Study On Preparation,Structure Characterization And Stability Of Millet Bran Peptide-Iron Chelate

Iron is an essential trace element,iron deficiency prone to various diseases.Foodborne peptide-iron chelate is an ideal iron supplement that it has been widely studied due to its higher absorption efficiency,stable and non-toxic side effects,etc.The protein content of millet bran is 4%?10%.It has high nutritional value and the advantages of hypoallergenic,anti-oxidation,and blood pressure reduction.It has greater development and utilization value.However,currently millet bran is mainly used as feed,causing resources waste.This study uses millet bran as the raw material to extract the protein components of millet bran,separate,purify and identify peptides from the component proteins with the highest iron chelation rate,prepare peptide-iron chelate,and optimize its preparation process.To discuss its chelating site and stability,the specific experimental content and results are as follows:(1)Screening of the protein components of millet bran.Extract component proteins from millet bran,and determine the Fe²⁺chelation rate and antioxidant capacity of each component protein.The results show that the chelation rate of millet bran globulin to Fe²⁺is the highest,43.2%±2.0%,and has a certain antioxidant capacity,of which the scavenging rate of hydroxyl radicals(·OH)is 2.73%±2.3%,the scavenging rate of superoxide iron radicals is2.41%±0.5%,and the 2,2'-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid(2,2'-azino-the clearance rate of bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS⁺)is 14.6%±1.1%,and the reducing power is 0.158±0.05.(2)Optimization of the proteolysis process of millet bran balls.Taking the degree of hydrolysis(DH)and Fe²⁺chelating ability of millet bran globulin as evaluation indicators,five enzymes were used:alkaline protease,flavor protease,papain,and complex enzyme I(alkaline protease:flavor protease=1:1),complex enzyme II(alkaline protease:flavor protease:papain=1:1:1)enzymatically hydrolyze millet bran globulin,select the best enzyme.The results showed that complex enzyme II is the best protease for preparing millet bran peptide-iron chelate,its hydrolysis degree is 10.77%±0.22%,and the chelation rate of millet bran ball proteolysis is 51.48%±0.77%.On this basis,with the degree of hydrolysis and chelation rate as evaluation indicators,the response surface method was used to optimize the preparation process of the proteolysate of millet bran ball.The response surface regression equation established is:DH=0.053–0.017A+0.026B–0.015 C+0.010D+0.024AB–0.057AC–(2.564E–003)AD–(3.205E–003)BC+(4.060E–003)BD–0.031CD+0.034A²–(5.288E–003)B²+0.030 C²–0.030D².Chelation rate=40.32+1.04A+1.73B+1.22C–1.67D–1.28AB+0.046AC–(6.393E–003)AD+2.43BC+1.36BD–6.39CD–16.75A²–8.67B²–8.12C²–6.58D².Among them,A represents p H,B represents temperature,C represents time,and D represents enzyme dosage.The optimal process conditions obtained are:p H 7.83,temperature 51.6?,enzyme dosage 8.17%,time 5.9 h,predicted hydrolysis degree and chelation rate are 14.04%and 40.15%,respectively,verify the degree of hydrolysis and chelation measured by the experiment.The coincidence rates were 13.95%and38.69%respectively,which were in good agreement with the predicted values.(3)Isolation,purification and sequence identification of millet bran globulin peptides.Sephadex G-25 was used to separate and purify the millet bran globules proteolysis product(MBGH)obtained under the above-mentioned optimal process conditions.As a result,three components A,B,and C were separated and determined respectively.Chelating rate,the result shows that the second component(MBGH-B)has the highest chelating rate(98.12%);MBGH-B is further separated and purified by Sephadex G-15.MBGH-B₁ and MBGH-B₂were separated,and their chelation rate was determined respectively.The results showed that the chelation rate of MBGH-B₂ was the highest(91.72%).The peptide sequence identification of MBGH-B₂ by ultra-high performance liquid phase and electrospray ionization mass spectrometry(ESI-MS/MS)showed that MBGH-B₂ contains two oligopeptides,namely Pro-Leu-Leu-Lys and Val-Ala-Val-Leu.(4)Process optimization of millet bran peptide-iron chelate.Taking the chelation rate as an indicator,using the separated and purified MBGH-B₂ to chelate with ferrous iron,using single factor and response surface to optimize the preparation process,the established response surface regression equation is:chelating rate Y=0.85+0.34X₁+0.097X₂–0.022X₃+0.17X₄–0.051X₁X₂–(9.106E–003)X₁X₃–0.015X₁X₄+0.016X₂X₃–0.017X₂X₄+0.026X₃X₄–0.25X₁²–0.036X₂²–0.093X₃²–0.11X₄²,where X₁ represents p H,X₂ represents time,X₃ represents temperature,and X₄ represents mass ratio.The optimal parameters of chelation rate obtained by optimization are:p H value is 7.67,reaction temperature is 28.66?,reaction time is 28.78 min,peptide and iron mass ratio is 433:1,and the predicted chelation rate reaches 100%.The measured chelation rate is 97.06%,which is in good agreement with the predicted value.Structural characterization of millet bran peptide-iron chelate.Through ultraviolet spectroscopy,Fourier infrared spectroscopy,Differential scanning calorimeter(differential scanning calorimeter,DSC)and X-ray diffraction(diffraction of x-rays,XRD)and other technologies,the millet bran peptide-iron chelate compound can be analyzed from various angles.Perform structural characterization.Among them,ultraviolet spectroscopy and fourier infrared spectroscopy analysis showed that the amino and carboxyl groups of the peptide were chelated with iron;the scanning electron microscope results showed that the surface microstructure and crystallinity of the peptides were chelated with iron;XRD indicates that the crystal structure of the peptide chelated with iron is changed;DSC indicates that the peptide-iron chelate has higher thermal stability than the peptide.(5)To study the effects of different pasteurization,p H and common food excipients(salt,sugar)on the stability and anti-oxidation of millet bran peptide-iron chelate,and to simulate gastrointestinal digestion to explore millet bran peptide-iron chelate.The stability of the compound.The results show that the millet bran peptide-iron chelate can maintain good stability and antioxidant capacity when heated at 100?for different time;the peptide-iron chelate has stability under different pasteurization treatments.It is good,but its antioxidant capacity is significantly reduced at 63?,69?,72?,75?,and 80?.At the same time,peptide-iron chelate has good stability and antioxidant capacity under different p H conditions(p H 4.0?p H 10.0);higher concentration of glucose has no significant effect on its stability and antioxidant capacity.However,Na Cl leads to a significant decrease in its antioxidant capacity,and the stability of the chelate is less affected.In addition,the in vitro simulated gastrointestinal digestion results show that the peptide-iron chelate is more stable than Fe SO₄and ferrous lactate.