CN106854672A - A kind of method that electron beam irradiation joint enzyme process isolates and purifies the wheat germ polypeptide of affine calcium high - Google Patents
A kind of method that electron beam irradiation joint enzyme process isolates and purifies the wheat germ polypeptide of affine calcium high Download PDFInfo
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- CN106854672A CN106854672A CN201710004653.0A CN201710004653A CN106854672A CN 106854672 A CN106854672 A CN 106854672A CN 201710004653 A CN201710004653 A CN 201710004653A CN 106854672 A CN106854672 A CN 106854672A
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The method that the wheat germ polypeptide of affine calcium high is isolated and purified the present invention relates to a kind of electron beam irradiation joint enzyme process.The present invention separates albumen as raw material with wheat germ, albumen is made into weakly alkaline suspension, the suspension is by after electron beam irradiation, wheat germ polypeptide is obtained using alkali protease solution, mixtures of polypeptides sequentially passes through the purification steps such as ultrafiltration, anion-exchange chromatography, gel filtration chromatography, RP HPLC, obtains the polypeptide Phe Val Asp Val Thr with highest chelating properties.The calcium chelating peptide that the present invention is obtained by transhipment, the mechanism of absorption of small peptide in the form of complex by small intestine active absorption, the utilization rate of calcium can be remarkably promoted.
Description
Technical field
The present invention relates to the technical field of preparation and the agricultural product intensive processing of Functional Polypeptides, more particularly, to a kind of wheat germ
The method of protein sources calcium chelating peptide.
Background technology
Because the ratio shared by vegetable food in the dietary structure of China is higher, calcium deficiency phenomenon is often produced, especially
Children, pregnant woman and old man, therefore replenish the calcium as the important topic in domestic scholars research.At present, calcium-supplementing preparation on the market
Predominantly inorganic calcium, organic calcium, amino acid calcium.Inorganic calcium (such as calcium carbonate, calcium phosphate, calcium chloride), this kind of calcium tonic is in stomach
Stability and dissolubility are poor in enteron aisle, and absorption rate is low, have stimulation to stomach；Organic calcium (such as calcium lactate, gluconic acid
Calcium, calcium citrate etc.), this kind of calcium tonic calcium content is low, and overall utilization is not high；Amino acid calcium (such as calcium glycine, calcium glutamate
Deng), this kind of calcium tonic calcium absorptivity is significantly improved, but is easily precipitated by phytic acid, the adverse effect for having the oxidation for promoting fat；Peptide-
Calcium, the calcium supplementing product as a new generation has obvious advantage, and calcium ion is by transhipment, the mechanism of absorption of small peptide with the shape of chelate
Formula not only accelerates the transhipment of calcium, and less energy intensive by intestinal absorption, remarkably promotes the utilization rate of calcium.At present, on peptide-calcium
Research focus primarily upon animal protein, the research to vegetable protein is less, but, animal protein high cost is present potential
Anaphylactogen, so being used for the preparation of peptide-calcium in the urgent need to developing the albumen of plant source.
Wheat embryo is the accessory substance (reserves reach 200~3,000,000 tons) of Machining Flour, containing abundant starch,
Protein, unsaturated fat, vitamin and mineral element, " the natural nutrition treasure-house of the mankind " is described as by nutritionist, wherein
Protein content accounts for 30% or so, is a kind of adequate proteins, it is necessary to which amino acid ligand connects substantially than the mode value recommended with WHO/FAO
Closely, nutritive value can match in excellence or beauty with Chicken Albumin.Wheat germ protein mainly includes albumin, globulin, glutelin and alcohol soluble protein.
Wherein, the dissolubility of globulin, glutelin and alcohol soluble protein is bad, and the comprehensive profit of wheat germ protein is limited to a certain extent
With causing the wasting of resources, environmental pollution.In order to save grain detraction, increase the added value of wheat germ, we attempt using electron beam irradiation
Treatment prepares the wheat germ polypeptide of affine calcium high with reference to enzyme process and the method for isolating and purifying.The wheat germ prepared based on the present invention is more
Peptide-calcium ion chelate, not only supplemented with calcium constituent and amino acid, and has carried out intensive processing and has had important to wheat embryo
Economy and theory significance.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the applicant separates pure there is provided a kind of electron beam irradiation joint enzyme process
The method for changing the wheat germ polypeptide of affine calcium high.The present invention can not only within a short period of time obtain substantial amounts of active peptide, promote wheat
The comprehensive utilization of embryo protein, and, the calcium chelating peptide prepared using wheat germ polypeptide can be small by the transhipment of small peptide, mechanism of absorption
The absorption of intestines rapid, high volume, significantly improves the utilization rate of calcium.
Technical scheme is as follows：
(1) a kind of electron beam irradiation joint enzyme process isolates and purifies the wheat germ polypeptide of affine calcium high, the amino acid sequence of polypeptide
It is Phe-Val-Asp-Val-Thr.
(2) a kind of electron beam irradiation joint enzyme process isolates and purifies the wheat germ polypeptide of affine calcium high, comprises the following steps：
(1) with defatted wheat germ as raw material, extract wheat germ with reference to the heavy method of alkali soluble acid and separate albumen, extraction conditions are：It is de-
Fat wheat germ powder is with solid-liquid ratio 1:10 are scattered in deionized water, and it is 9.0~9.5, constant temperature 40~45 that the pH of dispersion liquid is transferred into pH
DEG C stirring 2h, centrifugation obtain supernatant, supernatant pH is transferred to pH for 4.0~4.5 carry out protein precipitation, recalled to after albumen is centrifuged
PH is 7.0, obtains wheat germ separation albumen and freeze-drying is standby.
(2) wheat germ separates protein dry powder and is well mixed by solid-liquid ratio 10~40% (w/v) with deionized water, is formed suspended
Liquid, addition alkali lye is tuned into alkalescent (pH is 7.5~8.5), and suspension is sealed in transparent, radiotolerant hermetic bag, suspended
The thickness control of liquid is in 1~2cm.
(3) above-mentioned albumen suspension is modified using electronic beam irradiation technology, modified condition is：Irradiation dose is
1~10kGy, radiation parameter is 1.5~5.0MeV/20~40mA.
(4) protein after irradiated obtains wheat germ protein enzymolysis product by hydrolysis by novo.Hydrolysising condition
For：Protein concentration 2~5%, enzymolysis pH is that the weight proportion of 50~60 DEG C of 8.0~8.5, temperature, enzyme and albumen is 1:50~1:
100。
(5) enzymolysis product is separated first with ultra-filtration process, interception<1kDa, 1~3kDa,>The component of 3kDa, point
Each component is not collected.
(6) the calcium chelation percent highest component after ultrafiltration is collected, is carried out by the anion-exchange chromatographies of DEAE-FF 16/10
Separate, eluent is the Tris-HCl containing 0-0.5M NaCI for concentration gradient, and flow velocity is 5mL/min, and eluting peak is in 214nm
Under measure, dialysis desalting.
(7) collect by the isolated calcium chelation percent highest component of anion-exchange chromatography, by Superdex
The gel filtration chromatographies of Peptide 10/300 are separated, and eluent is deionized water, and flow velocity is 0.5mL/min, and eluting peak exists
It is measured under 214nm.
(8) collect and separate the calcium chelation percent highest component for obtaining by gel filtration chromatography, divided by RP-HPLC
From separation condition is that flow velocity lmL/min, the self-contained volume ratio of eluent is 5% as gradient eluent with 5-30% second eyeball solution
The mixed liquor of second eyeball and 95% water starts, and terminates to the mixed liquor that volume ratio is 30% second eyeball and 70% water, carries out gradient elution.
The present invention is beneficial to be had technical effect that：When being modified to protein suspension with electronic beam irradiation technology,
Electron beam can induce hydrone therein to crack, and generate free radical, ion, excite a large amount of active particles such as molecule.These
Active particle often has one or several unpaired electronics, therefore chemical property is highly unstable, easily attacks protein
Deng other biological macromolecular, promote protein molecular that deamination, decarboxylation, amino-acid oxidase, the fracture of disulfide bond or reconstruction, peptide chain occur
Degraded or crosslinking, hydrophobic group the series reaction such as expose so that it is poly- between the higher structure and protein molecular of protein molecular
Mode set changes.Modified protein can all produce very big change in the aspects such as viscosity, solubility, these changes
All will to a certain extent be conducive to the effect of biology enzyme, accelerate the hydrolysis of enzyme, shorten enzymolysis time, improve the hydrolysis of albumen
Degree, and then produce substantial amounts of Functional Polypeptides.These Functional Polypeptides are further isolated and purified can obtain the close calcium that chelation percent is significantly improved
Wheat germ polypeptide.
Brief description of the drawings
Fig. 1 is the analysis collection of illustrative plates of the anion-exchange chromatographies of DEAE-FF 16/10 in the embodiment of the present invention 3；
Fig. 2 is the analysis collection of illustrative plates of the gel filtration chromatographies of Superdex Peptide 10/300 in the embodiment of the present invention 3；
Fig. 3 is the separating spectrum of RP-HPLC in the embodiment of the present invention 3.
Fig. 4 is the mass spectrogram of Phe-Val-Asp-Val-Thr in the embodiment of the present invention 3.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
(1) wheat germ separates protein extraction：Defatted wheat germ powder (be purchased from Earthquake of Anyang station in Henan and avenge Co., Ltd all over the sky) is with solid-liquid ratio 1:
10 (w/v) are scattered in deionized water, and the acid-base value of dispersion liquid is constant in pH9.5, constant temperature 40 DEG C of stirrings 2h, 5000rpm from
Heart 10min obtains supernatant, and supernatant is transferred to pH4.0 protein precipitations, and albumen is washed 2 times and pH is transferred into neutrality, freeze-drying
Wheat germ is obtained afterwards separates albumen.
(2) wheat germ separated protein powder is mixed with deionized water by solid-liquid ratio 40% (w/v), adds alkali lye, forms alkalescent
Suspension (pH8.0), suspension is sealed in transparent hermetic bag, and thickness control is in 1~2cm.
(3) albumen suspension is placed on conveyer belt, in irradiation, irradiation dose 1kGy, radiation parameter under electron accelerator
5.0MeV/20mA。
(4) albumen after irradiated obtains wheat germ mixtures of polypeptides by hydrolysis by novo.Hydrolysising condition is：Egg
White concentration 5% (w/v), digests pH8.0, and temperature 50 C, enzyme is 1 with the weight proportion of albumen:100, hydrolyze 3h.Hydrolysis is completed
Afterwards, enzymolysis liquid boils 10min in boiling water, and 5000rpm centrifugations 10min, collects supernatant freeze-drying standby after cooling.
(5) enzymolysis product in (4) is separated by ultra-filtration process, ultra-filtration process is：Enzymolysis product is made after dissolving again
The polypeptide solution of 10mg/ml, the filter membrane for choosing 1kDa, 3kDa carries out ultrafiltration with PALL ultrafiltration systems, collects<1kDa, 1~
3kDa、>The component of 3kDa.
(6) calcium chelation percent highest component in (5) is placed in into the anion-exchange chromatographies of DEAE-FF 16/10 to be separated,
Eluent is the 20mM Tris-HCl (pH7.5) containing 0-0.5M NaCI for concentration gradient, and flow velocity is 5mL/min, eluting peak
Measured at 214nm.
(7) calcium chelation percent highest component in (6) is placed in into the gel filtration chromatographies of Superdex Peptide 10/300 to enter
Row is separated, and eluent is deionized water, and flow velocity is 0.5mL/min, and eluting peak is measured at 214nm.
(8) calcium chelation percent highest component in (7) is separated by RP-HPLC, separation condition is to use 5-30% second
Used as gradient eluent, flow velocity is lmL/min to eyeball solution, eluent is by the mixed liquor of 5% second eyeball and 95% water, to 30%
The mixed liquor of second eyeball and 70% water terminates, and carries out gradient elution, collects calcium chelation percent highest component, then using LC/MS liquid matter
Combination mass spectrometer draws polypeptide sequence.
Embodiment 2
(1) wheat germ separates protein extraction：Defatted wheat germ powder (be purchased from Earthquake of Anyang station in Henan and avenge Co., Ltd all over the sky) is with solid-liquid ratio 1:
10 (w/v) are scattered in deionized water, and the acid-base value of dispersion liquid is constant in pH9.5, constant temperature 40 DEG C of stirrings 2h, 5000rpm from
Heart 10min obtains supernatant, and supernatant is transferred to pH4.0 protein precipitations, and albumen is washed 2 times and pH is transferred into neutrality, freeze-drying
Wheat germ is obtained afterwards separates albumen.
(2) wheat germ separated protein powder is mixed with deionized water by solid-liquid ratio 10% (w/v), adds alkali lye, forms alkalescent
Suspension (pH8.5), suspension is sealed in transparent hermetic bag, and thickness control is in 1~2cm.
(3) albumen suspension is placed on conveyer belt, in irradiation, irradiation dose 5kGy, radiation parameter under electron accelerator
5.0MeV/20mA。
(4) albumen after irradiated obtains wheat germ mixtures of polypeptides by hydrolysis by novo.Hydrolysising condition is：Egg
White concentration 2% (w/v), digests pH8.5, and temperature 60 C, enzyme is 1 with the weight proportion of albumen:80, hydrolyze 3h.After the completion of hydrolysis,
Enzymolysis liquid boils 10min in boiling water, and 5000rpm centrifugations 10min, collects supernatant freeze-drying standby after cooling.
(5) enzymolysis product in (4) is separated by ultra-filtration process, ultra-filtration process is：Enzymolysis product is made after dissolving again
The polypeptide solution of 10mg/ml, the filter membrane for choosing 1kDa, 3kDa carries out ultrafiltration with PALL ultrafiltration systems, collects<1kDa, 1~
3kDa、>The component of 3kDa.
(6) calcium chelation percent highest component in (5) is placed in into the anion-exchange chromatographies of DEAE-FF 16/10 to be separated,
Eluent is the 20mM Tris-HCl (pH7.5) containing 0-0.5M NaCI for concentration gradient, and flow velocity is 5mL/min, eluting peak
Measured at 214nm.
(7) calcium chelation percent highest component in (6) is placed in into the gel filtration chromatographies of Superdex Peptide 10/300 to enter
Row is separated, and eluent is deionized water, and flow velocity is 0.5mL/min, and eluting peak is measured at 214nm.
(8) calcium chelation percent highest component in (7) is separated by RP-HPLC, separation condition is to use 5-30% second
Used as gradient eluent, flow velocity is lmL/min to eyeball solution, eluent is by the mixed liquor of 5% second eyeball and 95% water, to 30%
The mixed liquor of second eyeball and 70% water terminates, and carries out gradient elution, collects calcium chelation percent highest component, then using LC/MS liquid matter
Combination mass spectrometer draws polypeptide sequence.
Embodiment 3
(1) wheat germ separates protein extraction：Defatted wheat germ powder (be purchased from Earthquake of Anyang station in Henan and avenge Co., Ltd all over the sky) is with solid-liquid ratio 1:
10 (w/v) are scattered in deionized water, and the acid-base value of dispersion liquid is constant in pH9.5, constant temperature 40 DEG C of stirrings 2h, 5000rpm from
Heart 10min obtains supernatant, and supernatant is transferred to pH4.0 protein precipitations, and albumen is washed 2 times and pH is transferred into neutrality, freeze-drying
Wheat germ is obtained afterwards separates albumen.
(2) wheat germ separated protein powder is mixed with deionized water by solid-liquid ratio 25% (w/v), adds alkali lye, forms alkalescent
Suspension (pH7.5), suspension is sealed in transparent hermetic bag, and thickness control is in 1~2cm.
(3) albumen suspension is placed on conveyer belt, in irradiation, irradiation dose 10kGy, radiation parameter under electron accelerator
5.0MeV/20mA。
(4) albumen after irradiated obtains wheat germ mixtures of polypeptides by hydrolysis by novo.Hydrolysising condition is：Egg
White concentration 5% (w/v), digests pH8.0, and temperature 50 C, enzyme is 1 with the weight proportion of albumen:50, hydrolyze 3h.After the completion of hydrolysis,
Enzymolysis liquid boils 10min in boiling water, and 5000rpm centrifugations 10min, collects supernatant freeze-drying standby after cooling.
(5) enzymolysis product in (4) is separated by ultra-filtration process, ultra-filtration process is：Enzymolysis product is made after dissolving again
The polypeptide solution of 10mg/ml, the filter membrane for choosing 1kDa, 3kDa carries out ultrafiltration with PALL ultrafiltration systems, collects<1kDa, 1~
3kDa、>The component of 3kDa.
(6) calcium chelation percent highest component in (5) is placed in into the anion-exchange chromatographies of DEAE-FF 16/10 to be separated,
Eluent is the 20mM Tris-HCl (pH7.5) containing 0-0.5M NaCI for concentration gradient, and flow velocity is 5mL/min, eluting peak
Measured at 214nm.
(7) calcium chelation percent highest component in (6) is placed in into the gel filtration chromatographies of Superdex Peptide 10/300 to enter
Row is separated, and eluent is deionized water, and flow velocity is 0.5mL/min, and eluting peak is measured at 214nm.
(8) calcium chelation percent highest component in (7) is separated by RP-HPLC, separation condition is to use 5-30% second
Used as gradient eluent, flow velocity is lmL/min to eyeball solution, eluent is by the mixed liquor of 5% second eyeball and 95% water, to 30%
The mixed liquor of second eyeball and 70% water terminates, and carries out gradient elution, collects calcium chelation percent highest component, then using LC/MS liquid matter
Combination mass spectrometer draws polypeptide sequence.
Fig. 1 is wheat germ polypeptide DEAE-FF anion-exchange chromatography figures, and 3 peaks, wherein peak F2 are obtained through anion column purifying
Calcium ion it is sequestering most strong, collect the peak for the separation of gel column.Fig. 2 is wheat germ peptide-based gel filtering chromatogram figure, through solidifying
The purifying of glue post obtains 4 peaks, and the calcium ion of wherein peak F22 is sequestering most strong, collects the peak for the separation of RP-HPLC.
Fig. 3 is wheat germ polypeptide RPLC figure, and inverted post purifying is main to obtain 2 peaks, wherein the calcium ion chelating at peak 2
Property it is most strong, collect the peak for polypeptide sequencing.Fig. 4 schemes for LC-ESI/MS, by the result of LC-ESI/MS, identifies polypeptide
Amino acid sequence be Phe-Val-Asp-Val-Thr.The chelation percent of highest component calcium is as shown in table 1 in each step.
The chelation percent of highest component calcium in each purification procedures of table 1
The Phe-Val-Asp-Val-Thr obtained in embodiment 3 is improved than Hydrolyzing Protein in De fat Wheat Germ mixture, the chelation percent of calcium
86.37%.
《Ocean gelatine collagen oligopeptide calcium is isolated and purified and Structural Identification》(Liu Wenying, appoints the food industry such as Wei science and technology,
4th phase in 2015) in, there is provided after reverse chromatograms preliminary purification, calcium chelation percent is 51.38% ± 0.09% for it.The present invention
Chelating rate score to be far longer than the technology.
Above-described is only the preferred embodiment of the present invention, the invention is not restricted to above example.It is appreciated that this
Other improvement and change that art personnel directly derive or associate without departing from the spirit and concept in the present invention
Change, be considered as being included within protection scope of the present invention.
Claims (6)
1. a kind of method that electron beam irradiation joint enzyme process isolates and purifies the wheat germ polypeptide of affine calcium high, it is characterised in that：With wheat
It is raw material that embryo separates albumen, and wheat germ separation albumen is made into weakly alkaline suspension, and the suspension is by cold after electron beam irradiation
Freeze drying for standby, albumen separated using hydrolysis by novo wheat germ, obtain wheat germ mixtures of polypeptides, mixture by ultrafiltration,
Anion-exchange chromatography, gel filtration chromatography and RP-HPLC isolate and purify to obtain wheat germ polypeptide.
2. method according to claim 1, it is characterised in that：The preparation method of the suspension is：Wheat germ is separated into egg
Be well mixed for 10~40% according to feed liquid w/v ratios with deionized water in vain, add alkali lye, formed pH be 7.5~8.5 it is suspended
Liquid.
3. method according to claim 1, it is characterised in that：The method of the electron beam irradiation is：Suspension is sealed
In transparent, radiotolerant hermetic bag, in 1~2cm, electron irradiation dosage is 1~10kGy, irradiation to the thickness control of suspension
Condition is 1.5~5.0MeV/20~40mA.
4. method according to claim 1, it is characterised in that：The hydrolysis by novo condition is：Protein concentration is 2
~5%, enzymolysis pH are that 8.0~8.5,50~60 DEG C of temperature, enzyme and protein by weight proportioning are 1:50~1:100.
5. method according to claim 1, it is characterised in that：It is described isolate and purify concretely comprise the following steps wheat germ polypeptide first
Separated using ultra-filtration process, intercepted<1kDa, 1~3kDa,>The component of 3kDa；Calcium chelation percent highest component is collected to pass through
The anion-exchange chromatographies of DEAE-FF 16/10 are separated, and eluent is the Tris- containing 0~0.5M NaCI for concentration gradient
HCl, flow velocity is 5mL/min, and eluting peak is measured under 214nm；Collect calcium chelation percent highest component and pass through Superdex
The gel filtration chromatographies of Peptide 10/300 are separated, and eluent is deionized water, and flow velocity is 0.5mL/min, and eluting peak exists
It is measured under 214nm；Collect calcium chelation percent highest component to be separated by RP-HPLC, separation condition is with 5~30%
Used as gradient eluent, flow velocity is lmL/min to second eyeball solution, and the self-contained volume ratio of eluent is the mixing of 5% second eyeball and 95% water
Liquid starts, and terminates to the mixed liquor that volume ratio is 30% second eyeball and 70% water, carries out gradient elution, collects calcium chelation percent highest
Component.
6. method according to claim 1, it is characterised in that：The amino acid sequence of the polypeptide is Phe-Val-Asp-
Val-Thr。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107509996A (en) * | 2017-08-30 | 2017-12-26 | 常州市海若纺织品有限公司 | A kind of bone diet nutritional powder and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626847A (en) * | 2013-11-15 | 2014-03-12 | 江南大学 | Wheat germ protein source zinc phytochelatin and preparation method thereof |
| CN103937863A (en) * | 2014-04-17 | 2014-07-23 | 吉林大学 | Method for improving enzymolysis effect of soybean protein concentrate powder by means of irradiation technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626847A (en) * | 2013-11-15 | 2014-03-12 | 江南大学 | Wheat germ protein source zinc phytochelatin and preparation method thereof |
| CN103937863A (en) * | 2014-04-17 | 2014-07-23 | 吉林大学 | Method for improving enzymolysis effect of soybean protein concentrate powder by means of irradiation technology |
Non-Patent Citations (1)
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| CN107509996A (en) * | 2017-08-30 | 2017-12-26 | 常州市海若纺织品有限公司 | A kind of bone diet nutritional powder and preparation method thereof |
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