The Study Of Corn Peptide Preparation Technology And Its Antioxidant And Anti-inflammatory Activities

Corn,one of the three widely cultivated cereals in the world,which has the advantages of high yield,wide planting area and high nutritional value,etc.Corn protein,a main byproduct of the corn wet-milling process,is insoluble in water due to large amounts of hydrophobic amino acids.The poor solubility of corn protein and the special construction have created barrier for enzymes to attack the cleavage of the protein,which seriously limited the functional peptides'release and reduced the corn protein's bioavailability.Therefore,developing a more efficient technique to overcome these disadvantages mentioned above is in great demand.In this study,corn gluten meal was pretreated with ultrasound to improve the degree of hydrolysis,protein conversion and antioxidant activity of hydrolyzate.Additionally,the antioxidant activity and the physicochemical properties were investigated by simulating gastrointestinal tract digestion in vitro.Furthermore,the anti-inflammatory activity of corn peptides on colitis,endothelial cell inflammation and the NF-?B signaling pathway related to inflammatory on EA.hy926 cells were studied.The main research contents and results are as follows:?1?Preparation of corn antioxidant peptides by ultrasound-assisted corn proteolysis and the effect of ultrasound on its physicochemical properties and antioxidant activity were studied.An orthogonal design L₉?3⁴?was applied to ultrasonic-assisted enzymolysis in the preparation of corn antioxidant hydrolysates.Under the optimal ultrasound pretreatment conditions?5 W/L power,2s/2s on/off time,50°C temperature and 25 min time?,the DH and the conversation of con protein were24.70%and 62.85%,significantly increased by 28.17%and 29.93%,respectively.After ultrasound pretreatment,the content of<200 Da peptides,200–1000 Da peptides,1000-3000 Da peptides,>3000 Da peptides were 8.13%,63.31%,17.69%and 7.90%successively.Which showed ultrasound pretreatment caused an increase of11.84%and 21.29%for the fraction of 200–1000 Da peptides and 1000-3000 Da peptides,correspondingly.Meanwhile,the fraction of<200 Da,>3000 Da peptides decreased by 15.61%and 46.5%,respectively.The content of free amino acids in the hydrolyzates was 55.24?g/mL,which decreased by 5.63?g/mL.The content of peptides in the hydrolyzates was 777.18?g/m L,which increased by 85.66?g/m L.The hydrophobic amino acids in form of peptides were 391.95?g/m L,which increased by 55.17?g/m L.The antioxidant activity of zein hydrolyzate were increased remarkably,and the IC₅₀ of DPPH and·OH radical scavenging decreased from 2.41 mg/mL to 1.95 mg/mL,9.98 mg/m L to 7.61 mg/m L,respectively.?2?The objective of this chapter was to investigate the physicochemical properties and antioxidant activity of corn protein hydrolysates under simulated gastrointestinal digestion,which were prepared by ultrasound under the conditions optimized in Chapter 2.After simulated gastrointestinal?GI?digestion,the DH of corn protein significantly increased from 22.67%to 39.40%,77.51%peptides were still retained.Amino acid analysis showed some peptides containing special amino acids?Pro,Asp and Glu?had strong ability to resist digestion.Compared to the undigested CPH,the final CPH digest exhibited excellent antioxidant activity.The DPPH radical rate were increased from 33.51%to46.29%?12.78%increase?,hydroxyl radicals scavenging activity?4.12%increase?and reducing power?50.94%increase?,but lower inhibition rate of linoleic acid peroxidation?41.53%decrease?.Two GI resistant antioxidant peptides?Tyr-Pro-Gln,406.45 Da;Ala-Tyr-Leu,365.43 Da?were purified with multi-step chromatography and identified by UPLC-ESI-MS.The IC₅₀0 of DPPH radical scavenging activities of these two peptides were 10.251?g/mL and20.271?g/m L,respectively.?3?The anti-inflammatory activity of corn antioxidant peptides on intestinal inflammation was studied by cell experiment and mice model.corn antioxidant peptides digest significantly?p<0.05?decreased TNF-?-induced COX-2,INOS and IL-8 expression on Caco-2 cells.The MPO activity,the pro-inflammatory cytokine expression?TNF-?,IL-6?in the mouse colon,was significantly?p<0.05?down-regulated by corn antioxidant peptides after DSS treatment,but was inhibited upon PL administration.The corn antioxidant peptides supplementation was shown to prevent DSS-induced loss of weight,colitis symptoms,and shortening of colon length.The corn antioxidant peptides could largely improve the morphological characteristics of colonic tissue of colitis mice induced by DSS;reduce colonic tissue damage and inflammatory cell infiltration.?4?The objectives of this chapter were to investigate the anti-inflammatory activity on endothelial EA.hy926 cell and bioavailability of zein hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption,and to identify novel anti-inflammatory peptides after transport across Caco-2 cells.Zein hydrolysates were prepared by Alcalase,thermolysin and neutral protease,and further digested using gastrointestinal proteases;All three zein hydrolysates showed anti-inflammatory activity by significantly reducing the expression of tumor necrosis factor-??TNF-??induced VCAM-1 significantly?p<0.05?.Their transport was studied in Caco-2 cells.Anti-inflammatory activity was studied in endothelial EA.hy926 cells.The simulated gastrointestinal digestion has little effect on the anti-inflammatory activity of zein hydrolysates.The transported digest displayed stronger anti-inflammatory activity than that of digest by inhibiting VCAM-1 and ICAM-1 expression significantly?p<0.01?.The transport percent of thermolysin hydrolysate digest was 1.2±0.2%after 4 h's transport on Caco-2 cell monolayers.Three novel peptides PPYLSP,IIGGAL and FLPPVTSMG that could be absorbed through Caco-2 cells were identified and sequenced and showed strong anti-inflammatory activity at 0.2 mM.?5?The effect of corn peptides on TNF-?induced inflammation and NF-?B signal pathway on endothelial cell were investigated.Zein hydrolyzate?4 mg/m L?,three peptides PPYLSP,IIGGAL and FLPPVTSMG?0.4mM?could significantly inhibit TNF-?-induced ICAM-1 and VCAM-1 expression by 22.63%-62.73%and56.23%-76.62%respectively in EA.hy926 cell?p<0.01?.Both of them could significantly decrease TNF-?-induced ROS by 25.27%-36.18%?p<0.05?.Both of them could significantly reduce TNF-?-induced adhesion of EA.hy926 cells to monocytes U937 cells?p<0.01?;the effect of peptide FLPPVTSMG was significantly better than peptide IIGGAL.Zein hydrolyzate and three peptides could significantly inhibit TNF-?induced down-regulation of TNFR1 in EA.hy926?p<0.05?;while could not alter TNFR2 levels.Zein hydrolyzate and three peptides could significantly?p<0.01?attenuate TNF-?-induced p65 phosphorylation on EA.hy926 cell;while has little effect on TNF-?induced I?B?and I?B?degradation.The above results showed that the ultrasound-assisted corn gluten meal enzymolysis can effectively improve the conversion rate of corn protein,hydrolysis degree and antioxidant activity of corn hydrolysates;the antioxidant activity of corn peptide has good stability under simulated gastrointestinal digestion;The corn peptides has remark anti-inflammatory activity on intestinal inflammation and endothelial EA.hy926 cells.This study provided a novel idea and theoretical basis for the preparation of functional peptides with antioxidant activity and anti-inflammatory activity from corn gluten meal.