CN109439715A - Mung bean protein peptide-chelates of zinc preparation method - Google Patents
Mung bean protein peptide-chelates of zinc preparation method Download PDFInfo
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Abstract
The present invention relates to a kind of production methods of mung bean protein peptide-chelates of zinc, using mung bean protein as raw material, Semen phaseoli radiati polypeptide hydrolyzate is obtained after hydrolysis by novo, after the polypeptide freeze-drying in Semen phaseoli radiati polypeptide hydrolyzate, obtains mung bean protein peptide-chelates of zinc with zinc chelatropic reaction.The present invention is with mung bean protein peptide and Zinc Ions Chelated, the characteristics of being easy to intestinal absorption using mung bean protein small peptide, it can promote small intestine to the absorption of zinc and accumulate in vivo, improve the absorption rate of zinc, precipitating being generated in the intestinal environment of alkalinity to avoid zinc ion simultaneously and being lost, the zinc ion dissolution rate and dialysance of mung bean protein peptide chelates of zinc of the invention after gastro-intestinal digestion are above white vitriol.
Description
Technical field
The invention belongs to food processing fields, are related to a kind of preparation method of mung bean protein peptide-chelates of zinc.
Background technique
Mung bean is the seeds of leguminous plant of dual-purpose of drug and food, protein, starch, vitamin, minerals and people rich in
Body essential amino acid, wherein protein content is up to 19.5%-33.1%, 2-3 times higher than Cereal seed, and mung bean is rich in egg
Propylhomoserin, tryptophan, lysine, leucine, threonine complete protein, score according to WHO/FAO, the ammonia in mung bean protein matter
Base acid chemical score is all higher than recommendation, is a kind of good vegetable protein；Semen phaseoli radiati polypeptide is the enzymolysis product of mung bean protein, with
Based on the small-molecular peptides of 3-6 amino acid composition, also containing a small amount of macromolecular peptide, free amino acid, carbohydrate and inorganic salts etc. at
Point, the composition of amino acid mainly has methionine, tryptophan, tyrosine and an arginine, Semen phaseoli radiati polypeptide as functional food or
Food additives have broad application prospects than mung bean protein, still can because Semen phaseoli radiati polypeptide is under conditions of lower pH
Holding is increased compared with highly dissoluble, and with concentration, and viscosity variation is also little；In addition, the patient weaker for functions of intestines and stomach or to subtract
When light stomach burden, the nutrient source of protein can be used as, the low antigenicity of Semen phaseoli radiati polypeptide makes to cause allergic reaction after eating,
So can be used for the food of enteral nutrition complementary therapy or postoperative recovery patient.
Zinc has highly important physiological function, is mainly manifested in as one of the essential trace elements of the human body: (1) joining
With the metabolism of protein, carbohydrate, lipid and nucleic acid；(2) integrality of membrane structure is maintained；(3) promote body
Growth and development and regeneration；(4) promote the normal function of skin and bone；(5) promote the development of intelligence rate；(6) improve normal
Having a sharp sense of taste property；Zinc is particularly significant to the growth and development of children especially infant, and zinc-deficiency can make infant growth slow
Slowly, the depauperation of intelligence rate, immune function decline, human body is lower to the absorptivity of zinc in diet, and main cause is that zinc and phytic acid exist
Insoluble compound can be formed in small intestine, since human intestine lacks corresponding phytase, this insoluble zinc-phytic acid complexes
The bioavailability of zinc cannot be greatly reduced by human consumption and utilization；Chelates of zinc is structurally characterized in that by being coordinated altogether
The effect of valence chemical bond, the stable compound of the chemical property of formation, under suitable condition, N- Amino End Group, the end the C- carboxylic of peptide
The groups such as carbonyl and imino group in base, amino acid side chain and peptide chain can be used as ligand, provide electricity for metal zinc ion
Son forms stable compound, and in these compounds, zinc ion is located at the center of ring mostly, is not easy to be dissociated out, chemistry
Property is more stable；The uptake and transport of peptide chelates of zinc is to carry out by the uptake and transport mechanism of peptide, rather than pass through metal
What movement system was absorbed and utilized, chelate penetrates intestines according to the absorption and transport mechanism and feature of small peptide in whole form
Road mucous layer enters blood, avoids competition antagonism of the metal ion in intestinal absorption, and not only absorption is fast, energy consumption is low, but also
Many biochemical processes can be reduced, so that the bioavailability of trace element zinc is improved, therefore, using mung bean protein as raw material,
Make mung bean protein peptide and Zinc Ions Chelated, by small peptide absorption approach, has to the absorption rate for improving polypeptide and zinc ion
Important meaning.
Summary of the invention
The object of the present invention is to provide a kind of preparation methods of mung bean protein peptide-chelates of zinc, and existing zinc is overcome to mend preparation
The low disadvantage of absorptivity promotes absorption of the human body to zinc.
The present invention is achieved through the following technical solutions:
A kind of preparation method of mung bean protein peptide-chelates of zinc, is using mung bean protein as raw material, through hydrolysis by novo
Mung bean protein peptide hydrolysate is obtained afterwards, after the polypeptide freeze-drying in mung bean protein peptide hydrolysate, is obtained with zinc chelatropic reaction green
Legumin peptide-chelates of zinc.
Further, the protease is alkali protease.
Further, further include that ultrafiltration classification is carried out to mung bean protein peptide hydrolysate, it is subsequent to filter out the progress of 3-5ku peptide fragment
Chelatropic reaction.
Further, the condition of chelatropic reaction be polypeptide and zinc mass ratio 3:1-7:1, it is pH 4-8,30-70 DEG C of temperature, anti-
40-120min between seasonable.
Further, the condition of chelatropic reaction be peptide with zinc mass ratio 6.08:1, pH 6.31,50.32 DEG C of temperature, react
Time 80.99min.
Specifically, method includes the following steps: taking 50g mung bean protein to be dissolved in the deionized water of 1.25L, prior to 90
It is pre-processed 15 minutes in DEG C water-bath, 0.5mol/L NaOH tune pH to 9 is used after temperature is down to 55 DEG C, 4ml basic protein is added
Enzyme, keeping pH with the NaOH of 0.5mol/L is 9, hydrolyzes boiling water bath enzyme deactivation after 4h；Hydrolyzate is cooled to room temperature, 1mol/L_ is used
HCL adjusts pH value and is centrifuged 15min in 5000r/min to after 7, takes supernatant；By supernatant ultrafiltration under 0.25MPa pressure, according to
The secondary ultrafiltration membrane ultrafiltration for being 5,3ku through molecular mass, obtains the mung bean protein peptide filter liquor that molecular mass is 3-5ku, will
It is spare after peptide fragment permeate freeze-drying after classification；6.08g mung bean protein peptide is dissolved in 121.6ml deionized water to be made into substrate dense
The peptide liquid that degree is 5% adjusts pH to 6.31 with 1mol/L HCL, rear that 1g ZnSO is added4·7H2O is vortexed and mixes, chelated
Reaction, reaction temperature are 50.32 DEG C, and solution is cooled to room temperature by reaction time 80.99min after reaction, with three times
Dehydrated alcohol alcohol precipitation, precipitation to be precipitated are centrifuged 15min with 10000r/min, collect precipitating, washed with dehydrated alcohol to supernatant
It is non-discolouring that zinc indicator is added；Then baking oven elder generation temperature regulating drops to 30 DEG C of baking chelates to doing, is chelated to 100 DEG C of baking 1h
The high mung bean protein peptide-chelates of zinc of rate.
Further, mung bean protein peptide-chelates of zinc is promoting the application in zinc-iron alloy solution.
Good effect by adopting the above technical scheme: the present invention using mung bean protein as raw material, make mung bean protein peptide and zinc from
Son chelating is utilized mung bean protein peptide conducive to absorption of human body, can promote small intestine to the absorption of zinc and accumulate in vivo, to mention
The absorption rate of high zinc avoids zinc ion from generating precipitating in the intestinal environment of alkalinity and loses, mung bean protein peptide of the invention
The digestibility and retention rate of chelates of zinc are above inorganic zinc, while optimizing to hydrolysis process, chelating technique, into one
Step improves the preparation efficiency of chelate.
Detailed description of the invention
Fig. 1 is the metal-chelating rate of five kinds of enzymolysis products；
Fig. 2 be not classified and ultrafiltration after each fraction mung bean protein peptide chelation percent comparison；
Fig. 3 is influence of the peptide zinc mass ratio to 3-5ku mung bean protein peptide fragment chelation percent；
Fig. 4 is influence of the pH to 3-5ku mung bean protein peptide fragment chelation percent；
Fig. 5 is influence of the temperature to 3-5ku mung bean protein peptide fragment chelation percent；
Fig. 6 is influence of the time to 3-5ku mung bean protein peptide fragment mung bean protein peptide zinc chelation percent；
Fig. 7 is contour and response surface figure of each factor reciprocation to mung bean protein peptide zinc chelation percent；
Fig. 8 is the ultraviolet spectra of 3-5ku mung bean protein peptide and its chelates of zinc；
Fig. 9 is the infrared spectroscopy of 3-5ku mung bean protein peptide and its chelates of zinc, and first figure is the infrared of mung bean protein peptide
Spectrum, second figure are the infrared spectroscopy of mung bean protein peptide chelates of zinc；
Figure 10 is the comparison signal for simulating inorganic zinc and mung bean protein peptide-chelates of zinc zinc solubility and transmitance in enteron aisle
Figure.
Specific embodiment
Below with reference to embodiment and test example, the following further describes the technical solution of the present invention, but should not be construed as pair
Limitation of the invention:
Embodiment 1
This example demonstrates that mung bean protein peptide-chelates of zinc preparation process.
It takes 50g mung bean protein to be dissolved in the deionized water of 1.25L, is pre-processed 15 minutes in prior to 90 DEG C water-baths, to temperature
Degree uses 0.5mol/L NaOH tune pH to 9 after being down to 55 DEG C, and 4ml alkali protease is added, and keeps pH with the NaOH of 0.5mol/L
It is 9, hydrolyzes boiling water bath enzyme deactivation after 4h；Hydrolyzate is cooled to room temperature, with 1mol/L_HCL adjust pH value to after 7 in 5000r/
Min is centrifuged 15min, takes supernatant；It is successively 5,3ku's with through molecular mass by supernatant ultrafiltration under 0.25MPa pressure
Ultrafiltration membrane ultrafiltration, obtains the mung bean protein peptide filter liquor that molecular mass is 3-5ku, and standby is lyophilized in the peptide fragment permeate after classification
With；6.08g mung bean protein peptide is dissolved in and is made into the peptide liquid that concentration of substrate is 5% in 121.6ml deionized water, with 1mol/L HCL
PH to 6.31 is adjusted, it is rear that 1g ZnSO is added4·7H2O is vortexed and mixes, and carries out chelatropic reaction, and reaction temperature is 50.32 DEG C, reaction
Time is 80.99min, is after reaction cooled to room temperature solution, and with three times dehydrated alcohol alcohol precipitation, precipitation to be precipitated is used
10000r/min is centrifuged 15min, collects precipitating, is washed with dehydrated alcohol non-discolouring to supernatant addition zinc indicator；Baking oven is first
Then temperature regulating drops to 30 DEG C of baking chelates to doing, obtains the high mung bean protein peptide-chelates of zinc of chelation percent to 100 DEG C of baking 1h.
Embodiment 2
This example demonstrates that mung bean protein peptide-chelates of zinc preparation process.
It takes 50g mung bean protein to be dissolved in the deionized water of 1.25L, is pre-processed 15 minutes in prior to 90 DEG C water-baths, to temperature
Degree uses 0.5mol/L NaOH tune pH to 9 after being down to 55 DEG C, and 4ml alkali protease is added, and keeps pH with the NaOH of 0.5mol/L
It is 9, hydrolyzes boiling water bath enzyme deactivation after 4h；Hydrolyzate is cooled to room temperature, with 1mol/L HCL adjust pH value to after 7 in 5000r/
Min is centrifuged 15min, takes supernatant；It is successively 5,3ku's with through molecular mass by supernatant ultrafiltration under 0.25MPa pressure
Ultrafiltration membrane ultrafiltration, obtains the mung bean protein peptide filter liquor that molecular mass is 3-5ku, and standby is lyophilized in the peptide fragment permeate after classification
With；3g mung bean protein peptide is dissolved in and is made into the peptide liquid that concentration of substrate is 5% in 60ml deionized water, adjusts pH with 1mol/L_HCL
It is rear that 1g ZnSO is added to 44·7H2O is vortexed and mixes, and carries out chelatropic reaction, and reaction temperature is 30 DEG C, and the reaction time is
Solution is cooled to room temperature by 120min after reaction, and with three times dehydrated alcohol alcohol precipitation, 10000r/min is used in precipitation to be precipitated
It is centrifuged 15min, collects precipitating, is washed with dehydrated alcohol non-discolouring to supernatant addition zinc indicator；Baking oven elder generation temperature regulating is to 100
DEG C dry 1h, then drop to 30 DEG C of bakings chelates to do, obtain the high mung bean protein peptide-chelates of zinc of chelation percent.
Embodiment 3
This example demonstrates that mung bean protein peptide-chelates of zinc preparation process.
It takes 50g mung bean protein to be dissolved in the deionized water of 1.25L, is pre-processed 15 minutes in prior to 90 DEG C water-baths, to temperature
Degree uses 0.5mol/L NaOH tune pH to 9 after being down to 55 DEG C, and 4ml alkali protease is added, and keeps pH with the NaOH of 0.5mol/L
It is 9, hydrolyzes boiling water bath enzyme deactivation after 4h；Hydrolyzate is cooled to room temperature, with 1mol/L HCL adjust pH value to after 7 in 5000r/
Min is centrifuged 15min, takes supernatant；It is successively 5,3ku's with through molecular mass by supernatant ultrafiltration under 0.25MPa pressure
Ultrafiltration membrane ultrafiltration, obtains the mung bean protein peptide filter liquor that molecular mass is 3-5ku, and standby is lyophilized in the peptide fragment permeate after classification
With；7g mung bean protein peptide is dissolved in and is made into the peptide liquid that concentration of substrate is 5% in 140ml deionized water, is adjusted with 1mol/L_HCL
PH to 8, it is rear that 1g ZnSO is added4·7H2O is vortexed and mixes, and carries out chelatropic reaction, and reaction temperature is 70 DEG C, and the reaction time is
Solution is cooled to room temperature by 40min after reaction, with three times dehydrated alcohol alcohol precipitation, precipitation to be precipitated, with 10000r/min from
Heart 15min collects precipitating, is washed with dehydrated alcohol non-discolouring to supernatant addition zinc indicator；Baking oven elder generation temperature regulating is to 100 DEG C
1h is dried, 30 DEG C of baking chelates is then dropped to doing, obtains the high mung bean protein peptide-chelates of zinc of chelation percent.
Test example 1
This example demonstrates that the screening of experiment condition.
(1) using mung bean protein as raw material, alkali protease, neutral proteinase, papain, flavor albumen are selected
Enzyme, compound protease (flavor protease: alkali protease=1:1) this five kinds of protease, it is right under the conditions of respective peak enzymolysis-ability
Mung bean protein is digested (the enzymatic hydrolysis suitable condition such as table 1 of four kinds of protease), using chelation percent as index screening most suitable albumen out
Enzyme.
The enzymatic hydrolysis suitable condition of 1 four kinds of protease of table
The measuring method of chelation percent is as follows:
The measurement of Zinc Ions Chelated rate uses EDTA compleximetry.
The processing method of sample when total zinc-content determination: taking Semen phaseoli radiati polypeptide-zinc reaction solution 5mL, and deionized water is settled to 50
mL。
The processing method of the survey timed samples of chelating peptide Zn content: 5mL is separately extracted reaction solution, the anhydrous second of 3 times of volumes is added
Alcohol, 10000r/min centrifugation 15min, removes supernatant, precipitating is settled to 50mL with deionized water after being sufficiently mixed uniformly.
EDTA complexometry: xylenol orange indicator is added dropwise in the reaction solution 20mL after taking constant volume, and mass fraction is added dropwise and is
20% hexa to solution is in stable aubergine, 4mL hexa is added, with 0.01 mol/L
EDTA titration, it is terminal that solution, which becomes yellow from aubergine,.
In formula, the concentration of C:EDTA solution, mol/L；
VBlank: EDTA volume, mL needed for replacing prepare liquid with deionized water；
VAlways: the EDTA volume consumed when total zinc-content determination, mL；
VChelating: the EDTA volume consumed when chelating zinc-content determination, mL
As shown in Figure 1, five kinds of mung bean protein zymolytes have metal-chelating effect, hydrolysis chelation percent early period increases test result
It is larger, it is due to the growth with enzymolysis time, the peptide fragment being exposed is also more, promotes the progress of chelatropic reaction；Afterwards
Phase tends towards stability and on a declining curve, be because some polypeptides continue to be degraded to free amino acid and caused by chelation percent reduce
Reason；The metal-chelating rate of five kinds of zymolytes is all different, and the chelation percent of the enzymolysis liquid of alkali protease is higher than other four kinds,
This is because the enzyme site of different enzymes is different, the end amino acid of polypeptide after hydrolysis is caused, amino acid forms and puts in order
Have a certain difference, to affect the combination energy rate of peptide and zinc, alkali protease to the carboxylic end peptide bond of vegetable protein have compared with
Strong specificity.
(2) mung bean protein is digested with the chelation percent filtered out highest protease, before hydrolysising condition laboratory
The hydrolysising condition of phase optimization: 55 DEG C of temperature, substrate mass fraction 4%, enzyme additive amount 8%, pH value 9, continuous hydrolysis 4h, hydrolysis
After enzyme deactivation 10min in boiling water bath, 5000r/min is centrifuged 10min, it is spare to collect supernatant.
(3) supernatant for preparing (2) ultrafiltration under 0.25MPa pressure rate is successively 10,5 with molecular mass is penetrated,
The ultrafiltration membrane ultrafiltration of 3ku, respectively obtaining molecular mass is>10,5-10,3-5, and the mung bean protein peptide filter liquor of<3ku will digest
It is spare after the freeze-drying of the chelating peptide fragment permeate of liquid stoste and each fraction of classification, it is that index screening goes out chelation percent most with step (2)
High peptide fragment carries out following chelating experiment.
As a result as seen from Figure 2, the mung bean protein peptide after classification, chelation percent is different, this shows mung bean egg
The molecular weight of white peptide plays an important role for chelation percent, in addition to being greater than 10ku peptide fragment chelation percent lower than unassorted peptide fragment,
He significantly increases peptide fragment, this is because eliminating most high molecular weight protein, increases hydrophobic amino acid content, and causes
The growth of chelation percent, the chelation percent of 3-5ku fraction is maximum after ultrafiltration, has reached 54.34%, than improving 22.15% before not being classified,
Therefore select penetrate molecular mass for 3-5ku ultrafiltration membrane as work film.
(4) it weighs a certain amount of mung bean protein peptide to be dissolved in deionized water, ZnSO is added4·7H2O is made into peptide zinc mass ratio
The peptide zinc solution of (3:1,4:1,5:1,6:1,7:1) sufficiently dissolves, and is vortexed and mixes, is chelated after being adjusted to pH (4,5,6,7,8)
Reaction；Reaction temperature is (30,40,50,60,70 DEG C), and the reaction time is (40,60,80,120min), is cooled to room after taking-up
Temperature is precipitated, precipitation to be precipitated with three times dehydrated alcohol, and 10000g is centrifuged 15 minutes, collects precipitating, and multiple alcohol is washed till supernatant and adds
It is non-discolouring to enter zinc carboxylic acid indicator；First high temperature dries baking oven, after drop to 30 DEG C of baking chelates to dry, investigate each factor to mung bean
The influence of protein peptides zinc chelating energy rate.
The measurement of Zinc Ions Chelated rate uses EDTA compleximetry.
The processing method of sample when total zinc-content determination: taking Semen phaseoli radiati polypeptide-zinc reaction solution 5mL, and deionized water is settled to 50
mL。
The processing method of the survey timed samples of chelating peptide Zn content: 5mL is separately extracted reaction solution, the anhydrous second of 3 times of volumes is added
Alcohol, 10000r/min centrifugation 15min, removes supernatant, precipitating is settled to 50mL with deionized water after being sufficiently mixed uniformly.
EDTA complexometry: xylenol orange indicator is added dropwise in the reaction solution 20mL after taking constant volume, and mass fraction is added dropwise and is
20% hexa to solution is in stable aubergine, 4mL hexa is added, with 0.01 mol/L
EDTA titration, it is terminal that solution, which becomes yellow from aubergine,.
The concentration of C:EDTA solution, mol/L；
VBlank: EDTA volume, mL needed for replacing prepare liquid with deionized water；
VAlways: the EDTA volume consumed when total zinc-content determination, mL；
VChelating: the EDTA volume consumed when chelating zinc-content determination, mL
In formula: the concentration (mol/L) of C:EDTA；V is the volume (mL) for consuming titrating solution；M is the quality for weighing chelate
(g)。
As a result as follows:
As can be seen from Figure 3, when mung bean protein peptide zinc mass ratio is 3:1, since zinc ion content is excessive, a large amount of zinc ions are simultaneously
Do not participate in chelating cause chelate energy rate it is lower, but peptide zinc mass ratio be 7:1 when, chelation percent slowly reduces instead, this is because zinc
Ion has reached saturation state, continues growing peptide zinc than reducing peptide utilization rate instead；When ratio is 6:1, mung bean protein peptide zinc
Chelating energy rate is up to 64.87mg/g, the ratio range 5:1-7:1 of final choice mung bean protein peptide and zinc.
As shown in Figure 4, pH influences chelating energy rate more significant, this is because pH is that protein peptides and microelement are formed and chelated
An important factor for object, chelation percent is weaker when pH is lower, increases pH chelation percent and slowly rises, and when pH is 6, chelation percent is maximum, continues
Increase the decline of pH chelation percent, this may be OH under alkaline condition-Metal ion can be competed with electron-donating group ultimately form hydrogen-oxygen
Change zinc precipitating, final choice 5-7.
As can be seen from Figure 5, appropriate heating can promote the generation of chelate, and mung bean protein peptide zinc chelating energy rate is maximum at 50 DEG C,
But excessive temperature rise can make polypeptide that carbonyl ammonia occur and reduce chela rate, therefore high temperature is unfavorable for the generation of chelate, final choice temperature
Range is 30-70 DEG C.
As can be seen from Figure 6, the time influences zinc chelating energy rate not significant, chelates the time within the scope of 40-80min, chelating
Rate is in rising trend, and 80min chelates energy rate maximum, gradually decreases more than 80min mung bean protein peptide zinc chelating energy rate, final choice
Range is in 40-120min.
On the basis of single factor experiment, pH (X is chosen1), peptide zinc mass ratio (X2), time (X3) and temperature (X4) 4 because
Element is used as independent variable, and it is response that mung bean protein peptide zinc, which chelates energy rate (Y), carries out the experiment of four factors, three horizontal respone face, horizontal
Coding is shown in Table 2, and analysis the results are shown in Table 3 and table 4.
2 Box-Behnken experimental factor of table and level code
3 center rotation combination experimental design of table and result
The variance analysis of 4 regression equation of table
Note: R2=0.9787, RAdj2=0.9575；* .P < 0.05, significant difference；* .P < 0.01, difference is extremely significant.
Recurrence is fitted to test data using mung bean protein peptide chelated zinc energy rate as response, regression model side can be obtained
Journey is 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 for testing foundation is extremely significant (P<0.01), loses quasi- item except not significant (P>0.05), it was demonstrated that
Models fitting degree is preferable, R2=0.9787, RAdj2=0.9575, illustrate that the model can preferably explain the variation of response,
It can be used for analyzing and predicting mung bean protein peptide-zinc chelating technique.
Fig. 7 shows the significant three-dimensional curve of pairwise interaction and contour map between 4 factors, in conjunction with variance analysis
It is found that influence chelating energy rate sequencing are as follows: pH value > peptide zinc mass ratio > time > temperature, reaction pH and the time, pH with
There are reciprocal effects to act on (P < 0.05) for temperature, peptide zinc mass ratio and time, time and temperature, without aobvious between remaining each factor
Write reciprocal effect effect.
The optimal conditions that mung bean protein peptide-zinc chelates technique can be obtained by model are as follows: pH value 6.31, mass ratio are
Under the conditions of 6.08:1, reaction time 80.99min, temperature are 50.33 DEG C, the predicted value of mung bean protein peptide chelated zinc energy rate is
81.24%, it carries out three groups of parallel laboratory tests to obtain the actual value of mung bean protein peptide chelation percent being 81.04% according to above-mentioned condition, with
Predicted value is close, and therefore, the experiment condition after being optimized by response surface analysis can be used to instruct mung bean protein peptide-zinc chelating
The actual production of technique has certain realistic meaning.
Test example 2
This test example illustrates mung bean protein peptide-chelates of zinc structural characterization.
1, mung bean protein peptide-zinc chelating front and back ultraviolet spectral analysis
Weigh 1mg mung bean protein peptide respectively and its chelate be dissolved in 1ml distilled water after be put into ultraviolet specrophotometer progress
Full spectral scan, wave-length coverage are 200~400nm.
As shown in Figure 8, peptide has apparent absorption peak at 228nm and 272nm, and the maximum absorption band of its chelate divides
It does not move at 230nm and 278nm, for the maximum absorption band of peptide in 228nm or so, this is by (C=O) n → π of carbonyl on peptide bond * electricity
Sub- transition as a result, after chelatropic reaction occurs for peptide and zinc ion, Zn2+After forming fit key with N, O in peptide, peptide is affected
Carbonyl (C=O) n → π * electron transition on key, to bring it about blue shift；Polypeptide has weaker absorption peak at 272nm wavelength,
It is herein usually the UV absorption of phenylalanine, and blue shift has occurred in the absorption peak herein of its chelate, this is mung bean egg
Amido bond and Zn in white peptide2+Caused by forming ground state complex compound, ultraviolet spectra demonstrates the generation of chelate.
2, mung bean protein peptide-zinc chelating front and back infrared spectroscopy
Mung bean protein peptide and its chelate, KBr are placed first it is dry to requiring in baking oven, weigh 2mg sample with
200mgKBr is ground in the agate mortar, after mixed sample is pressed into transparent sheet with tablet press machine, uses infrared light
Spectrometer is scanned analysis, obtains spectrum.
From fig. 9, it can be seen that the bands of a spectrum of chelate are displaced compared with mung bean protein peptide, increase or disappear, this is
Zinc ion in conjunction with the amino acid residue of peptide caused by infrared spectroscopy variation, it was demonstrated that the generation of chelate；Mung bean peptide
In infrared spectrogram, 3385.50cm-1Wide absorption peak place appearance-OH Chong Die with-NH stretching vibration frequency, and mung bean peptide zinc chela
It closes wave number in the spectrogram of object and increases to 3404.53cm-1, and remitted its fury, it may be possible to Zn2+Occur with-OH or-NH group anti-
It answers, cloud density is caused to increase.
The absorption peak of the upper-C=O of-NH-C=O is in 1666.21cm in mung bean peptide infrared spectrogram-1Do not have behind place, with zinc chelating
Too big variation, and-COOH is symmetrically changed with anti-title stretching vibration, illustrates Zn2+The position chelated may be-COOH
On-OH, the absorption peak of the upper-NH of-NH-C=O is in 1151.75cm-1Place vanishes from sight with zinc chelating, illustrates and Zn2+Chelating
Position may be the-NH group on-NH-C=O；Peptide chelates of zinc end-NH2In-plane bending vibration and out-of-plane bending vibration
Blue shift, and intensity abruptly increase all have occurred compared with the peptide not chelated, it may be possible to Zn2+With-NH2Caused by reacting.
3, mung bean protein peptide-chelates of zinc in vitro digestion
In deionized water by peptide chelates of zinc and Zinc vitriol (control) dissolution, it is adjusted with the HCl of 1mol/L molten
Liquid pH value is added a certain proportion of pepsin, is uniformly mixed, 37 DEG C of shaking water bath 2h to 2.0；After reaction, to mixing
A certain proportion of pancreatin and bile extract are added in system, this mixture is moved into bag filter by regulation system pH value to 7.2
In (6000Da), 37 DEG C of shaking water bath 2h.
The measurement of zinc ion dissolution rate
In simulation digestion process, supernatant is taken after stomach cardia digests 2h and after pepsin-pancreatin digestion 4h, is utilized
EDTA complexometric titration wherein zinc ion content, to indicate the zinc ion dissolution rate of different digestion phases.
V1: EDTA liquor capacity required for zinc ion, mL in titration supernatant；
V2: titrate EDTA liquor capacity, mL required for zinc ion in isometric solution；
The concentration of C:EDTA solution, mol/L.
The measurement of zinc ion dialysance
After pancreatin digestion, the aqueous solution outside bag filter is taken, is contained using EDTA complexometry measurement aqueous zinc
Amount, to indicate the zinc ion content through simulation enteron aisle.
V1: EDTA liquor capacity required for zinc ion, mL in titration supernatant；
V2: titrate EDTA liquor capacity, mL required for zinc ion in isometric solution；
The concentration of C:EDTA solution, mol/L.
The many because being known as of Measurements of Zinc in Foods utilization rate are influenced, human body is lower to the absorptivity of zinc in diet, and main cause is
Zinc and phytic acid can form insoluble compound in small intestine, since human intestine lacks corresponding phytase, this insoluble zinc-
Phytic acid complexes cannot greatly reduce the bioavailability of zinc by human consumption and utilization, be disappeared using in-vitro simulated gastrointestinal tract
Change-dialysis can evaluate the bioavailability of mineral matter element, the dialysance of peptide chelates of zinc and Zinc vitriol and molten
Solution property is measured according to the experimental method of the descriptions such as Wolfgor and Wang, after pipe intestinal digesting, the dissolution rate of two kinds of substances
It is as shown in Figure 10 with dialysance.
According to the experimental results, in peptide chelates of zinc dissolution rate of the zinc ion after pipe intestinal digesting be respectively 98.91%,
55.69% (P > 0.05)；And dissolution rate of the zinc ion after pipe intestinal digesting is respectively 97.8%, 33.15% in inorganic zinc salt
(P<0.05)；The dialysance of zinc ion is respectively 43.00%, 23.41% in peptide chelates of zinc and inorganic zinc salt, peptide chelates of zinc
The dialysance of middle zinc ion is significantly higher than the dialysance of zinc ion in inorganic zinc salt, although inorganic zinc salt is present in stomach with solubilised state
It in portion's environment, but is remarkably decreased into its dissolubility after intestinal environment, therefore the zinc ion in peptide chelates of zinc is easier to enter
Into intestinal environment, this shows compared with inorganic zinc salt, and the bioavailability of peptide chelates of zinc is higher, and main cause may
Have two aspects: on the one hand, intestinal environment category weakly alkaline environment, is the major absorption site of metal ion, and zinc ion is formed with peptide
Chelate bonds it is more more stable than the ionic bond in inorganic zinc salt, after pipe intestinal digesting, peptide chelates of zinc still exists with molecular forms,
Zinc ion is set to be hardly formed zinc hydroxide precipitation；On the other hand, the small peptide chelates of zinc of number molecular weight is straight with molecular forms
It is absorbed to connect intestinal wall (bag filter), and the biggish Whey Protein Concentrate peptide chelates of zinc of number molecular weight can be in stomach
Pepsin under the action of continue to be broken down into small molecule, reenter and by intestinal absorption.
The present invention has the advantage that
(1) hydrolysis by novo is used, hydrolyzate metal-chelating rate is higher than other protease；
(2) optimization of mung bean protein hydrolase polypeptide technique, the degree of hydrolysis after optimization are higher；
(3) ultrafiltration segmentation has been carried out to the hydrolyzate of protein peptides, chelatropic reaction is carried out with the highest peptide fragment of chelation percent；
(4) chelating technique is also optimized, and polypeptide is enable to chelate to the greatest extent with zinc；
(5) greatly increase the bioavailability of metal ion, just compensate for the defect of some zinc supplementation agent, with zinc from
Centered on son, five yuan or six-membered ring structure are closely formed in conjunction with amino and carbonyl, avoid zinc ion in the enteron aisle ring of alkalinity
Border generates precipitating and loses；
(6) peptide-chelate of metal ion is the principal mode of body absorption and transhipment metal ion, and is body synthesis egg
The intermediate material of white process, infiltration rate is fast and can be much less biochemical process, saves human body energy consumption.
The present invention makes mung bean protein peptide and Zinc Ions Chelated using mung bean protein as raw material, and mung bean protein peptide is utilized and is conducive to
Absorption of human body can promote small intestine to the absorption of zinc and accumulate in vivo, to improve the absorption rate of zinc, avoid zinc ion
Precipitating is generated in the intestinal environment of alkalinity and is lost, the digestibility and retention rate of mung bean protein peptide chelates of zinc of the invention
It is above organic zinc and zinc carbonate；Hydrolysis process, chelating technique are optimized simultaneously, further increase the preparation effect of chelate
Rate.
Claims (7)
1. a kind of preparation method of mung bean protein peptide-chelates of zinc, it is characterised in that: be using mung bean protein as raw material, through albumen
Mung bean protein peptide hydrolysate is obtained after enzyme hydrolysis, after the polypeptide freeze-drying in mung bean protein peptide hydrolysate, with zinc chelatropic reaction
Obtain mung bean protein peptide-chelates of zinc.
2. the preparation method of mung bean protein peptide-chelates of zinc according to claim 1, it is characterised in that: the protease
For alkali protease.
3. the preparation method of mung bean protein peptide-chelates of zinc according to claim 1 or 2, it is characterised in that: further include pair
Mung bean protein peptide hydrolysate carries out ultrafiltration classification, filters out 3-5ku peptide fragment and carries out subsequent chelatropic reaction.
4. the preparation method of mung bean protein peptide-chelates of zinc according to claim 1, it is characterised in that: the chelating
The condition of reaction is polypeptide and zinc mass ratio 3:1-7:1, pH4-8,30-70 DEG C of temperature, reaction time 40-120 min.
5. the preparation method of mung bean protein peptide-chelates of zinc according to claim 4, it is characterised in that: the chelating
The condition of reaction is peptide and zinc mass ratio 6.08:1, pH6.31,50.32 DEG C of temperature, 80.99 min of reaction time.
6. the preparation method of mung bean protein peptide-chelates of zinc according to claim 1, it is characterised in that: this method includes
Following steps: taking 50 g mung bean proteins to be dissolved in the deionized water of 1.25 L, first and in 90 DEG C of water-baths pre-processes 15 minutes,
With 0.5 mol/L NaOH tune pH to 9 after temperature is down to 55 DEG C, 4ml alkali protease is added, with the NaOH of 0.5 mol/L
Keeping pH is 9, hydrolyzes boiling water bath enzyme deactivation after 4 h, hydrolyzate is cooled to room temperature, with 1mol/L HCL adjust pH value to after 7 in
5000r/min is centrifuged 15 min, takes supernatant；By supernatant ultrafiltration under 0.25 MPa pressure, successively with through molecular mass
For the ultrafiltration membrane ultrafiltration of 5,3ku, the mung bean protein peptide filter liquor that molecular mass is 3-5ku is obtained, the peptide fragment after classification is penetrated
It is spare after liquid freeze-drying；6.08g mung bean protein peptide is dissolved in and is made into the peptide liquid that concentration of substrate is 5% in 121.6 ml deionized waters, is used
1mol/L HCL adjusts pH to 6.31, rear that 1g ZnSO is added4•7H2O is vortexed and mixes, and carries out chelatropic reaction, and reaction temperature is
50.32 DEG C, the reaction time is 80.99 min, is after reaction cooled to room temperature solution, with three times dehydrated alcohol alcohol precipitation, to
Precipitation is centrifuged 15min with 10000r/min, collects precipitating, washed with dehydrated alcohol to supernatant and zinc indicator is added not
Discoloration；Then baking oven elder generation temperature regulating drops to 30 DEG C of baking chelates to doing, obtains the high mung bean protein of chelation percent to 100 DEG C of baking 1h
Peptide-chelates of zinc.
7. the preparation method of mung bean protein peptide-chelates of zinc according to claim 1, it is characterised in that: the mung bean
Protein peptides-chelates of zinc is promoting the application in zinc-iron alloy solution.
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| CN110951812A (en) * | 2019-12-31 | 2020-04-03 | 河南工业大学 | Preparation method of peanut peptide-zinc chelate |
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| CN114686552A (en) * | 2022-04-02 | 2022-07-01 | 吉林农业大学 | Preparation method of camellia seed meal polypeptide zinc chelate |
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