Optimization Of Enzymatic Hydrolysis Conditions Of Highland Barley Glutenin And Study On Antioxidant And Anti-inflammatory Activities Of Enzymatic Hydrolysates

Highland barley(HordeumvulgareL.)is not only a necessary food for Tibetan people,but also an important cash crop in Qinghai-Tibet Plateau.Its output accounts for the highest proportion of food crops in Tibet,and it contains various nutrients.Highland barley possesses high level of protein and various essential amino acids.If the protein could be separated and extracted from highland barley and the enzymatic highland barley protein hydrolysates could be prepared,these will provide a theoretical basis for the development of functional foods and a new idea for the deep processing of highland barley.In this study,in order to further utilize highland barley,the glutenin of highland barley was extracted by Osborne method,and then the glutenin of highland barley was hydrolyzed by compound proteases.Then the enzymatic hydrolysis conditions of highland barley glutenin was optimized by response surface method,and the optimal parameters were obtained.Finally,the antioxidant and anti-inflammatory activities of enzymatic highland barley glutenin hydrolysates were evaluated by in vitro experiments.The main results are as follows:1.Osborne method was used to extract glutenin from highland barley.The enzymatic hydrolysis conditions of highland barley glutenin was optimized by single factor test,Plackett-Burman test and Box-Behnken response surface test.The optimum technological conditions of enzymatic hydrolysis were determined as follows:the ratio of alkaline protease to flavor protease was 1:1(enzyme activity ratio),enzyme addition was 18900 U/g,temperature was 58?,substrate concentration was 8%,pH=9.0 and enzymatic hydrolysis time was 180 min.Under these conditions,the actual polypeptide concentration was 19.345 ± 0.252 mg/mL,which was close to the predicted value 19.877 mg/mL.The results showed that the model established by response surface is credible and can better predict the concentration of polypeptide.The concentration of polypeptide was significantly higher than that of single factor test.2.Enzymatic highland barley glutenin hydrolysates(HBP)were divided into four components including molecular weight less than 3 kDa,3?10 kDa,10?30 kDa and 30?100 kDa(named HBP-1,HBP-2,HBP-3 and HBP-4 respectively).The in vitro antioxidant activities of HBP were determined by testing DPPH-,ABTS-and hydroxyl radical-scavenging ability.The oxidative damage model was established by H2O2-treated RAW264.7 cells to explore the effects of HBP on the level of intracellular reactive oxygen species(ROS),the activities of lactate dehydrogenase(LDH)and intracellular superoxide dismutase(SOD).The experimental results showed that different concentrations of HBP had certain scavenging effects on DPPH,ABTS and hydroxyl radical.Different components of HBP could significantly reduce the relative content of ROS and the activity of LDH in RAW264.7 cells,and HBP-2 could significantly increase the activity of SOD in RAW264.7 cells.HBP had certain protective effects on RAW264.7 cells damaged by H2O2,reduced the oxidative damage of cells,showing certain antioxidant activity.3.The oxidative damage model was established by H2O2 treatment.The Griess method was used to detect the effects of HBP on the content of Nitric oxide(NO)in the supernatant.Quantitative real-time PCR was used to detect the effects of HBP on the mRNA levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6),interleukin-10(IL-10)and transforming growth factor-?(TGF-?).The results showed that different components of HBP could significantly reduce the amount of NO and the mRNA relative expression of COX-2,iNOS,TNF-?,IL-6,and IL-10.HBP-2 and HBP-3 could significantly reduce the mRNA relative expression of IL-1?.These results showed that HBP had certain anti-inflammatory potential.