Studies On Preparation Of Antioxidants From Milk Thistle Meals And Its Activity

Lipid oxidation is the main cause of deterioration of fat food during processing and storage.Adding antioxidants can reduce off flavor,inhibit nutrition loss and prolong shelf life.However,synthetic antioxidants have potential damage.This leads to develop natural antioxidants.Meanwhile,because of people's pursue for health,supply of exogenous antioxidants becomes very necessary.These make people research for high efficiency,safety and multifunctional natural antioxidants.Milk thistle meal,residue of herb milk thistle seeds after extracting silymarin, containing abundant protein,carbohydrates and flavonoids,and it has great nutrition value.The objective of this study was to prepare antioxidants,extracting total flavonoids from milk thistle meal,separated by macroporous resin;extracted polysaccharide from the residue of milk thistle meal;then prepared antioxidative peptides by fermenting the residue of milk thistle meal with Bacillus Natto,and desiccated bacterial sludge to powder.This work achieved comprehensive utilization, extended manufacture chain,and improved added value of agricultural product.A method for evaluating oxidation status of refrigerated cooked ground pork was established by synchronous fluorescence.10mL0.02%Vc solution was added to 0.5g cooked ground pork,and then extracted by ultrasonic with strong class,△λwas 70nm, excitation slit width was 10nm,emission slit width was 10nm,determine synchronous fluorescence intensity of samples at 326nm.In order to improve screening efficiency,a method for determining inhibitory rate of antioxidants on lipid oxidation of refrigerated cooked ground pork was established,stored at 4℃for 3 days,inhibited synchronous fluorescence intensity as index.This method was simple,sensitive and accuracy.Milk thistle meal total fiavonoids were extracted by 60%ethanol and the extraction rate was 91.9%.Macroporous resin DM-130 was chosen to purity the total flavonoids by static adsorption and desorption.The best separated technology was that 200mL total flavonoids solution was loaded at 2mL/min rate,8 times of bed volume water was used to wash impurity,then eluated at flow rate 0.9mL/min by 4 times of bed volume 80%ethanol.As a result,its purity increased from 24.32%to 78.14%.The EC₅₀ of milk thistle meal crude flavonoids and refined flavonoids inhibitory rate on lipid oxidation of refrigerated cooked ground pork were respectively 0.2659mg/mL and 0.1331mg/mL. Polysaccharide was extracted from the residue of milk thistle meal assisted by pulsed ultrasonic.The best result of extraction ratio 4.06%was obtained by 2—level Factorial Design and Response Surface Methodology under the optimized condition, granularity was 60 mesh.temperature was 45℃,the ratio of liquid to solid was 25,the extracting time was 43 minutes,,ultrasonic power was 550 W.The deproteinization technology was achieved by comparing the percentage of the deproteinization and losing polysaccharide and the capacity of polysaccharide solution scavenging DPPH free radical.The results showed that,adjusting the pH value of 1%polysaccharide solution to 2 by HCl,the sample was placed stably for a night and then centrifugaled, repetitive operation twice,the percentage of the deproteinization was 99.9%,the percentage of losing polysaccharide was 18.1%and the capacity of polysaccharide solution scavenging DPPH free radical was 40.3%.The crude polysaccharide was fractionated with ethanol repeatedly and four fractions were obtained.The EC₅₀ of milk thistle polysaccharide inhibitory rate on lipid oxidation of refrigerated cooked ground pork was 11.028mg/mL.The inhibitory rate of all fractions were lower than that of milk thistle polysaccharide。Milk thistle antioxidative peptides were prepared by fermentation method at first time.The best composition of culture medium was obtained by single factor and orthogonal design which composed of 10%the residue of milk thistle meal,0.06% glucose,0.03%CaC12 and 0.05%MgSO4.The best result of the peptide conversion ratio 60.96%was obtained by Plackett-Burman design and Response Surface Methodology under the optimized condition,the shaking speed was 180rpm,seed culture time was 16h,inoculation volume was 5%,the volume of medium in 500ml flask was 60ml,temperature was 36.5℃,initial pH value was 6.9,fermentation time was 28.5h.The peptide conversion rate was 62.47%by 5 liter fermentor.The ultrafiltration results showed that the inhibitory rate of peptides whose molecular weight were less than 10k Da on lipid oxidation of refrigerated cooked ground pork was 75.16%,while that of peptides having a molecular weight of less than 3k Da was 77.59%.Consider synthetically,the peptide having a molecular weight of less than 10k Da was chosen as antioxidative peptide.Antioxidative peptides were made into powder by spray drying technic,where peptide content was 95.14%,and it's inhibitory rate on lipid oxidation of refrigerated cooked ground pork was 73.28%and EC₅₀ was 5.28mg/L.The results of in vitro experiments showed that the EC₅₀ of scavenging DPPH radical of milk thistle meal purified flavonoids,polysaccharides and peptides were 0.992,0.527,9.468 and 4.811mg/mL,total antioxidative capacity of 10mg/mL BHT, milk thistle meal purified flavonoids,polysaccharides and peptides were equal to 12.77,9.27,1.86,2.94mmol/L FeSO4 respectively,chelating metal ions capacity of 10mg/mL BHT,milk thistle meal purified flavonoids,polysaccharides and peptides were equal to 0.049,0.028 and 0.115mg/mL Na₂EDTA respectively.The results of cooked ground pork experiment during 7 days refrigerated storage showed that the inhibitory effect on oxidation of adding 0.04%milk thistle meal purified flavonoids,3%polysaccharides and 1.5%peptides was equivalent to that of 0.02%BHT.TBARS content,carbonyls content,non heme iron content had great correlation with synchronous fluorescence intensity,and this proved that synchronous fluorescence intensity could evaluate the oxidative stress of cold-storage cooked ground pork.A method for evaluating protein oxidation damage in vitro by synchronous fluorescence was established.△λwas 30nm,excitation slit width was 20nm,emission slit width was 20nm,determined synchronous fluorescence intensity of BSA solution at 404nm.The method was simple,fast and sensitive.The results of inhibitory effect on protein oxidative damage in vitro showed milk thistle peptide inhibited oxidative damage by chelating metal ions and scavenging free radical,while milk thistle purified flavonoids inhibited oxidative damage by scavenging free radical.The inhibitory effect of milk thistle polysaccharide was weak.The inhibitory effect on protein oxidative damage of 0.03%milk thistle meal purified flavonoids and 1%peptides was equivalent to that of 0.03%Vc.