Study On The Preparation Of Corn ACE Inhibition Peptides By Ultrasound Assisted Enzymatic Hydrolysis-membrane Separation With Gradient Feeding Substrate And Its Fe²⁺ Chelating Properties

The purpose of this research was to increase the yield of corn peptides,achieve a continuous and efficient production method of ACE inhibition peptides,and develop a novel iron supplement.Corn meal is a byproduct of corn processing and was used as the raw material in this study.The optimal feeding and ultrasonic assisting conditions were determined based on the different feeding methods and the ultrasound assisted methods.In addition,the continuous production method of corn ACE inhibition peptides by dual-frequency ultrasound assisted enzymatic hydrolysis-membrane separation with gradient feding substrate further improved the yield of corn peptides.And the ultrasound influencing mechanism on the structures of protein-retaining and peptide-transmitting also was explored.Furthermore,a new type of ferrous iron supplement was prepared by chelation reaction of corn ACE inhibition peptide and ferrous iron.The chelation reaction mechanism of corn peptides and iron and the stability of chelate were further explored at the molecular level.The main research conclusions were as follows:(1)Through the comparative tests of the feeding method and the ultrasoundassisted method,it was concluded that the gradient dilution feeding method and the 20/28 k Hz dual-frequency focused ultrasound-assisted method were more suitable,which significantly improved the yield of peptides.The optimized process parameters were obtained as follows:substrate concentration 10.86 mg/m L,added enzyme amount 7329 U/g,4 times of ultrasound,enzymolysis time 300 min.Under these conditions,the peptide yield and ACE inhibition rate were 68.11%and 86.09%,respectively.Compared with the non-ultrasonic distributed enzymatic membrane separation mode,the peptide yield and protein conversion rate were increased by 58.72%and 41.59%,respectively.It can be concluded that the dual-frequency ultrasound-assisted gradient dilution feeding enzyme membrane coupling preparation method was an efficient and continuous method for preparing corn ACE inhibition peptides.(2)Through the analysis of ultraviolet-visible(UV)spectra,fluorescence spectra,surface hydrophobicity,sulfhydryl and disulfide bond contents,and circular dichroism(CD)results,it was found that ultrasonic treatment changed the structure of corn protein and peptide.In terms of protein,the intensity of UV characteristic absorption peak was increased,the intensity of fluorescence was decreased,the surface hydrophobicity and the content of disulfide bonds were decreased,and the intermolecular/intramolecular hydrogen bonding strength was increased.In terms of peptide,the surface hydrophobicity and the content of sulfhydryl groups were increased,and the intermolecular/intramolecular hydrogen bonding strength was enhanced.However,it was found that ultrasonic treatment had no significant effect on the secondary structure of corn protein and peptide.Scanning electron microscopy(SEM)analysis revealed that ultrasonic treatment made the apparent structure of corn protein and peptide uneven,which was helpful to accelerate the enzymolysis reaction and increase the yield of peptides.Atomic force microscopy(AFM)results showed that the size of corn protein particles was significantly reduced by the ultrasonic treatment,the total number of protein particles was significantly increased,and the distribution become more concentrated,which was also conducive to improving the yield of peptides.On the other hand,the size of corn peptides was significantly larger,and the aggregation phenomenon of peptides was obvious.Finally,the result of amino acid composition showed that ultrasonic treatment had no significant effect on the amino acid composition of corn peptides.(3)Through single factor and response surface tests,the optimal preparation parameters of corn ACE inhibition peptide-iron chelate were obtained as follows:ascorbic acid concentration 2%,peptide concentration 10 mg/m L,peptide iron ratio 5:1,p H 10,reaction temperature 20?,and chelating time 40 min.Under these conditions,the chelation rate was 66.39%,the chelate yield was 82.87%,and the ACE inhibition rate was 82.21%.Iron chelation had no significantly effect on the ACE inhibitory activity of corn peptides.Furthermore,the existence of peptide-iron chelate was confirmed by the qualitative detection of Na₂S.Thus an efficient preparation method of corn ACE inhibition peptide-iron supplement was obtained.(4)The results of UV spectra showed that the corn ACE inhibition peptide did combine with ferrous ion to form a chelate,which made the absorption peak blue shift and the peak intensity increased significantly.FTIR results further confirmed the formation of chelate,and determined that carboxyl,amino,and carbonyl were the main chelating reaction sites of corn peptide with ferrous ion.The results of SEM and EDS showed that the Fe peak was found in the chelate,which proved that the chelation reaction between ferrous iron and corn peptide did occur,and the chelate showed a compact and porous apparent structure.Atomic force microscopy and particle size distribution results showed that the chelation reaction made the particle size of the chelate significantly smaller and the distribution was more uniform and concentrated.The result of zeta potential analysis showed that the negative charge on the chelate surface was significantly reduced,which further proved that the carboxylic acid group was one of the binding sites with ferrous ion in the peptide side chain.XRD results indicated that the chelate formed a new diffraction peak at 2?=12.22°which further proved the formation of chelate.The analysis of the amino acid composition showed that the acidic amino acids Asp and Glu(total content of 44.02%)and the basic amino acids Lys and Arg(total content of 14.57%)in the peptide were mainly involved in ferrous iron chelation.(5)The stability test results showed that the corn ACE inhibition peptide-iron chelate had a good thermal decomposition stability,and the maximum thermal decomposition temperature was 195?.The corn ACE inhibition peptide-iron chelate had a good stability(retention rate?95.47%)under neutral and alkaline conditions(p H6?10)and a high thermal stability(retention rate?85.10%)in a wide temperature range(25?70?).In additon,the chelate had a good resistance to gastrointestinal enzyme digestibility(retention rate of 79.18%).Thus corn ACE inhibition peptide-iron chelate was a new type of iron supplement with good stability.