CN100569795C - A kind of oxidation resistance zymolyte that is rich in pollen active peptide - Google Patents
A kind of oxidation resistance zymolyte that is rich in pollen active peptide Download PDFInfo
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- CN100569795C CN100569795C CNB2007100977153A CN200710097715A CN100569795C CN 100569795 C CN100569795 C CN 100569795C CN B2007100977153 A CNB2007100977153 A CN B2007100977153A CN 200710097715 A CN200710097715 A CN 200710097715A CN 100569795 C CN100569795 C CN 100569795C
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Abstract
The present invention relates to a kind of oxidation resistance zymolyte that is rich in pollen active peptide, be to be raw material with the bee pollen, with broken wall of melissa pollen, after the degreasing, add 8～25 times of amount deionized waters, adjusting the pH value is 6～10, be heated to 30～60 ℃, add proteolytic enzyme and carry out enzyme digestion reaction, temperature is controlled at 20～80 ℃, insulated and stirred 30～180min, proceed to protein content more after a little while at enzyme digestion reaction, heat to more than 80 ℃ insulated and stirred 10～30min, enzymolysis reaction rapidly, reduce to room temperature then rapidly, carry out solid-liquid separation, obtain clear liquid, separate with the ultra-filtration membrane of holding back relative molecular weight＞10000D, get filtered solution and separate, get the surplus liquid drying of filter with ultra-filtration membrane or the nanofiltration membrane of holding back relative molecular weight＜400D.This zymolyte has the anti-oxidant effect of Denging, can be made into pulvis, tablet, capsule, electuary etc.The present invention does not contain any chemical reagent, and great exploitation potential for its is having good prospects for application aspect healthcare products, makeup and the foodstuff additive.
Description
Technical field
The present invention relates to a kind of bee pollen zymolyte and preparation method thereof, this zymolyte has the feature that significantly improves the bee pollen resistance of oxidation, is rich in pollen active peptide.The invention belongs to natural product deep processing and technical field of bioengineering, be specifically related to medicine biological technique or functional foodstuff processing technology, particularly be rich in the method for the oxidation resistance zymolyte of pollen active peptide with the bee pollen preparation.
Background technology
Bee pollen is that honeybee is gathered the pollen of returning, except that the composition that general pollen is arranged, also contain a small amount of nectar and the secretory product when gathering, added, has stronger physiologically active, can strengthen immune function of human body, antitumor, anti-constipation, delay senility, having beautification function, is a kind of ideal natural nutriment of worth research and development.
Why bee pollen has medical care effect, except wherein containing numerous nutritive ingredients, also is undivided with containing miscellaneous biologically active substance in the bee pollen.These activeconstituentss have activated protein, bioactive peptide, flavonoid compound, active polysaccharide and trace element etc.Protein content is quite abundant in the bee pollen, accounts for 7%～35% of its dry weight, and amino acid accounts for more than 20% of total amount, total free aminoacids 1%～2%.Protein content in rape (the B rassica campestris) bee pollen is about 27%, but water-soluble protein only accounts for about 30% of pollen total protein, all the other are water-insoluble and insoluble albumen, human body is difficult to digest and assimilate, in addition, contain some in the pollen and can cause protein hypersensitive, influenced some people enjoying bee pollen.In order better utilised the Nature to give human rarity, this patent utilizes different proteolytic enzyme that pollen protein is degraded to micromolecular bioactive peptide, has improved the bioavailability of pollen, has reduced to take pollen and cause possibility hypersensitive.The preparation of being rich in the oxidation resistance zymolyte of pollen active peptide does not at home and abroad appear in the newspapers.
Summary of the invention
One of purpose of the present invention is poor for the edible palatability that solves existing bee pollen existence, and anaphylaxis such as existence has a stomach-ache and low dose, short period of time were taken bee pollen, the inapparent problem of its biopotency after part crowd took; A kind of oxidation resistance zymolyte that is rich in pollen active peptide that is derived from bee pollen is provided.
Two of purpose of the present invention provides a kind of preparation method who is rich in the oxidation resistance zymolyte of pollen active peptide, this method adopts suitable enzyme solution, insoluble high molecular weight protein in the bee pollen is decomposed into the solubility small-molecular peptides, obtains being rich in the enzymolysis product of pollen active peptide.Experiment in vitro shows that this zymolyte has very strong antioxygenation, has good scavenging(action) to lipid peroxidation, hydroxy radical qiao, ultra-oxygen anion free radical.
For achieving the above object, the technical solution used in the present invention is as follows:
With broken wall of melissa pollen, after the degreasing, add 8～25 times of amount deionized waters, preferred 10～20 times of amount deionized waters, use alkali to regulate pH value to 6～10, preferred 7～9, preferred alkali metal hydroxide (the sodium hydroxide that uses, potassium hydroxide etc.), alkaline carbonate (yellow soda ash, salt of wormwood etc.), alkali metal hydrocarbonate (sodium bicarbonate, saleratus etc.) alkali remains on pH the scope of regulation, add proteolytic enzyme (neutral protease, papoid, trypsinase, Sumizyme MP etc.), preferred Sumizyme MP, temperature is controlled at 20～80 ℃, preferred 30～50 ℃, insulated and stirred 30～180min, preferred 120～180min carries out enzymolysis, make the insoluble protein in the pollen be converted into soluble peptide, to improve the extraction yield of pollen peptide.Reaction finishes the back and heats up 70～90 ℃, insulated and stirred 10～30min enzyme that goes out, reduce to room temperature then rapidly, filter or the centrifugal precipitation of removing, collect clear liquid, separate with the ultra-filtration membrane of holding back relative molecular weight＞10000D, get filtered solution and separate, get the surplus liquid freeze-drying of filter and preserve with ultrafiltration or the nanofiltration membrane of holding back relative molecular weight＜400D.This zymolyte is pollen active peptide and amino acid whose mixture, is rich in the peptide section of all size, also contains biologically active substances such as pollen flavones.
The present invention is under the condition of particular concentration, temperature, time, with specific enzyme bee pollen albumen is carried out enzymolysis, with the film in different apertures enzymolysis solution is separated enrichment with effective constituent, obtain being rich in the enzymolysis product of pollen active peptide, can significantly improve the antioxygenation of bee pollen.
The oxidation resistance zymolyte that is rich in pollen active peptide of gained of the present invention can be cooked foodstuff additive, immunomodulator, also can add edible, medicinal or the used for cosmetic auxiliary material is made healthcare products, cosmetic formulations.
The invention has the advantages that:
1, raw material is a crude substance, wide material sources, and extract obtained any objectionable impurities and the chemical assistant of not containing has anti-oxidant function.
2, according to proteinic characteristic in the Brassica campestris L pollen, select suitable enzyme solution, the water-insoluble pollen protein is degraded to the soluble peptide of different molecular weight, has increased substantially the content of bioactive peptide, wherein contained abundant small molecule active peptide and nutritive substance easily are absorbed by the body.
3, solvability is good, extraction yield is high, the zymolyte solubleness in water that is rich in pollen active peptide is big, and bioavailability is good, and extraction yield is about 60%, and the extraction yield of common aqueous extract only is about 30%, by zymolysis technique the extraction yield of pollen is significantly increased.
4, the enzyme solution technological process is simple, pollution-free, noresidue, and reaction substrate, reagent and reaction environment are safe from harm, and be easy to operate, and the efficient height is suitable for suitability for industrialized production, and application and development prospect is preferably arranged.
5, contain a certain amount of pollen flavones in the pollen enzymolysis product, play synergy, improve biological activity, the especially anti-oxidant activity of bee pollen with pollen active peptide.
6, the functional factor (pollen active peptide, pollen flavones) that obtains by enzymolysis process is compared with bee pollen and is had more significant, specific medical health care function, the allergic phenomena of having avoided the vegetable-protein in the bee pollen that human body is produced simultaneously.Contain hardly in the pollen zymolyte among the present invention or do not contain fully and can be used as antigenic macro-molecular protein, therefore, can avoid anaphylaxis.
9, the antioxidation activity in vitro experiment shows that bee pollen zymolyte of the present invention has anti peroxidation of lipid, removes the effect of ultra-oxygen anion free radical and hydroxy radical qiao.
Description of drawings
Fig. 1 is the simple process flow diagram of the inventive method.
Fig. 2 is the comparison that pollen zymolyte and pollen aqueous extract are removed the hydroxy radical qiao effect
Fig. 3 is the comparison that pollen zymolyte and pollen aqueous extract are removed ultra-oxygen anion free radical
Fig. 4 is the comparison of pollen zymolyte and the oxidation of pollen aqueous extract lipotropism matter
In order to understand essence of an invention better, describe technology contents of the present invention in detail with embodiment below, should be understood that these embodiment only are used to the present invention is described and are not used in to limit the scope of the invention.
Embodiment 1
The preparation of zymolyte: take by weighing a certain amount of broken wall Brassica campestris L pollen, adding pH value with material-water ratio 1: 10 is in 9 the buffer system, mixing, and 50 ℃ of attemperation are that 1500U/g adding Sumizyme MP is hydrolyzed in enzyme amount and concentration of substrate ratio.Behind the reaction 3h, 90 ℃ of enzyme 10min that go out, centrifugal 15min under 4000rpm, supernatant liquor removes macromolecular cpds such as deproteinize with the ultra-filtration membrane of holding back relative molecular weight＞10000D, film permeation parts (being the micromolecular compound part) is removed monose, amino acid, ion component etc. with ultrafiltration or the nanofiltration membrane of holding back relative molecular weight＜400D, and it is dry to get the surplus liquid cooling freeze-drying of filter.After testing, Brassica campestris L pollen is behind hydrolysis by novo and membrane sepn, and the content of free state nitrogen is brought up to more than 60% by 3% in the propollen aqueous extract, and the pollen protein major part is degraded to micromolecular polypeptide, is dissolved in the enzymolysis solution.
The preparation of zymolyte: take by weighing a certain amount of broken wall Brassica campestris L pollen, adding pH value with material-water ratio 1: 10 is in 6.5 the buffer system, mixing, and 50 ℃ of attemperation are that 10000U/g adding papoid is hydrolyzed by the enzyme-to-substrate concentration ratio.Behind the reaction 3h, 90 ℃ of enzyme 10min that go out, centrifugal 15min under 4000rpm, supernatant liquor removes macromolecular cpds such as deproteinize with the ultra-filtration membrane of holding back relative molecular weight＞10000D, film permeation parts (being the micromolecular compound part) is removed monose, amino acid, ion component etc. with ultrafiltration or the nanofiltration membrane of holding back relative molecular weight＜400D, and it is dry to get the surplus liquid cooling freeze-drying of filter.
Embodiment 3
The preparation of zymolyte: take by weighing a certain amount of broken wall Brassica campestris L pollen, adding pH value with material-water ratio 1: 10 is in 7 the buffer system, mixing, and 40 ℃ of attemperation are hydrolyzed by enzyme-to-substrate concentration ratio 800U/g adding neutral protease.Behind the reaction 3h, 90 ℃ of enzyme 10min that go out, centrifugal 15min under 4000rpm, supernatant liquor removes macromolecular cpds such as deproteinize with the ultra-filtration membrane of holding back relative molecular weight＞10000D, film permeation parts (being the micromolecular compound part) is removed monose, amino acid, ion component etc. with ultrafiltration or the nanofiltration membrane of holding back relative molecular weight＜400D, and it is dry to get the surplus liquid cooling freeze-drying of filter.
Embodiment 4 pollen zymolytes and pollen aqueous extract are removed the comparison of hydroxy radical qiao
Take by weighing a certain amount of broken wall Brassica campestris L pollen, with material-water ratio 1: 10, mixing, 50 ℃ of attemperation, behind the reaction 3h, centrifugal 15min under 4000rpm, the supernatant liquor lyophilize obtains the pollen aqueous extract.The pollen zymolyte is pressed embodiment 1 preparation, carries out the clearance test of external hydroxy radical qiao.The PBS 0.4ml of experimental procedure: 50mmol/lPH 7.4,0.1ml certain density sample A (contrast replaces with the solvent of molten sample), 1.04mmol/L EDTA 0.1ml, 1mmol/l FeSO4 0.1ml, 1mmol/l Vc 0.1ml, 10mmol/L H2020.1ml, 60mmol/l ribodesose 0.05ml (the A sample does not add, and replaces with distilled water).Constant volume is to 1.0ml.37 ℃ of heating in water bath 60min add the TCA-TBA-HCl mixed solution of 2.0ml then.100 boiling water heating 15min, the absorbancy at mensuration 532nm place.
Inhibiting rate=[A contrast-(A mensuration-A sample)]/A contrast
A contrast-replace sample with solvent
A mensuration-adding DR and sample
A sample-replace DR with distilled water adds sample
Brassica campestris L pollen zymolyte and aqueous extract are removed the hydroxy radical qiao test-results and are shown, both all have the ability of removing hydroxy radical qiao, and dose-effect relationship is arranged, i.e. the effect of removing hydroxy radical qiao along with the increase of sample concentration is strengthened gradually.When 25mg/mL concentration, zymolyte reaches 83.1% to the inhibiting rate of hydroxy radical qiao, and aqueous extract only is 46.9% to the inhibiting rate of hydroxy radical qiao.The EC that compares the two
50(i.e. 50% effective concentration of removing free radical) as can be known, zymolyte EC
50Be 3.35mg/mL aqueous extract EC
50Be 24.23mg/mL, by enzymolysis, the ability that pollen extract is removed hydroxy radical qiao is more than 7 times of aqueous extract, this explanation, and the enzymolysis Brassica campestris L pollen can significantly improve its ability of removing hydroxy radical qiao.
The pollen zymolyte of employing embodiment 1 preparation and the pollen aqueous extract of embodiment 4 preparations carry out the clearance test of external ultra-oxygen anion free radical, adopt Nanjing to build up the anti-ultra-oxygen anion free radical testing cassete of bio-engineering research institute, XOD reaction system generation ultra-oxygen anion free radical in the simulation human body.According to testing cassete explanation carrying out application of sample, fully mixing places 37 ℃ of water bath with thermostatic control 40min, adds the 2.0mL developer respectively, leaves standstill 10min behind the mixing, with the distilled water zeroing, measures the absorbancy at 550nm place.
The anti-ultra-oxygen anion free radical test-results of Brassica campestris L pollen zymolyte and aqueous extract shows, both all have the ability of removing ultra-oxygen anion free radical, and there is dose-effect relationship, the removing effect increases with the increase of sample concentration, the trend of removing ultra-oxygen anion free radical is identical with the trend of removing hydroxy radical qiao, the aqueous extract inhibiting rate is lower, and inhibiting rate has only 40.56% when 30mg/mL, and zymolyte has reached 73.51%.The resistance of oxidation that compares the two, the EC of zymolyte
50EC for the 9.92mg/mL aqueous extract
50Be 53.27mg/mL, the ability that zymolyte is removed ultra-oxygen anion free radical is more than 5 times of aqueous extract.This shows, can significantly improve the ability of its anti-ultra-oxygen anion free radical behind the bee pollen enzymolysis.
The comparison of embodiment 6 pollen zymolytes and the oxidation of pollen aqueous extract lipotropism matter
Adopt the pollen zymolyte of embodiment 1 preparation and the pollen aqueous extract of embodiment 2 preparations to carry out the test of external anti peroxidation of lipid, liposome dispersion system: 300mg is dissolved in 30ml 10mmol/L pH 7.4PBS, the ice bath concussion.Tricholroacetic Acid-thiobarbituricacid-hydrochloric acid mixed solution: 15g TCA, 0.375gTBA, the 2.1ml concentrated hydrochloric acid is put into 100ml water successively.Determination step: (the A sample does not add with adding 0.2ml Yelkin TTS in the sample hose successively, replace with damping fluid), certain density sample 1.0ml (A contrast with the solvent replacement of molten sample), 50ulFeSO4 (347.5mg ferrous sulfate, the 35mg xitix, constant volume is to 25ml) add PBS damping fluid constant volume to 3ml, 37 ℃ of heating in water bath 40min, every 5min shakes up once.The TCA-TBA-HCl mixed solution that adds 2.0ml then.100 boiling water heating 15min, the centrifugal 10min of 5000rpm makes blank with distilled water, measures the absorbancy at 532nm place.
Inhibiting rate=[A contrast-(A mensuration-A sample)]/A contrast
A contrast-replace sample with solvent
A mensuration-adding Yelkin TTS and sample
A sample-replace Yelkin TTS with PBS adds sample
Brassica campestris L pollen zymolyte and aqueous extract suppress the lipid peroxidation test-results and show that both all have the ability of very strong inhibition lipid peroxidation, and trend is basic identical, and inhibiting rate increases with the increase of sample concentration, presents dose-effect relationship.When 30mg/mL concentration, zymolyte and aqueous extract have all reached 90% to the inhibiting rate of lipid peroxidation.The EC of the two
50Be respectively 1.71mg/mL and 3.31mg/mL, it is very capable that zymolyte suppresses lipid peroxidation, is about 2 times of aqueous extract, illustrate that ability of its inhibition lipid peroxidation has strengthened behind the pollen enzymolysis.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (3)
1, a kind of oxidation resistance zymolyte that is rich in pollen active peptide, it is characterized in that, with the Brassica campestris L pollen is raw material, with broken wall of melissa pollen, after the degreasing, add 8～25 times of amount deionized waters, adjusting the pH value is 6～10, be heated to 30～60 ℃, add proteolytic enzyme and carry out enzyme digestion reaction, described proteolytic enzyme is neutral protease, papoid, trypsinase or Sumizyme MP, temperature are controlled at 30～50 ℃, insulated and stirred 30～180min, carry out enzymolysis, make the insoluble protein in the pollen be converted into soluble peptide, heat to 90 ℃ then rapidly, insulated and stirred 10～30min enzyme that goes out, enzymolysis reaction, reduce to room temperature then rapidly, centrifugal 15min carries out solid-liquid separation with the speed of 4000rpm, and supernatant liquor is crossed the ultra-filtration membrane of holding back relative molecular weight＞10000D, get filtered solution and separate, get the surplus liquid drying of filter with the film of holding back relative molecular weight＜400D.
2, zymolyte as claimed in claim 1 is characterized in that, the described film of holding back relative molecular weight＜400D is ultra-filtration membrane or nanofiltration membrane.
3, zymolyte as claimed in claim 1 is characterized in that, described drying is lyophilize.
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| CN101978864B (en) * | 2010-09-28 | 2013-04-10 | 中国农业科学院蜜蜂研究所 | Rape bee-pollen extract and use thereof |
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| CN102697038B (en) * | 2012-05-29 | 2013-10-16 | 宁夏农林科学院 | Medlar bee pollen tablet |
| CN102940653A (en) * | 2012-11-30 | 2013-02-27 | 扬州大学 | Bee pollen water extract buccal tablet for preventing and treating alcoholic fatty liver |
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| CN104593461A (en) * | 2014-12-29 | 2015-05-06 | 唯美度科技（北京）有限公司 | Low-molecular-weight bee-pollen protein peptide and preparation method thereof |
| CN104862364B (en) * | 2015-04-28 | 2018-06-05 | 辽宁天增祥生物科技有限公司 | Bee pollen form cole Oligopeptide Compositions and preparation method thereof |
| CN105767820A (en) * | 2016-03-03 | 2016-07-20 | 扬州大学 | Bee pollen polypeptide drink and preparation method thereof |
| CN106947797A (en) * | 2017-03-05 | 2017-07-14 | 赵德润 | A kind of rape pollen active peptide and preparation method thereof |
| CN107802595A (en) * | 2017-12-24 | 2018-03-16 | 姚佑灿 | A kind of moisturizing anti-aging elite |
| CN111494294B (en) * | 2020-06-02 | 2021-06-15 | 中国海洋大学 | Bee pollen mask and preparation method thereof |
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| CN1165637A (en) * | 1996-05-20 | 1997-11-26 | 平阳县威华食品饮料有限公司 | Pollen beverage and its prodn. tech. |
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Patent Citations (4)
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|---|---|---|---|---|
| CN1165637A (en) * | 1996-05-20 | 1997-11-26 | 平阳县威华食品饮料有限公司 | Pollen beverage and its prodn. tech. |
| CN1137270C (en) * | 2000-03-01 | 2004-02-04 | 刘树军 | Preparation method for producing small molecular weight titanium by using soybean separated protein and its equipment |
| CN1392155A (en) * | 2002-07-23 | 2003-01-22 | 江南大学 | Desalting and concentrating of oligopeptide solution by nano filtering technology |
| CN1274361C (en) * | 2004-07-16 | 2006-09-13 | 吉林大学 | Soybean peptide, its preparation and application |
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