highest（0.048 U/m L）.The strain was identified as Aspergillus niger.It was named Aspergillus niger SD14.The Genbank accession number of ITS gene sequence was KY860764.In order to improve the ability of producing enzyme,A.niger SD14 was mutated by ARTP mutagenesis and the fermentation conditions was optimized.Six positive mutant strains were screened out.The enzyme activity of A.niger SD14.721 was the highest（0.081 U/mL）,increased by 69% than the original strain.It has a good genetic stability.Under the condition of optimization,the CGA hydrolase was produced with enzyme activity of 0.139 U/mL,which was 1.72 times before optimization and 2.90 times of original strain.Secondly,crude enzyme was purified by ultrafiltration concentration,ammonium sulfate precipitation,DEAE Sepharose FF ion chromatography and Superdex G200 gel filtration chromatography.The specific activity of purified CGA hydrolase was 4.35 U/mg.The SDS-PAGE analysis of the purified enzyme gave a single band corresponding to the relative molecular mass of 96.7 k.Enzymatic properties of the purified CGA hydrolase were studied.It had optimal pH of 7.0,temperature 60 ℃.The enzyme activity was inhibited by metal ions Cu²⁺,Fe²⁺,Al³⁺ and Fe³⁺.Kinetic studies showed that Michaelis constant Km is 1.85 μM,Vmax is 0.38 μmol/min for CGA hydrolase.Then,the application of CGA hydrolase in sunflower seeds by aqueous extraction process was carried out.With the dephenolized sunflower seeds as raw material,the effects of the adding amount of CGA hydrolase,temperature and time on the hydrolasis rate of remained CGA were studied.The optimum conditions for hydrolasis the remained CGA of dephenolized sunflower seeds were: enzyme added quantity 1.0 U/g,temperature 30℃,time 3 h.Under this condition,the hydrolysis rate of residual CGA was 93.09%.Finally,treated with self-made CGA hydrolase and a commercial cellulase "Amano" 3（containing CGA hydrolysis activity）respectively,Sunflower seed proteins powder was prepared.The properties of four kinds of sunflower seed protein,such as the color,in vitro digestibility and solubility,water/oil holding capacity and emulsifying capacity,were compared and analyzed.After the hydrolysis of CGA,the color of sunflower seed protein changed from green to pale gray,sensory quality enhanced.The solubility was markedly improved after enzymatic hydrolysis,80.4% and 87.7% respectively.The water binging capacity and emulsification stability increased markedly and the protein in vitro digestibility increased to 88.79% and 89.27% respectively.Amino acids analysis showed that the amino acid structure of sunflower seed protein was balanced,with high nutritional value.