Study The Preparation Of Tannins From Walnut Milk Residues

China is one of the big countries for walnut planting and production,walnut is a kind of economic crops resource using as both medicine and food,and it has been received more and more abroad attention in recent years.The study found that walnut has a lot of tannins and has a series of physiological and biological activities,it can delay chronic diseases and it has been widely used in medicine and cosmetic industry.This subject mainly aimed at the walnut milk production of waste walnut milk residues as the test material and it provided by Yangyuan company in hebei province.Through the analysised of walnut milk residues,it were mixtures of walnut kernel pellicle and chopped walnuts,dry material contains protein and fat,and rich in tannins,so this material is worth further developing and utilizing.The objectives of this project were to optimize the extracting processes of tannins from walnut milk residues;purification tannins from walnut milk residues by macroporous resins;use high performance liquid chromatography(HPLC)to detection walnut milk residues tannins of free phenols were studied;in-vitro and in vivo antioxidant and antimicrobial properties of tannins from walnut milk residues.For walnut products and its by-products provides the basis for the comprehensive utilization,the ultimate aim was to advance the economic value of walnut milk residues.This topic firstly studied the basic ingredients of walnut milk residues.Tannins were obtained from the walnut residue by the method of ultrasonic wave.The characteristics of this method has high efficiency and easy operation.The optmium extraction condition was investigated by single factors and orthogonal analysis experiments.The optmium extracting conditions were the ultrasonic time of 60 minutes,the ratio of material to solution as 1:70,the supersonic temperature of 70?,the ultrasonic power of 50 W,60%ethanol solution(pH 1.5).Three extraction to get tannic acids content was 51.34 mg/g.The types of tannins from walnut milk residue were defined as the hydrolysable tannins through the performance experiment.This study had used ethanol and chloroform organic solvents for walnut milk residue at the beginning of tannic acids solution purification.According to the nature of the tannins,then choosed the nonpolar,weak polarity and medium polarity resin,then used the static adsorption experiments made by HPD-450?HPD-0450?AB-8?D-001?D-201?XDA-1?XDA-10 for screening optimum macroporous resin to purification of tannins from walnut residues,used dynamic adsorption method to study optimum macroporous resin conditions of purification of tannins from walnut residues.The results indicated that the bestest macroporous resin adsorption on tannins from walnut residues was the XDA-1.The optimal adsorption conditions were the concentration of the raw material was 0.3 mg/mL when flow velocity was 3 mL/min.The optimal elution conditions were elution velocity was 1 mL/min with 70%ethanol solution(pH 1).The eluent was collected and concentrated and vacuum drying to get powder with total tannins content 82%.In this study,an efficient high-performance liquid chromatographic gradient method for the determination of tannins in walnut residues was developed and validated.The chromatographic conditions of tannins were investigated and established.The methanol solution was chromatographed on an Inertsil ODS-SP C18(250×4.6 mm)column,with a gradient of mobile phase:2%acetic acid glacial-acetonitrile,the flow rate of 0.5 mL/min and tannins were detected at 280 nm.The result showed that the method existed fairly good linearity,the recovery rate was between 80.66%and 108.17%,and the RSD of repeatability,stability and precision were all lower than 10%.The method was high sensitivity,conveniency and accuracy.In the present study,tannins from walnut milk residue were evaluated for its in vitro and in vivo antioxidant activity.In vivo study,used the D-galactose induced Kunming mouse subacute aging model,and for its in vitro scavenging effects on the DPPH,ABTS,superoxide anions.The reducing power and inhibition of lipid peroxidation of mouse liver were also evaluated.The results showed that tannins from walnut milk residue had good antioxidant activity,and its concentration was positively correlation.The percent scavenging activity of tannins against DPPH was 85%,in the 0.5 mg/mL,the superoxide anions clearance rate was below 60%,weak in DPPH radical scavenging capacity.In the 0.5 mg/mL,tannins have the strongest scavenging activity to ABTS,the clearance rate was 93%.Meanwhile its can inhibition lipid peroxidation of mouse liver.In the 1 mg/mL,the Inhibition rate wass below 20%.Furthermore,tannins from walnut milk residues improve the antioxidant enzyme activities,including Superoxide dismutase(SOD)and Glutathione peroxide enzyme(GSH-PX),decreased malondialodehyde(MDA)levels in liver and brain and improved total antioxidant capability in D-galactose-treated mice.This study detectioned the antimicrobial property of tannins from walnut milk residue.The results showed the strong ability to inhibit the growth of Staphylococcus aureus,Escherichia coli,Bacillus subtilis,Waxy bacillus at 0.6 mg/mL.Meanwhile,inhibited the growth of Aspergillus oryzae,Mucor but Aspergillus Niger at 1 mg/mL.