Study On The Preparation Of Foxtail Millet Prolamins Peptide And Its Antioxidant And Anti-inflammatory Activities

Foxtail millet is a good source of plant protein because of its hypoallergenicity and richness in essential amino-acids.The main component of millet protein is alcohol-soluble protein,which is called millet gliadin or millet prolamins?MP?.As MP contains a large number of hydrophobic amino acids,it is difficult to dissolve in water.Therefore,issues such as low hydrolysis degree and low protein conversion rate exist in the preparation of bioactive peptides by enzymatic hydrolysis.Besides,studies on the activity of millet protein peptide mainly focus on antioxidant,immune regulation,lowering blood pressure,etc.However,studies on other physiological activities are inadequate,and evaluation of its activity in cell level and animal body is relatively insufficient.Firstly,the secondary structure of MP was investigated.Secondly,peptides with antioxidant activity were prepared by enzymatic hydrolysis of MP assisted by ultrasound and heat pre-treatment.Moreover,its structure was characterized.Thirdly,the stability during food process and in vitro digestion was assessed.Fourthly,the antioxidant and anti-inflammatory activities in cells as well as its possible mechanism was studied.Finally,these actives were re-evaluated by animal tests.The main results were as follows:?1?The structure of MP was studied.Sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?results showed there were 3 main molecular weight?MW?distribution zones of MP from 11 to 25 kDa,especially in 17 kDa.X ray diffraction results indicated MP was amorphous in structure and high diffraction intensity at 2?of 20 implied high proportion of?-helices.The shape of MP was like cylindrical and its outer surface was smooth and wrapped.Circular dichroism?CD?analysis showed MP belongs to all-?secondary structure and most protein molecules were concealed in helical structures unfavorable for the enzymatic hydrolysis.The amino acids with higher content in the MP were glutamic acid,leucine,aspartic acid and phenylalanine,which accounted for 59.1%of the total amino acids.In addition,hydrophobic amino acids accounted for 44.8%of the total.?2?Ultrasound and heat treatment was used to change the secondary structure of MP,in order to increase its water solubility and hydrolysis by protease.Ultrasound treatment could alter part of its?-helical structure into?structures.Ultrasound treatment combined with heat treatment could lead to a decrease of helical structure by 22%and an increase of?structures by 11.3%.Besides,the solubility of MP surpassed 60%and degree of hydrolysis?DH?was over 30%both significantly higher than control?p<0.05?.?3?Ultrafiltration,gel chromatography and RP-HPLC were used to separate and purify the antioxidative active peptide from millet prolamins hydrolysate?MPH?.The amino acid sequence was identified and the stability of its activity was further investigated.Two novel antioxidant peptide:PFLF and IALLIPF were isolated and identified from MPH with MW<1kDa by alcalase?MPH-A-I?,whose 1-diphenyl-2-trinitrophenylhydrazine?DPPH?free radical scavenging ability were 149 and 134?M Trolox Equivalents?TE?/g protein,Oxygen radical absorbance capacity?ORAC?were 1180 and 700?M TE/g protein.Effects of processing conditions,food materials,metal ions and simulated gastrointestinal digestion on the stability of antioxidant activity of MPP were studied.The activity of MPP decreased significantly as temperature rose up to 85?.When the temperature reaches 100?,the DPPH free radical activity of MPH-A-I decreased by 30%compared with that at 25?.Extreme pH?<6 or>8?affected the activity stability of the peptide.When pH was 6?8,the DPPH free radical scavenging activity retention rates of the three peptides were greater than 85%.NaCl could improve the DPPH radical scavenging capacity of MPP.Sucrose at the concentration of 2-10mg/mL had no significant effect on the antioxidant activity of MPP.Citric acid had synergistic effect on the antioxidant activity of MPP.When the citric acid mass fraction was 0.2 mg/m L,the DPPH radical scavenging rate of the three peptides increased by 4%,11%and 15%,respectively.Potassium sorbate and sodium benzoate in the concentration range of 0.04-0.20mg/mL had little effect on the stability,and its DPPH radical scavenging activity maintenance rate was above 85%.The effect of Cu²⁺and Zn²⁺on the antioxidant activity of MPP was greater than K⁺,Mg²⁺and Ca²⁺.The free radical scavenging activity decreased after simulated gastrointestinal digestion in vitro,but the activity retention rate remained above 83%.?4?HaCaT cells induced by H₂O₂ and rat macrophages?RAW264.7?induced by lipopolysaccharide?LPS?was used to assess anti-oxidant and anti-inflammation capacity of three MPP?MPH-A-I,PFLF,IALLIPF?.MPP could reduce oxidative damage of HaCaT cells induced by H₂O₂ and reduce reactive oxygenspecies?ROS?from oxidative injured HaCaT cells in a certain concentration range.The activity of superoxide dismutase?SOD?and catalase?CAT?as well as glutathione?GSH?significantly increased with the increase of peptides concentration,leading to a significant decrease of malondialdehyde?MDA??p<0.05?.Inflammation in RAW264.7 induced by LPS was relieved by MPP.Cell differentiation was effectively prevented by peptide IALLIPF at the concentration of 250?g/m L.And the cell morphology was improved as well as control group.MPP exhibited anti-inflammatory effect by inhibiting the production of NO and pro-inflammatory cytokines including Tumornecrosis factor??TNF-??,Interleukin 6?IL-6?and Interleukin 1??IL-1??.The inhibition effect of IALLIPF was better than that of MPH-A-I and PFLF.?5?Nuclear factor kappa-B?NF-?B?as well as mitogen-activated protein kinase?MAPK?signal pathway was investigated to learn the mechanism of anti-inflammatory activity.MPP significantly?p<0.05?reduced the phosphorylation level of the inhibitor of the NF-?B?I?B?and the nuclear transport of the p65 protein,suggesting that the NF-?B signaling pathway played an important role in the MPP's suppression of the level of pro-inflammatory cytokines in RAW264.7 macrophages induced by LPS.In addition,MPP could significantly?p<0.05?reduce the phosphorylation of three mitogen-activated protein kinase?MAPK?family proteins:p38,Erk1/2 and JNK in RWA264.7 cells,suggesting that the bioactive peptide inhibited the expression levels of the pro-inflammatory factors TNF-?,Il-6 and Il-1?through the MAPK signaling pathway.?6?D-Galactose-induced oxidative damaged mice and Dextran sodium sulfate?DSS?-induced mice model was used to assess in vivo bioactivity of IALLIPF.IALLIPF exhibited anti-oxidant ability in D-Galactose-induced oxidative damaged mice.After 6weeks of continuous administration of MP peptide,compared with model group,the serum level of total antioxidant capacity?T-AOC??an increase of 53%?,and SOD?an increase of59%-147%?,CAT?an increase of 30%-94%?,GSH-Px?an increase of 35%-126%?in main organs including brain,heart and liver of high dosage group?500 mg/kg·d?were significantly improved?p<0.05?.In addition,MDA level significantly?p<0.05?decreased to42%-64%as much as modle group.IALLIPF improved the clinical symptoms of colitis in DSS-induced mice and down-regulated myeloperoxidase?MPO?activity and the expression level of TNF-?and IL-6 in colon.Compared to control,MPO activity,TNF-?and IL-6 level in high dosage group?500 mg/kg·d?decreased by 40%,59%and 56%,respectively.