CN109439715B - Preparation method of mung bean protein peptide-zinc chelate - Google Patents
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Abstract
The invention relates to a preparation method of mung bean protein peptide-zinc chelate, which takes mung bean protein as a raw material, obtains mung bean polypeptide hydrolysate after alkaline protease hydrolysis, and obtains mung bean protein peptide-zinc chelate after polypeptide in the mung bean polypeptide hydrolysate is subjected to freeze drying and is subjected to chelation reaction with zinc. According to the invention, the mung bean protein peptide is chelated with zinc ions, and the characteristic that the mung bean protein small peptide is easy to absorb by intestinal tracts is utilized, so that the zinc absorption and accumulation in the body by the small intestine can be promoted, the absorption utilization rate of zinc is improved, and the loss caused by the precipitation of zinc ions in an alkaline intestinal tract environment can be avoided.
Description
Technical Field
The invention belongs to the field of food processing, and relates to a preparation method of a mung bean protein peptide-zinc chelate.
Background
The mung bean is a leguminous plant seed used as both medicine and food, contains rich protein, starch, vitamins, mineral substances and amino acids necessary for human bodies, wherein the content of the protein is as high as 19.5-33.1 percent and is 2-3 times higher than that of cereal grains, the mung bean is rich in complete protein of methionine, tryptophan, lysine, leucine and threonine, and the chemical scores of the amino acids in the mung bean protein are all higher than the recommended value according to WHO/FAO score, so that the mung bean is high-quality plant protein; the mung bean polypeptide is an enzymolysis product of mung bean protein, mainly contains micromolecule peptide consisting of 3-6 amino acids, and also contains a small amount of macromolecular peptide, free amino acids, saccharides, inorganic salt and other components, wherein the amino acids mainly comprise methionine, tryptophan, tyrosine and arginine, and the mung bean polypeptide has a wide application prospect as a functional food or food additive compared with the mung bean protein, because the mung bean polypeptide still can keep higher solubility under the condition of lower pH and has small viscosity change along with the rise of concentration; in addition, the mung bean polypeptide can be used as a nutrient source of protein for patients with weak gastrointestinal functions or for reducing gastrointestinal burden, and can be used as a food for enteral nutrition adjuvant therapy or postoperative recovery patients because the mung bean polypeptide has low antigenicity and does not cause allergic reactions after eating.
Zinc is one of the essential trace elements of human body, has very important physiological functions, and is mainly expressed in that: (1) involved in the metabolism of proteins, carbohydrates, lipids and nucleic acids; (2) maintaining the integrity of the cell membrane structure; (3) promoting the growth and development of organisms and tissue regeneration; (4) promoting normal function of skin and bone; (5) promoting intelligence development; (6) improving normal taste sensitivity; zinc is very important for the growth and development of children, particularly infants, the zinc deficiency can lead the growth and development of the infants to be slow, the intelligence rate is poor, the immunologic function is reduced, the absorption rate of a human body to zinc in diet is low, the main reason is that zinc and phytic acid can form an insoluble compound in small intestines, and the insoluble zinc-phytic acid compound cannot be absorbed and utilized by the human body due to the lack of corresponding phytase in the intestinal tract of the human body, so the bioavailability of the zinc is greatly reduced; the zinc chelate has the structural characteristics that through the action of a coordination covalent chemical bond, a compound with stable chemical properties is formed, under a proper condition, N-terminal amino, C-terminal carboxyl, amino acid side chains, carbonyl groups, imino groups and other groups in the peptide chain can be used as ligands to provide electrons for metal zinc ions, and a stable compound is formed, wherein in the compound, most of zinc ions are positioned in the center of a ring, are not easy to dissociate, and have more stable chemical properties; the absorption and operation of the peptide zinc chelate are carried out according to the absorption and operation mechanism of the peptide, but not by the absorption and utilization of a metal operation system, the chelate enters blood through an intestinal mucosa layer in an integral form according to the absorption and transportation mechanism and characteristics of small peptide, the competitive antagonism of metal ions during intestinal absorption is avoided, the absorption is fast, the energy consumption is low, and a plurality of biochemical processes can be reduced, so that the bioavailability of trace element zinc is improved.
Disclosure of Invention
The invention aims to provide a preparation method of mung bean protein peptide-zinc chelate, which overcomes the defect of low absorptivity of the existing zinc supplement preparation and promotes the absorption of zinc by a human body.
The invention is realized by the following technical scheme:
a method for preparing mung bean protein peptide-zinc chelate compound takes mung bean protein as raw material, the mung bean protein peptide hydrolysate is obtained after alkaline protease hydrolysis, and the mung bean protein peptide-zinc chelate compound is obtained after polypeptide in the mung bean protein peptide hydrolysate is frozen and dried and is subjected to chelation reaction with zinc.
Further, the protease is an alkaline protease.
Further, the method also comprises the steps of carrying out ultrafiltration and classification on the mung bean protein peptide hydrolysate, and screening out 3-5ku peptide fragments for subsequent chelation reaction.
Further, the chelating reaction conditions are that the mass ratio of the polypeptide to the zinc is 3:1-7:1, the pH value is 4-8, the temperature is 30-70 ℃, and the reaction time is 40-120 min.
Further, the chelating reaction conditions are that the mass ratio of the peptide to the zinc is 6.08:1, the pH is 6.31, the temperature is 50.32 ℃, and the reaction time is 80.99 min.
Specifically, the method comprises the following steps: dissolving 50g of mung bean protein in 1.25L of deionized water, pretreating for 15 minutes in a 90 ℃ water bath kettle, adjusting the pH to 9 with 0.5mol/L NaOH after the temperature is reduced to 55 ℃, adding 4ml of alkaline protease, keeping the pH to 9 with 0.5mol/L NaOH, hydrolyzing for 4 hours, and inactivating enzyme in a boiling water bath; cooling the hydrolysate to room temperature, adjusting the pH value to 7 with 1mol/L _ HCL, centrifuging at 5000r/min for 15min, and collecting supernatant; ultrafiltering the supernatant under 0.25MPa, sequentially ultrafiltering with ultrafiltration membrane with molecular weight of 5, 3ku to obtain mung bean protein peptide filtrate with molecular weight of 3-5ku, and freeze drying the fractionated peptide fragment filtrate for use; 6.08g of mung bean protein peptide is dissolved in 121.6ml of deionized water to prepare a peptide solution with the substrate concentration of 5%, and the pH is adjusted to 6 by using 1mol/L HCL.31, then 1g of ZnSO was added4·7H2O, vortex mixing uniformly, carrying out chelation reaction at 50.32 ℃ for 80.99min, cooling the solution to room temperature after the reaction is finished, precipitating with triple absolute ethyl alcohol, centrifuging for 15min at 10000r/min after precipitation, collecting the precipitate, washing with absolute ethyl alcohol until the supernatant is added with a zinc indicator and does not change color; the temperature of the oven is firstly adjusted to 100 ℃ and the oven is baked for 1h, then the temperature is reduced to 30 ℃ and the chelate is baked till the chelate is dried, and the mung bean protein peptide-zinc chelate with high chelation rate is obtained.
Furthermore, the mung bean protein peptide-zinc chelate is applied to promoting zinc absorption.
Adopt above-mentioned technical scheme's positive effect: the mung bean protein is used as a raw material, the mung bean protein peptide is chelated with zinc ions, the mung bean protein peptide is beneficial to absorption by a human body, and can promote zinc absorption by small intestines and accumulation in the body, so that the absorption and utilization rate of zinc is improved, and the zinc ions are prevented from being precipitated and lost in an alkaline intestinal environment.
Drawings
FIG. 1 is the metal chelation rates of five enzymatic products;
FIG. 2 is a comparison of chelation rates of unfractionated and post-ultrafiltrated fractions of mung bean protein peptides;
FIG. 3 is the effect of peptide zinc mass ratio on chelating rate of 3-5ku mung bean protein peptide;
FIG. 4 is a graph showing the effect of pH on the chelation rate of 3-5ku mung bean protein peptide fragments;
FIG. 5 is a graph showing the effect of temperature on the chelation rate of 3-5ku mung bean protein peptide fragments;
FIG. 6 is a graph showing the effect of time on the chelating rate of zinc in mung bean protein peptides of 3-5ku mung bean protein peptide fragments;
FIG. 7 is a graph of contour lines and response surface of interaction of various factors on the mung bean protein peptide zinc chelation rate;
FIG. 8 is an ultraviolet spectrum of 3-5ku mung bean protein peptide and its zinc chelate;
FIG. 9 is an infrared spectrum of a mung bean protein peptide and its zinc chelate at 3-5ku, the first being an infrared spectrum of a mung bean protein peptide and the second being an infrared spectrum of a mung bean protein peptide zinc chelate;
fig. 10 is a comparative graphical representation of simulated inorganic zinc and mung bean protein peptide-zinc chelate zinc solubility and permeability in the gut.
Detailed Description
The following embodiments and test examples are further described, but should not be construed as limiting the present invention:
example 1
This example illustrates the preparation of a mung bean protein peptide-zinc chelate.
Dissolving 50g of mung bean protein in 1.25L of deionized water, pretreating for 15 minutes in a 90 ℃ water bath kettle, adjusting the pH to 9 with 0.5mol/L NaOH after the temperature is reduced to 55 ℃, adding 4ml of alkaline protease, keeping the pH to 9 with 0.5mol/L NaOH, hydrolyzing for 4 hours, and inactivating enzyme in a boiling water bath; cooling the hydrolysate to room temperature, adjusting the pH value to 7 with 1mol/L _ HCL, centrifuging at 5000r/min for 15min, and collecting supernatant; ultrafiltering the supernatant under 0.25MPa, sequentially ultrafiltering with ultrafiltration membrane with molecular weight of 5, 3ku to obtain mung bean protein peptide filtrate with molecular weight of 3-5ku, and freeze drying the fractionated peptide fragment filtrate for use; dissolving 6.08g mung bean protein peptide in 121.6ml deionized water to prepare peptide solution with substrate concentration of 5%, adjusting pH to 6.31 with 1mol/L HCL, and adding 1g ZnSO4·7H2O, vortex mixing uniformly, carrying out chelation reaction at 50.32 ℃ for 80.99min, cooling the solution to room temperature after the reaction is finished, precipitating with triple absolute ethyl alcohol, centrifuging for 15min at 10000r/min after precipitation, collecting the precipitate, washing with absolute ethyl alcohol until the supernatant is added with a zinc indicator and does not change color; the temperature of the oven is firstly adjusted to 100 ℃ and the oven is baked for 1h, then the temperature is reduced to 30 ℃ and the chelate is baked till the chelate is dried, and the mung bean protein peptide-zinc chelate with high chelation rate is obtained.
Example 2
This example illustrates the preparation of a mung bean protein peptide-zinc chelate.
Dissolving 50g of mung bean protein in 1.25L of deionized water, and performing water bath at 90 DEG CPretreating in a pot for 15 minutes, adjusting the pH to 9 with 0.5mol/L NaOH after the temperature is reduced to 55 ℃, adding 4ml of alkaline protease, maintaining the pH to 9 with 0.5mol/L NaOH, hydrolyzing for 4 hours, and inactivating the enzyme in boiling water bath; cooling the hydrolysate to room temperature, adjusting pH to 7 with 1mol/L HCL, centrifuging at 5000r/min for 15min, and collecting supernatant; ultrafiltering the supernatant under 0.25MPa, sequentially ultrafiltering with ultrafiltration membrane with molecular weight of 5, 3ku to obtain mung bean protein peptide filtrate with molecular weight of 3-5ku, and freeze drying the fractionated peptide fragment filtrate for use; dissolving 3g of mung bean protein peptide in 60ml of deionized water to prepare peptide solution with substrate concentration of 5%, adjusting pH to 4 with 1mol/L _ HCL, and adding 1g of ZnSO4·7H2O, vortex mixing uniformly, carrying out chelation reaction at the reaction temperature of 30 ℃ for 120min, cooling the solution to room temperature after the reaction is finished, precipitating with three times of absolute ethyl alcohol, centrifuging for 15min at 10000r/min after precipitation, collecting the precipitate, washing with absolute ethyl alcohol until supernate is added with a zinc indicator and does not change color; the temperature of the oven is firstly adjusted to 100 ℃ and the oven is baked for 1h, then the temperature is reduced to 30 ℃ and the chelate is baked till the chelate is dried, and the mung bean protein peptide-zinc chelate with high chelation rate is obtained.
Example 3
This example illustrates the preparation of a mung bean protein peptide-zinc chelate.
Dissolving 50g of mung bean protein in 1.25L of deionized water, pretreating for 15 minutes in a 90 ℃ water bath kettle, adjusting the pH to 9 with 0.5mol/L NaOH after the temperature is reduced to 55 ℃, adding 4ml of alkaline protease, keeping the pH to 9 with 0.5mol/L NaOH, hydrolyzing for 4 hours, and inactivating enzyme in a boiling water bath; cooling the hydrolysate to room temperature, adjusting pH to 7 with 1mol/L HCL, centrifuging at 5000r/min for 15min, and collecting supernatant; ultrafiltering the supernatant under 0.25MPa, sequentially ultrafiltering with ultrafiltration membrane with molecular weight of 5, 3ku to obtain mung bean protein peptide filtrate with molecular weight of 3-5ku, and freeze drying the fractionated peptide fragment filtrate for use; dissolving 7g of mung bean protein peptide in 140ml of deionized water to prepare peptide solution with substrate concentration of 5%, adjusting pH to 8 with 1mol/L _ HCL, and adding 1g of ZnSO4·7H2And O, vortex mixing uniformly, carrying out chelation reaction at 70 ℃ for 40min, cooling the solution to room temperature after the reaction is finished, and carrying out reaction with three times of anhydrousPrecipitating with ethanol, centrifuging at 10000r/min for 15min after precipitation, collecting precipitate, washing with anhydrous ethanol until supernatant is discolored after adding zinc indicator; the temperature of the oven is firstly adjusted to 100 ℃ and the oven is baked for 1h, then the temperature is reduced to 30 ℃ and the chelate is baked till the chelate is dried, and the mung bean protein peptide-zinc chelate with high chelation rate is obtained.
Test example 1
This example illustrates screening of experimental conditions.
(1) The mung bean protein is used as a raw material, five proteases, namely alkaline protease, neutral protease, papain, flavourzyme and compound protease (flavourzyme: alkaline protease: 1), are selected, the mung bean protein is subjected to enzymolysis under respective optimal enzymolysis conditions (the appropriate enzymolysis conditions of the four proteases are shown in table 1), and the optimal protease is screened by taking the chelation rate as an index.
TABLE 1 conditions suitable for the enzymatic hydrolysis of four proteases
The chelation rate was determined as follows:
the chelating rate of zinc ions is determined by EDTA complexation titration.
The method for processing the sample when the total zinc content is measured comprises the following steps: 5mL of mung bean polypeptide-zinc reaction liquid is taken, and the volume of deionized water is adjusted to 50 mL.
The method for processing the sample when determining the content of the chelated peptide zinc comprises the following steps: and adding 3 times of volume of absolute ethyl alcohol into another 5mL of reaction solution, fully and uniformly mixing, centrifuging at 10000r/min for 15min, removing supernatant, and fixing the volume of the precipitate to 50mL by using deionized water.
EDTA complexometric titration: and (3) taking 20mL of reaction liquid with constant volume, dropwise adding a xylenol orange indicator, dropwise adding 20% by mass of hexamethylenetetramine until the solution is in a stable mauve color, then adding 4mL of hexamethylenetetramine, titrating by using 0.01mol/L EDTA, and finally obtaining the end point when the solution is changed from the mauve color to yellow color.
In the formula, C: the concentration of EDTA solution, mol/L;
Vblank space: replacing the required volume of EDTA (ethylene diamine tetraacetic acid) of the solution to be detected by using deionized water, wherein the volume is mL;
Vgeneral assembly: the volume of EDTA consumed in the determination of the total zinc content is mL;
Vchelation: volume of EDTA consumed in determining the amount of Zinc chelate, mL
The test result is shown in fig. 1, five kinds of mung bean protein zymolytes have metal chelation, the chelation rate is greatly increased in the early stage of hydrolysis, and the exposed peptide segments are more along with the increase of the enzymolysis time, so that the chelation reaction is promoted; the later period tends to be flat and downward, because the chelation rate is reduced because some polypeptides are continuously degraded into free amino acids; the metal chelating rates of the five zymolytes are different, the chelating rate of the enzymolysis liquid of the alkaline protease is higher than that of the other four zymolytes, because the cutting sites of different enzymes are different, the terminal amino acid of the hydrolyzed polypeptide, the composition and the arrangement sequence of the amino acid have certain difference, thereby influencing the binding energy rate of the peptide and the zinc, and the alkaline protease has stronger specificity to the carboxyl terminal peptide bond of the plant protein.
(2) Carrying out enzymolysis on the mung bean protein by using the screened protease with the highest chelating rate, wherein the hydrolysis conditions are optimized in the early stage of a laboratory: continuously hydrolyzing for 4h at the temperature of 55 ℃, the mass fraction of the substrate being 4%, the enzyme adding amount being 8% and the pH value being 9, inactivating the enzyme in a boiling water bath for 10min after the hydrolysis is finished, centrifuging for 10min at the speed of 5000r/min, and collecting the supernatant for later use.
(3) And (3) ultrafiltering the supernatant prepared in the step (2) under the pressure rate of 0.25MPa, sequentially ultrafiltering by using ultrafiltration membranes with the permeation molecular weights of 10,5 and 3ku to respectively obtain mung bean protein peptide filtrate with the molecular weights of more than 10,5 to 10, 3 to 5 and less than 3ku, freeze-drying the stock solution of the enzymolysis solution and the chelate peptide fragment permeate of each fraction for later use, and screening out the peptide fragment with the highest chelate rate by using the step (2) as an index to perform the following chelate experiment.
As can be seen from the results in FIG. 2, the chelating rates of the fractionated mung bean protein peptides are different, which indicates that the molecular weight of the mung bean protein peptides plays an important role in the chelating rate, the chelating rate of the peptide fragments larger than 10ku is lower than that of the peptide fragments not fractionated, and the other peptide fragments are significantly increased, because most of macromolecular proteins are removed, the hydrophobic amino acid content is increased, so that the chelating rate is increased, the chelating rate of the fraction 3-5ku after ultrafiltration is the largest, and reaches 54.34%, which is 22.15% higher than that of the fraction not fractionated, so that the ultrafiltration membrane with the molecular weight of 3-5ku is selected as the working membrane.
(4) Weighing a certain amount of mung bean protein peptide, dissolving in deionized water, adding ZnSO4·7H2Preparing a peptide zinc solution with the mass ratio of peptide zinc (3:1,4:1,5:1,6:1,7:1) by using O, fully dissolving, uniformly mixing by using a vortex, adjusting the pH value to (4,5,6,7,8), and then carrying out chelation reaction; the reaction temperature is (30,40,50,60,70 ℃), the reaction time is (40,60,80,120min), the mixture is taken out and cooled to room temperature, three times of absolute ethyl alcohol is used for precipitation, after precipitation, 10000g of absolute ethyl alcohol is centrifuged for 15min, the precipitate is collected, and the mixture is washed by alcohol for many times until the supernatant is added with a zinc carboxylic acid indicator to be free from color change; drying the oven at high temperature, then cooling to 30 ℃ to dry the chelate, and investigating the influence of various factors on the zinc chelating capacity of the mung bean protein peptide.
The chelating rate of zinc ions is determined by EDTA complexation titration.
The method for processing the sample when the total zinc content is measured comprises the following steps: 5mL of mung bean polypeptide-zinc reaction liquid is taken, and the volume of deionized water is adjusted to 50 mL.
The method for processing the sample when determining the content of the chelated peptide zinc comprises the following steps: and adding 3 times of volume of absolute ethyl alcohol into another 5mL of reaction solution, fully and uniformly mixing, centrifuging at 10000r/min for 15min, removing supernatant, and fixing the volume of the precipitate to 50mL by using deionized water.
EDTA complexometric titration: and (3) taking 20mL of reaction liquid with constant volume, dropwise adding a xylenol orange indicator, dropwise adding 20% by mass of hexamethylenetetramine until the solution is in a stable mauve color, then adding 4mL of hexamethylenetetramine, titrating by using 0.01mol/L EDTA, and finally obtaining the end point when the solution is changed from the mauve color to yellow color.
C: the concentration of EDTA solution, mol/L;
Vblank space: replacing the required volume of EDTA (ethylene diamine tetraacetic acid) of the solution to be detected by using deionized water, wherein the volume is mL;
Vgeneral assembly: the volume of EDTA consumed in the determination of the total zinc content is mL;
Vchelation: volume of EDTA consumed in determining the amount of Zinc chelate, mL
In the formula: c: concentration of EDTA (mol/L); v is the volume of consumed titration solution (mL); m is the mass (g) of the chelate compound.
The results are as follows:
as can be seen from fig. 3, when the mass ratio of the mung bean protein peptide to the zinc is 3:1, a large amount of zinc ions do not participate in chelation due to excessive content of zinc ions, so that the chelation energy rate is low, but when the mass ratio of the peptide to the zinc is 7:1, the chelation rate is slowly reduced, because the zinc ions are saturated, the peptide to zinc ratio is continuously increased, and the peptide utilization rate is reduced; when the ratio is 6:1, the maximum zinc chelation capacity of the mung bean protein peptide is 64.87mg/g, and the ratio of the mung bean protein peptide to zinc is selected to be 5:1-7: 1.
As can be seen from FIG. 4, pH has a significant effect on the chelating energy rate because pH is an important factor for the formation of chelate complexes between protein peptides and trace elements, and the chelating rate is weak when pH is low, and increases slowly, and when pH is 6, the chelating rate is the greatest, and continues to increase, and the chelating rate decreases, which may be the case when OH is in alkaline condition-Can compete with electron donating groups for metal ions to eventually form zinc hydroxide precipitate, and finally 5-7 is selected.
As can be seen from FIG. 5, the chelate formation is accelerated by a suitable temperature rise, the mung bean protein peptide zinc chelating ability is the greatest at 50 ℃, but the polypeptide is degraded by ammonia carbonyl due to an excessive temperature rise, so the chelate formation is not favored by a high temperature rise, and the final selection temperature range is 30-70 ℃.
As can be seen from FIG. 6, the time has no significant effect on the zinc chelation energy rate, the chelation rate is in an increasing trend when the chelation time is in the range of 40-80min, the chelation energy rate is the largest when the chelation time is 80min, the zinc chelation energy rate of the mung bean protein peptide is gradually reduced when the mung bean protein peptide exceeds 80min, and the final selection range is 40-120 min.
Selecting pH (X) based on single factor test1) Peptide zinciumQuantitative ratio (X)2) Time (X)3) And temperature (X)4)4 factors are used as independent variables, the mung bean protein peptide zinc chelating energy rate (Y) is used as a response value, a four-factor three-level response surface experiment is carried out, the level codes are shown in a table 2, and the analysis results are shown in a table 3 and a table 4.
TABLE 2 Box-Behnken test factors and horizontal coding
TABLE 3 center rotation combination test design and results
TABLE 4 analysis of variance of regression equation
Note: r2＝0.9787，RAdj20.9575; *. P is less than 0.05, the difference is significant; p < 0.01, the difference was very significant.
Fitting regression is carried out on test data by taking the mung bean protein peptide chelated zinc energy rate as a response value, and the regression model equation can be obtained as follows:
Y＝80.15+6.25X1+1.99X2+1.66X3-0.47X4-1.36X1X2-2.46X1X3+2.77X1X4-3.31X2X3+0.44X2X4-2.16X3X4-9.86X1 2-8.55X2 2-5.54X3 2-4.83X4 2
as can be seen from Table 4, the model established by the experiment was extremely significant (P)<0.01), denotation (P) is not significant>0.05), the model fitting degree is proved to be better, R2＝0.9787，RAdj20.9575, which shows that the model can better explain the change of response value, can be used for analyzing and predicting the mung bean protein peptide-zinc chelation process.
Fig. 7 shows three-dimensional curves and contour plots with remarkable pairwise interaction among 4 factors, and the sequence influencing the chelation energy rate is known to be by combining variance analysis: the pH value is more than the peptide zinc mass ratio and more than the time is more than the temperature, the interaction influence (P is less than 0.05) exists between the reaction pH and time, between the reaction pH and the reaction temperature, between the reaction pH and the reaction time, between the reaction time and the reaction time, and between the reaction time and the reaction temperature, and the other factors have no obvious interaction influence.
The optimal conditions for obtaining the mung bean protein peptide-zinc chelation process through the model are as follows: the predicted value of the mung bean protein peptide chelating zinc rate is 81.24% under the conditions that the pH value is 6.31, the mass ratio is 6.08:1, the reaction time is 80.99min and the temperature is 50.33 ℃, three groups of parallel experiments are carried out according to the conditions to obtain the actual value of the mung bean protein peptide chelating rate which is 81.04% and is similar to the predicted value, so that the optimized experimental conditions obtained through response surface analysis can be used for guiding the actual production of the mung bean protein peptide-zinc chelating process, and certain practical significance is achieved.
Test example 2
This experimental example illustrates the structural characterization of the mung bean protein peptide-zinc chelate.
1. Ultraviolet spectrum analysis before and after chelation of mung bean protein peptide-zinc
Respectively weighing 1mg of mung bean protein peptide and the chelate thereof, dissolving in 1ml of distilled water, and putting into an ultraviolet spectrophotometer for full spectrum scanning, wherein the wavelength range is 200-400 nm.
As can be seen from FIG. 8, the peptide has distinct absorption peaks at 228nm and 272nm, while the maximum absorption peaks of the chelate are shifted to 230nm and 278nm, respectively, and the maximum absorption peak of the peptide is around 228nm, which is the result of the electron transition from carbonyl (C ═ O) n → π on the peptide bond, when the peptide and zinc ion have a chelating reaction, Zn is added2+After forming a complex bond with N, O in the peptide, it affects the carbonyl (C ═ O) n → pi electron transition on the peptide bond, thereby causing it to undergo a blue shift; the polypeptide has a weak absorption peak at 272nm, where it is usually the ultraviolet absorption of phenylalanine, and the chelate complex thereof isThe absorption peak of the peptide undergoes blue shift, which is the amido bond and Zn in the mung bean protein peptide2+The formation of chelates is evidenced by the formation of ground-state complexes by ultraviolet spectroscopy.
2. Mung bean protein peptide-zinc chelating front and back infrared spectrum
Firstly, placing mung bean protein peptide and chelate thereof and KBr in an oven to be dried to the requirement, weighing 2mg of sample and 200mg of KBr in an agate mortar to be ground and uniformly mixed, pressing the mixed sample into a transparent sheet by a tablet press, and then scanning and analyzing by an infrared spectrometer to obtain a spectrum.
As can be seen from FIG. 9, the chelate band is shifted, increased or disappeared as compared with the mung bean protein peptide, which is a change in the infrared spectrum caused by the binding of zinc ions to the amino acid residue of the peptide, confirming the formation of the chelate; in the infrared spectrogram of mung bean peptide, 3385.50cm-1A wide absorption peak with overlapped-OH and-NH stretching vibration frequency appears, and the wave number in the spectrogram of the mung bean peptide zinc chelate is increased to 3404.53cm-1And weakened in strength, possibly Zn2+Reaction with-OH or-NH groups results in an increase in electron cloud density.
The absorption peak of-C ═ O on-NH-C ═ O in the mung bean peptide infrared spectrogram is 1666.21cm-1Here, there was not much change after chelation with zinc, but there was a change in both-COOH symmetrical and asymmetric stretching vibration, indicating that Zn2+The site of chelation may be-OH on-COOH, -NH-C ═ O-NH, with an absorption peak at 1151.75cm-1Here, no disappearance of chelation with Zn indicates that Zn is involved2+The chelating position may be an-NH group on-NH-C ═ O; peptide Zinc chelate terminal-NH2Both the in-plane bending vibration and the out-of-plane bending vibration of (2) are blue-shifted and sharply increased in strength, probably Zn, compared to the unchelated peptide2+and-NH2Caused by the occurrence of a reaction.
3. Simulated digestion of mung bean protein peptide-zinc chelate in vitro
Dissolving a peptide zinc chelate and zinc sulfate heptahydrate (contrast) in deionized water, adjusting the pH value of the solution to 2.0 by using 1mol/L HCl, adding pepsin in a certain proportion, uniformly mixing, and carrying out oscillation water bath at 37 ℃ for 2 hours; after the reaction is finished, adding pancreatin and bile extract in a certain proportion into the mixed system, adjusting the pH value of the system to 7.2, transferring the mixture into a dialysis bag (6000Da), and shaking the water bath at 37 ℃ for 2 h.
Determination of Zinc ion dissolution Rate
In the simulated digestion process, after pepsin is digested for 2 hours and pepsin-pancreatin is digested for 4 hours, supernatant is taken, and the content of zinc ions in the supernatant is determined by an EDTA (ethylene diamine tetraacetic acid) complexation titration method so as to represent the dissolution rate of the zinc ions in different digestion stages.
V1: titrating the volume of EDTA solution required by zinc ions in the supernatant, namely mL;
V2: titrating the volume of EDTA solution required by zinc ions in the equal volume of the solution, wherein the volume is mL;
c: concentration of EDTA solution, mol/L.
Determination of the Zinc ion dialysance
After the digestion by pancreatin, taking the water solution outside the dialysis bag, and measuring the content of zinc ions in the water solution by EDTA complexometric titration to show the content of the zinc ions penetrating through the simulated intestinal tract.
V1: titrating the volume of EDTA solution required by zinc ions in the supernatant, namely mL;
V2: titrating the volume of EDTA solution required by zinc ions in the equal volume of the solution, wherein the volume is mL;
c: concentration of EDTA solution, mol/L.
Factors influencing the utilization rate of zinc in diet are many, the absorption rate of zinc in diet is low in human body, the main reason is that zinc and phytic acid can form an insoluble complex in small intestine, the insoluble zinc-phytic acid complex can not be absorbed and utilized by human due to lack of corresponding phytase in human intestinal tract, the bioavailability of zinc is greatly reduced, the bioavailability of mineral elements can be evaluated by using in vitro simulated gastrointestinal tract digestion-dialysis method, the dialysis rate and solubility of peptide zinc chelate and zinc sulfate heptahydrate are determined according to the experimental method described by Wolfgor and Wang, and the dissolution rate and dialysis rate of two substances after gastrointestinal tract digestion are shown in figure 10.
The experimental result shows that the dissolution rates of zinc ions in the peptide zinc chelate after gastrointestinal tract digestion are respectively 98.91% and 55.69% (P is more than 0.05); the dissolution rates of zinc ions in the inorganic zinc salt after gastrointestinal tract digestion are respectively 97.8% and 33.15% (P is less than 0.05); the dialysis rates of zinc ions in the peptide zinc chelate and the inorganic zinc salt are respectively 43.00% and 23.41%, the dialysis rate of the zinc ions in the peptide zinc chelate is significantly higher than that of the zinc ions in the inorganic zinc salt, although the inorganic zinc salt exists in a stomach environment in a dissolved state, the solubility of the inorganic zinc salt is significantly reduced after the inorganic zinc salt enters an intestinal environment, and therefore the zinc ions in the peptide zinc chelate can more easily enter the intestinal environment, which shows that the bioavailability of the peptide zinc chelate is higher compared with the inorganic zinc salt, and the main reason of the method can be two-sided: on one hand, the intestinal environment belongs to a weak alkaline environment and is a main absorption part of metal ions, a chelate bond formed by zinc ions and peptide is more stable than an ionic bond in inorganic zinc salt, and after gastrointestinal tract digestion, peptide zinc chelate still exists in a molecular form, so that zinc ions are difficult to form zinc hydroxide precipitate; on the other hand, some peptide zinc chelates with small molecular weight are directly absorbed through intestinal wall (dialysis bag) in molecular form, while some whey protein concentrate peptide zinc chelates with larger molecular weight are further decomposed into small molecules under the action of pepsin in stomach, and then enter and are absorbed by intestinal tract again.
The invention has the following advantages:
(1) hydrolyzing with alkaline protease, wherein the metal chelating rate of hydrolysate is higher than that of other protease;
(2) the mung bean protein peptide hydrolysis process is optimized, and the optimized hydrolysis degree is higher;
(3) performing ultrafiltration segmentation on hydrolysate of the protein peptide, and performing chelation reaction on the peptide segment with the highest chelation rate;
(4) the chelating process is also optimized, so that the polypeptide can be chelated with zinc to the maximum extent;
(5) greatly improves the bioavailability of metal ions, exactly makes up for the defects of some zinc supplementing agents, takes zinc ions as the center, and is tightly combined with amino and carbonyl to form a five-membered or six-membered ring structure, thereby avoiding the zinc ions from generating precipitation in the alkaline intestinal environment and losing;
(6) the peptide-metal ion chelate is a main form for absorbing and transporting metal ions by organisms and is an intermediate substance in the process of synthesizing protein by the organisms, the absorption speed is high, a plurality of biochemical processes can be reduced, and the energy consumption of the organisms is saved.
The mung bean protein is used as a raw material, the mung bean protein peptide is chelated with zinc ions, the mung bean protein peptide is beneficial to absorption by a human body, and can promote zinc absorption by small intestines and accumulation in the body, so that the absorption utilization rate of zinc is improved, and the zinc ions are prevented from being precipitated and lost in an alkaline intestinal environment; meanwhile, the hydrolysis process and the chelation process are optimized, so that the preparation efficiency of the chelate is further improved.
Claims (3)
1. A preparation method of mung bean protein peptide-zinc chelate is characterized by comprising the following steps: the method comprises the following steps of (1) hydrolyzing mung bean protein serving as a raw material by using protease to obtain mung bean protein peptide hydrolysate, freeze-drying polypeptide in the mung bean protein peptide hydrolysate, and carrying out chelation reaction on the polypeptide and zinc to obtain mung bean protein peptide-zinc chelate, wherein the protease is alkaline protease; performing ultrafiltration and grading on the mung bean protein peptide hydrolysate, screening 3-5ku peptide fragments for subsequent chelation reaction, wherein the chelation reaction conditions are that the mass ratio of the polypeptide to the zinc is 3:1-7:1, the pH is 4-8, the temperature is 30-70 ℃, and the reaction time is 40-120 min; the method comprises the following steps: dissolving 50g of mung bean protein in 1.25L of deionized water, pretreating with a 90 ℃ water bath kettle for 15 minutes, adjusting the pH to 9 with 0.5mol/L NaOH after the temperature is reduced to 55 ℃, adding 4ml of alkaline protease, keeping the pH to 9 with 0.5mol/L NaOH, carrying out hydrolysis for 4 hours, inactivating enzyme in a boiling water bath, cooling the hydrolysate to room temperature, adjusting the pH to 7 with 1mol/L HCL, centrifuging at 5000r/min for 15 minutes, and taking supernatant;ultrafiltering the supernatant under 0.25MPa, sequentially ultrafiltering with ultrafiltration membrane with molecular weight of 5, 3ku to obtain mung bean protein peptide filtrate with molecular weight of 3-5ku, and freeze drying the fractionated peptide fragment filtrate for use; dissolving 6.08g mung bean protein peptide in 121.6ml deionized water to prepare peptide solution with substrate concentration of 5%, adjusting pH to 6.31 with 1mol/L HCL, and adding 1g ZnSO4•7H2O, vortex mixing uniformly, carrying out chelation reaction at 50.32 ℃ for 80.99min, cooling the solution to room temperature after the reaction is finished, precipitating with triple absolute ethyl alcohol, centrifuging for 15min at 10000r/min after precipitation, collecting the precipitate, washing with absolute ethyl alcohol until the supernatant is added with a zinc indicator and does not change color; the temperature of the oven is firstly adjusted to 100 ℃ and the oven is baked for 1h, then the temperature is reduced to 30 ℃ and the chelate is baked till the chelate is dried, and the mung bean protein peptide-zinc chelate with high chelation rate is obtained.
2. The method for preparing mung bean protein peptide-zinc chelate according to claim 1, wherein: the chelating reaction conditions are that the mass ratio of the peptide to the zinc is 6.08:1, the pH value is 6.31, the temperature is 50.32 ℃, and the reaction time is 80.99 min.
3. The method for preparing mung bean protein peptide-zinc chelate according to claim 1, wherein: the mung bean protein peptide-zinc chelate is applied to promoting zinc absorption.
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