| Millet resources are very rich and used to be the staple food in China.Bioactive peptides derived from grains play an important role in the prevention and treatment of oxidative stress and inflammation-related chronic diseases.Germination is an economical and feasible processing method to improve the nutritional value of grains.However,germinated grains are still raw materials that need to be heated and cooked before consumption,while heat treatment is also a feasible method to release bioactive peptides from food proteins.In addition,gastrointestinal digestion is an inevitable process of food proteins in human body and plays an important role in the biological activity of peptides.Nevertheless,the potential role of germination and heating(cooking and microwaving)of foxtail millet as a source of dual antioxidant and anti-inflammatory peptides after digestion in the gastrointestinal tract is not available yet.In this study,the aim was to investigate if digestion of germinated and heated foxtail millet proteins might release peptides with a positive role on the oxidative stress and protecting from inflammation after it was digested in an internationally standardized static gastrointestinal digestion model in vitro.This work is divided into four parts,mainly including:?Screening of ultrafiltrated fraction and RP-HPLC subfraction of foxtail millet and germinated foxtail millet protein digests with the highest activity based on chemical methods;?Effects of processing on the activity of RP-HPLC subfraction of of foxtail millet and germinated foxtail millet protein digests based on cell assay;?Assessment of absorption and transport capacity of RP-HPLC subfraction of foxtail millet and germinated foxtail millet protein digests;?Identification of the structure of the highest active RP-HPLC subfraction of foxtail millet and germinated foxtail millet protein digests.The main methods,results and conclusions are presented as follows.1.Four chemical antioxidant methods(ABTS radical scavenging ability,oxygen radical absorbance capacity,ferrous ion chelating ability and ferric reducing antioxidant power)and two chemical anti-inflammatory methods(nitric oxide production inhibition,BSA denaturation inhibition)were used to screen the ultrafiltrated fraction(>10 kDa,3-10 kDa,and<3 kDa)of foxtail millet and germinated foxtail millet protein digests,and it was shown that different antioxidant methods lead to different results,where<3 kDa fraction had a better ability of radical scavenging and chelating ability,and>10 kDa fraction had the highest reducing capacity.In addition,both anti-inflammatory methods show that<3 kDa fraction had a better inhibition rate.Accordingly,<3 kDa fraction was selected as the optimal fraction for further separation.2.The RP-HPLC for<3 kDa fraction was separated into four subfractions(F1,F2,F3 and F4)according to the flowing time,followed by determining the antioxidant and anti-inflammatory activity of subfractions of foxtail millet and germinated foxtail millet protein digests by using the same chemical methods.It was shown that the F4 subfraction had the highest antioxidant activity in all antioxidant methods,while different anti-inflammatory methods also showed the F4 subfraction has the highest anti-inflammatory activity.Finally,the F4 subfraction was regarded as the optimal subfraction for further evaluation.3.The oxidative stress model of Caco-2 cells induced by hydrogen peroxide was used to test the antioxidant activity of F4 subfraction in foxtail millet treated by different processing.The results showed that hydrogen peroxide decreased the cell activity to 70.84±1.94%,increased intracellular ROS to 140.71±8.32%,decreased GSH content from 90.17 ± 2.47 to 66.73±0.18 ?mol/g protein,and reduced SOD activity from 0.28±0.05 to 0.17 ± 0.02 U/mg protein.However,heat treatment,especially the boiled germinated millet,would increase ROS content,reduce GSH content and SOD activity,thereby reducing antioxidant activity.Germination can increase ROS production,but increase GSH content and SOD activity,so the effect of germination on oxidative stress of caco-2 cells is not clear,but all samples showed antioxidant activity.4.The inflammation model of RAW264.7 macrophage induced by lipopolysaccharide was used to test the anti-inflammatory effect of F4 subfraction in foxtail millet treated by different processing,and the results showed that LPS increased NO content from 4.43± 0.07 to 24.22±0.56 ?M,TNF-? content increased from 68.11 ± 2.30 to 138.06 ± 2.19 ng/mL,IL-6 content increased from 119.64±6.88 to 418.79± 19.91 pg/mL,and PGE2 content increased from 18.94 ± 0.54 to 21.18 ±0.34 pg/mL.However,boiling treatment could reduce the contents of NO,IL-6,TNF-? in the supernatant of RAW264.7 cells,thus enhancing the anti-inflammatory activity of F4 subfraction.However,the anti-inflammatory effect of germination and microwave treatment was not clear because germination increases NO and TNF-? but decreases IL-6 content,while microwave treatment had the opposite effect.In addition,all samples failed to inhibit PGE2 production.Therefore,the F4 subfractions of cooked samples(RB and GRB)were selected for further structural identification.5.The transport ability of F4 subfraction was assessed in Caco-2 cell monolayer model,and it was shown that the apparent permeability coefficient(Papp)decreases gradually with the transit times.After transport for 4 h,Papp values were around 4×10-5 cm/s,which were greater than 2×10-6 cm/s,suggesting that all F4 subfraction had a good ability of transport.6.The peptide sequence of the most active F4 subfraction(RB and GRB)was identified by LC-MS/MS and it was shown that eleven kinds of peptide sequence were identified from F4 subfraction of RB samples.Intersetingly,most of the peptides were rich of glycine residues(G).Moreover,seven kinds of peptides were identified from GRB sample,and most of the peptides had glutamic acid(E)and aspartic acid residues(D).7.This study simulated the consumption of foxtail millet and germinated foxtail millet by human,that is,the raw material-cooking-digestion process.The chemical and cytological methods were used to test the bioactivity,and it was shown that foxtail millet protein may play an antioxidant and anti-inflammatory role in this process.The results show that boiling was the most suitable method for cooking,and the identified peptides contributing to bioactivity were MAAAFSL?AVWCYLRF?AMDAGGGGSGSGQL?IINEPTAAAIAYGLDK?ISGLIYEETR?AMLEGKSEPECK?AVFPSIVGRPR?MGGGGGGAGR?GGGDDDDDADK?MMGDSTYR?KLSIIISK in raw millet and SEDDPFD?REEGVLIF?EAGNKGTLSF?MGPIPSTL?EDDQMDPMAK?QNWDFCEAWEPCF?MSHRGACGCEK in germinated millet.Furthermore,we found that glycine residues(G)in raw millet,and glutamic acid(E)and aspartic acid residues(D)in germinated millet played an important role in these peptides.Therefore,this study can provide theoretical basis for the development of bioactive peptides and the choice of cooking methods of foxtail millet and germinated foxtail millet,thus,promoting the rational and high-value utilization of foxtail millet resources. |
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