CN104862364A - Oilseed rape bee pollen oligopeptide composite and preparation method thereof - Google Patents
Oilseed rape bee pollen oligopeptide composite and preparation method thereof Download PDFInfo
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- CN104862364A CN104862364A CN201510208254.7A CN201510208254A CN104862364A CN 104862364 A CN104862364 A CN 104862364A CN 201510208254 A CN201510208254 A CN 201510208254A CN 104862364 A CN104862364 A CN 104862364A
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Abstract
The invention relates to a method for extracting the oligopeptide composite from oilseed rape bee pollen and provides the oligopeptide composite that is obtained by extraction by the method. According to the oilseed rape bee pollen oligopeptide composite, the bioavailability is high, and the composite is quite safe in terms of immunity. The invention further provides foods, health care products or cosmetics composites that contain the oilseed rape bee pollen oligopeptide composite.
Description
Technical field
The present invention relates to biological technical field.Concrete, the invention provides and a kind ofly from Brassica campestris L pollen, extract the method that obtains Oligopeptide Compositions and extracted the composition obtained by the method.The invention still further relates to the application of composition in healthcare products and makeup containing oligopeptides of extracting from Brassica campestris L pollen and obtaining.
Background technology
Rape, another name rape flower or rape, Latin literary fame Brassica rapa L, country of origin is with in Europe and the Central Asia one, phytology belongs to annual herb plant, Cruciferae.Rape is now comparatively wide in Chinese cultivated, is the rural economy crop of establishing in large scale.Current rape Main Cultivation (kind) type comprises turnip type rape (Brassica rapa (campestris) L.), mustard type rape (Brassica juncea L.), swede type rape (Brassica napus L.).
Bee pollen (bee pollen) refers to and adds the pollen that honeybee gathers in flowering nectariferous plant flower pesticide nectar and saliva through honeybee to its inside, be mixed into irregular oblateness, above with the embedding dough of holding vestige under the arm of honeybee hind leg.Bee pollen is usually containing 200 multiple nutritional components, wherein containing 20 multiple amino acids, more than 100 kind of enzyme and coenzyme, 10 multivitamins, more than 30 kind of major element and trace element, the nucleic acid of about 2% and multiple organic acid, unsaturated fatty acids, flavonoid compound and hormone etc.Thus, bee pollen is described as " concentrated nutrition library " " concentrated miniature natural medicament " and " nutritional complete food products " etc. by people.Modern medicine proves, bee pollen has following effect: (1) regulates functions of intestines and stomach: pollen has many sterilization components, can kill intestinal bacteria etc.; (2) human body comprehensive immunologic function is improved; (3) brain cardiovascular disease is prevented and treated: the flavonoid compound in pollen effectively can remove the deposition of fat on vessel wall, thus plays vessel softening and reducing blood-fat; (4) anti-aging, beautification function: belong to the cosmetics of trophicity, the compositions such as the VE in pollen, superoxide dismutase (SOD), selenium can moisten nutrition skin, the elasticity of recovery skin and bright and clean; (5) nervous system regulation, promotes sleep etc.
Oligopeptides refers to the polypeptide that amino acid number is less, and such as amino acid is less than the polypeptide of 10, particularly containing 1,2 or 3 amino acid whose polypeptide.In human body, oligopeptides higher efficiency can be rapidly absorbed into the recycle system and is utilized by tissue.
In Brassica campestris L pollen, protein content is quite abundant, and account for 7% ~ 35% of its dry weight, wherein water-soluble protein is less than 30%, and protein exists with macromole state, and bioavailability is low, and easily causes the anaphylaxis of part population.At nutrition and cosmetic industry, carrying out the more highly purified effective constituent material of extraction process acquisition to Brassica campestris L pollen is conventional means.The extracting method reported is mechanical means, microwave method, fermentation process and enzyme solution mainly.In the cole melissa powder preparation adopting existing method to obtain, protein also retains almost complete macromole form usually.Complete protein molecular brings unwanted immunomodulatory phenomenon sometimes.
CN102746413B reports a kind of bee pollen polysaccharide leach extraction method, wherein by bee pollen mechanical disintegration, adds cellulase, forms the bee pollen aqueous solution in the pasty state after broken wall, then uses the lixiviate of 400W ultrasonic power.
CN101401821B reports the method mechanically with cellulase and protease treatment bee pollen, to obtain a kind of bee pollen peptide slice.
In " bee pollen peptide separation purifying and the immunocompetence research " of Sun Peilong etc., Food science, 3rd phase in 2008, in Vol.29, the means such as Superose-12 gel chromatography, DEAE Sepharose F.F. ion exchange chromatography that have employed are to process Brassica campestris L pollen, obtain albumen wherein, and find that this albumen has the effect affecting immunizing power.
This area still needs to have in immune angle safer, namely the cole melissa powder product that the molecular weight of contained protein or polypeptide is lower, such as protein wherein or polypeptide be major part for containing length lower than five amino acid, be preferably one or two amino acid whose small-molecular peptides.
Summary of the invention
The invention provides a kind of method obtaining Oligopeptide Compositions from Brassica campestris L pollen extraction, wherein to adopting combined-enzyme method to be hydrolyzed after bee pollen pre-treatment again, obtain the composition containing small molecules oligopeptides, it is characterized in that, the molecular weight of described oligopeptides is mainly distributed in below 500D, is preferably at below 300D.In one aspect of the invention, described composition middle-molecular-weihydroxyethyl be the Gaussian distribution peak area percent of the oligopeptides of below 500D more than 60%, preferably greater than 70%.Of the present invention in another, described Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 300D is more than 50%.Also namely, the Gaussian distribution peak area percent containing 1 or 2 or 3 amino acid whose peptide in described Oligopeptide Compositions is more than 50%.
In the present invention, adopt and first pre-treatment is carried out to Brassica campestris L pollen, then by the method for combined-enzyme method hydrolysis Brassica campestris L pollen.Wherein, carrying out pre-treatment to Brassica campestris L pollen is alkaline purification, processes the regular hour by Brassica campestris L pollen in the solution of about pH9-11, such as more than 2-3 hour.Of the present invention in one of them, carrying out pre-treatment to Brassica campestris L pollen is that Brassica campestris L pollen is made solution, and its pH is adjusted to about 9-11 (such as with NaOH or Na
2cO
3or NaHCO
3or the bases such as its arbitrary combination regulates pH), keep more than 2 hours under this pH, such as about 3-8 hour.Preferably, described alkaline purification, at about 30-60 DEG C, such as, is carried out at about 40-50 DEG C.
In the present invention, be hydrolyzed Brassica campestris L pollen more after the pre-treatment with combined-enzyme method, the enzyme that wherein combined-enzyme method adopts is the combination of more than 3 kinds or 3 kinds lytic enzymes.Sumizyme MP, neutral protease and stomach en-must be comprised in the combination of described lytic enzyme.
Bee pollen has complicated structure and composition, comprises the saliva etc. of nectar and honeybee.There is outside bee pollen hard outer wall, its inside is the inclusion of the complexity containing various nutritive substance and sexual cell, the chemical component of its complexity causes adopting the method for enzymic hydrolysis to run into a lot of technical problem, such as, adopt the combination treatment method of single enzyme or some enzyme in prior art, in any case extend the amount of enzymolysis time and increase enzyme, the ratio containing one or more (2-3) amino acid whose polypeptide in protein ingredient all can not be made to increase.
Pre-treatment Brassica campestris L pollen first being carried out to alkaline purification that the present invention adopts, and then carry out the method that combined-enzyme method hydrolysis obtains Oligopeptide Compositions, prepare the Oligopeptide Compositions that its middle-molecular-weihydroxyethyl is mainly distributed in below 500D, described composition middle-molecular-weihydroxyethyl be the Gaussian distribution peak area percent of the oligopeptides of below 500D more than 70%, and described Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 300D is more than 50%.Do not report in prior art and obtained the composition that above-mentioned molecular weight is mainly distributed in the oligopeptides of below 500D from Brassica campestris L pollen.
Of the present invention in another, when adopting described combined-enzyme method to be hydrolyzed to Brassica campestris L pollen, the order adding enzyme is Sumizyme MP, stomach en-and neutral protease.In the method for the invention, according to the described order adding enzyme, the technique of the inventive method can be improved, reduce the time of a lot of processing step and operation, and economize in raw materials and intermediate materials.
Provided by the inventionly a kind ofly extract from Brassica campestris L pollen the method obtaining Oligopeptide Compositions and comprise the following steps:
A) Brassica campestris L pollen is ground into 40-150 order fine powder;
B) described fine powder is mixed by 1:20-1:40 with deionized water;
C) mixed solution alkali is adjusted to pH9-11,40-50 DEG C, keeps 3-8 hour;
D) pretreated mixed solution is warming up to 50-55 DEG C, adds 500,000 u/g Sumizyme MPs by 0.1 % by weight-5 % by weight of Brassica campestris L pollen dry weight, regulate pH to 8-9, insulation 2-4h;
E) adjust pH to 3-4, add 50,000 u/g stomach en-s, 50-60 DEG C by Brassica campestris L pollen dry weight 1 % by weight-3 % by weight, keep 2-4h;
F) regulate pH to 7-8, add 150,000 u/g neutral proteases, 40-50 DEG C by Brassica campestris L pollen dry weight 1 % by weight-3 % by weight, keep 2-4h;
G) coarse filtration;
H) gac of 1-3 % by weight is added, 50-55 DEG C of decolouring 0.5-1h;
I) clear, pale yellowish liquid is filtered to obtain with diatomite filter;
J) concentrated, carry out spraying dry or lyophilize obtains yellow to brown ceramic powder.
This method one of them in, by above-mentioned steps a)-j) molecular weight of oligopeptides has following Gaussian distribution in the composition that obtains:
In aforesaid method of the present invention, step a) in preferably order number be 50-150 order, step b) in preferred proportion be 1:20-1:30, step c) in preferable temperature be about 90 DEG C; Time is about 10min, steps d) in preferably add enzyme amount be the 1-1.2 % by weight of sub-Brassica campestris L pollen fine powder dry weight; Preferable temperature is about 50 DEG C; Preferred pH8.2-8.5; Hold time as 3-3.2h.Step e) in preferably add enzyme amount be the 1-3 % by weight of Brassica campestris L pollen fine powder dry weight, most preferably be 1 % by weight of sporophore dry weight; Preferably ph 3.6, preferable temperature is 55 DEG C, and the time is 3-3.2h.Step f) in preferably add enzyme amount be the 1-1.2 % by weight of Brassica campestris L pollen fine powder dry weight; Preferable temperature is about 50 DEG C; Preferred pH7.3; Hold time as 3-3.2h.Step g) middle preferable temperature 95 DEG C, time 15min.The amount of the use enzyme adopted in the optimal technical scheme in method of the present invention and hydrolysis time more effectively can obtain the oligopeptides extract needed, namely molecular weight is mainly distributed in the oligopeptides of below 500D, in one aspect of the invention, in described composition the Gaussian distribution peak area percent of the oligopeptides of 300D more than 50%.
The invention provides and extract by aforesaid method the composition obtained from Brassica campestris L pollen, wherein to adopting combined-enzyme method to be hydrolyzed after bee pollen pre-treatment again, obtaining the composition containing oligopeptides, it is characterized in that, the molecular weight of described oligopeptides is mainly distributed in below 500D, is preferably at below 300D.In one aspect of the invention, described composition middle-molecular-weihydroxyethyl be the Gaussian distribution peak area percent of the oligopeptides of below 500D more than 60%, preferably greater than 70%.Of the present invention in another, described Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 300D is more than 50%.Also namely, the Gaussian distribution peak area percent containing 1 or 2 or 3 amino acid whose peptide in described Oligopeptide Compositions is more than 50%.
Of the present invention in another, the molecular weight of the oligopeptides of described composition small molecular has following Gaussian distribution:
Provided by the invention extraction the composition obtained from Brassica campestris L pollen also contains the nutritive ingredients such as carbohydrate, VITAMIN and mineral substance.Method of the present invention in extract except oligopeptides, also can retain other active substance of above Brassica campestris L pollen effectively.
Brassica campestris L pollen provided by the invention can be widely used in food, healthcare products, cosmetic field.
The invention provides and extract from Brassica campestris L pollen the Oligopeptide Compositions obtained as elucidated before and preparing the application food, healthcare products, makeup.
In one aspect of the invention, provide a kind of make-up composition, it extracts containing, for example foregoing the Oligopeptide Compositions obtained from Brassica campestris L pollen.
In one aspect of the invention, provide a kind of Halth-care composition, it extracts containing, for example foregoing the Oligopeptide Compositions obtained from Brassica campestris L pollen.
In one aspect of the invention, provide a kind of food compositions, it extracts containing, for example foregoing the Oligopeptide Compositions obtained from Brassica campestris L pollen.
The formulation of food provided by the invention, healthcare products, make-up composition comprises for liquid or powder, i.e. oral liquid, capsule, tablet, ball etc.
The present invention can be used for food, healthcare products, makeup additive and can suitability for industrialized production be carried out.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, but should not limit the scope of the invention with this.
Embodiment 1
1. the preparation of Brassica campestris L pollen powder
Brassica campestris L pollen crusher is ground into 100 order fine powders.
2. the pre-treatment of Brassica campestris L pollen
Brassica campestris L pollen fine powder is mixed by 1:20 with deionized water, regulates mixed solution to pH9-11 with NaOH, at 45 DEG C, keep 6-8 hour.
3. the hydrolysis of Brassica campestris L pollen
Be warming up to 50-55 DEG C after insulation terminates, add 500,000 u/g Sumizyme MPs of 1 % by weight of Brassica campestris L pollen fine powder dry weight, regulate pH to 8.5 with 10%NaOH solution, 50 DEG C of insulation 3h.After insulation terminates, add 50,000 u/g stomach en-s of 1 % by weight of Brassica campestris L pollen fine powder dry weight, then adjust pH to 3.5 with 10%HCl solution, 50 DEG C keep 3h.After insulation terminates, add 150,000 u/g neutral proteases of 1 % by weight of Brassica campestris L pollen fine powder dry weight, regulate pH to 7.3 with 10%NaOH solution, 50 DEG C of insulation 3h.After insulation terminates, be warming up to 95 DEG C, keep 20min, inactivation treatment is carried out to enzyme.
After deactivation, with coarse sand cloth filtering reacting liquid.Then in filtrate, add the powdered active carbon of liquid 3%, 50 DEG C are stirred decolouring 0.5h, then filter with diatomite filter, obtain clear, pale yellowish liquid.Filter with 0.25-0.5 μm of nuclepore membrane filter, then carry out ultrafiltration by the daltonian film of throttling molecular weight 10000, collect permeate.
Concentrated, lyophilize obtains yellow to brown ceramic powder.
4. the measurement of pair Brassica campestris L pollen extract oligopeptides
Detect the JY/T 024-1996 of the molecular weight distribution of small-molecular peptides according to high efficient liquid phase analysis method conventional in the industry, the peptide molecular weight distribution of QB/T2879-2007 standard to extract measures, and obtains its Gaussian distribution result as follows:
Embodiment 2
1. the preparation of Brassica campestris L pollen powder
Brassica campestris L pollen crusher is ground into 100 order fine powders.
2. the hydrolysis of Brassica campestris L pollen
Brassica campestris L pollen fine powder is mixed by 1:20 with deionized water, is warming up to 50-55 DEG C, add 500,000 u/g Sumizyme MPs of 1 % by weight of Brassica campestris L pollen fine powder dry weight, regulate pH to 8.5 with 10%NaOH solution, 50 DEG C of insulation 6h.After insulation terminates, add 50,000 u/g stomach en-s of 1 % by weight of Brassica campestris L pollen fine powder dry weight, then adjust pH to 3.5 with 10%HCl solution, 50 DEG C keep 3h.After insulation terminates, add 150,000 u/g neutral proteases of 1 % by weight of Brassica campestris L pollen fine powder dry weight, regulate pH to 7.3 with 10%NaOH solution, 50 DEG C of insulation 3h.After insulation terminates, be warming up to 95 DEG C, keep 20min, inactivation treatment is carried out to enzyme.
After deactivation, with coarse sand cloth filtering reacting liquid.Then in filtrate, add the powdered active carbon of liquid 3%, 50 DEG C are stirred decolouring 0.5h, then filter with diatomite filter, obtain clear, pale yellowish liquid.Filter with 0.25-0.5 μm of nuclepore membrane filter, then carry out ultrafiltration by the daltonian film of throttling molecular weight 10000, collect permeate.
Concentrated, lyophilize obtains yellow to brown ceramic powder.
3. the measurement of pair Brassica campestris L pollen extract oligopeptides
By the powder prepared, detect the JY/T 024-1996 of the molecular weight distribution of small-molecular peptides according to high efficient liquid phase analysis method conventional in the industry, the peptide molecular weight distribution of QB/T2879-2007 standard to extract measures, and obtains its Gaussian distribution result as follows:
Rich in protein in Brassica campestris L pollen, and protein exists with macromole state, bioavailability is low, and easily causes the anaphylaxis of part population.The present invention carries out pre-treatment by alkaline process, and by the method for compound bio-enzyme mild hydrolysis, obtain Brassica campestris L pollen Oligopeptide Compositions, macro-molecular protein is made to be degraded to the higher oligopeptides of bioavailability, do not destroy the original activity of bee pollen, safer in immune angle, retain other nutritive ingredient as polysaccharide, mineral substance, VITAMIN etc. simultaneously, which in turn improve smell and the mouthfeel of product, add its applicability.
Unless otherwise noted, practice of the present invention will use the routine techniques of biotechnology and organic chemistry, inorganic chemistry etc., obviously except describing especially in above-mentioned explanation and embodiment, other mode can also realize the present invention.Other technical scheme within the scope of the present invention will be apparent to those skilled in the art in the invention with improving.According to instruction of the present invention, many changes and change are feasible, therefore all within the scope of the present invention.
Claims (10)
1. one kind is extracted the method obtaining Oligopeptide Compositions from Brassica campestris L pollen, wherein pre-treatment is carried out to Brassica campestris L pollen, then obtain Oligopeptide Compositions with combined-enzyme method hydrolysis, in described Oligopeptide Compositions, the molecular weight of oligopeptides is mainly distributed in below 500D, is preferably at below 300D.
2. the method for claim 1, wherein said Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 500D is more than 60%, preferably greater than 70%, preferred, described Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 300D is more than 50%.
3. the method for claim 1 or 2, wherein said pre-treatment is processed in the solution of about pH9-11 by Brassica campestris L pollen.
4. the method for claim 1 or 2, is characterized in that, wherein said combined-enzyme method is hydrolyzed the enzyme adopted and comprises Sumizyme MP, stomach en-and neutral protease, and preferably, the order adding enzyme is Sumizyme MP, stomach en-and neutral protease.
5. the method for claim 1, said method comprising the steps of:
A) Brassica campestris L pollen is ground into 40-150 order fine powder;
B) described fine powder is mixed by 1:20-1:40 with deionized water;
C) mixed solution alkali is adjusted to pH9-11,40-50 DEG C, keeps 3-8 hour;
D) pretreated mixed solution is warming up to 50-55 DEG C, adds 500,000 u/g Sumizyme MPs by 0.1 % by weight-5 % by weight of Brassica campestris L pollen dry weight, regulate pH to 8-9, insulation 2-4h;
E) adjust pH to 3-4, add 50,000 u/g stomach en-s, 50-60 DEG C by Brassica campestris L pollen dry weight 1 % by weight-3 % by weight, keep 2-4h;
F) regulate pH to 7-8, add 150,000 u/g neutral proteases, 40-50 DEG C by Brassica campestris L pollen dry weight 1 % by weight-3 % by weight, keep 2-4h;
G) coarse filtration;
H) gac of 1-3 % by weight is added, 50-55 DEG C of decolouring 0.5-1h;
I) clear, pale yellowish liquid is filtered to obtain with diatomite filter;
J) concentrated, carry out spraying dry or lyophilize obtains yellow to brown ceramic powder,
In the composition obtained, the molecular weight of oligopeptides has following Gaussian distribution:
6. the Brassica campestris L pollen Oligopeptide Compositions prepared by the method for claim 1, it is characterized in that, be hydrolyzed containing Brassica campestris L pollen the oligopeptides obtained in described composition, in described Oligopeptide Compositions, the molecular weight of peptide is mainly distributed in below 500D, is preferably at below 300D.
7. the Oligopeptide Compositions of claim 6, its middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 500D is more than 60%, preferably greater than 70%, preferred, described Oligopeptide Compositions middle-molecular-weihydroxyethyl is that the Gaussian distribution peak area percent of the oligopeptides of below 300D is more than 50%.
8. the Oligopeptide Compositions of claim 7, wherein the molecular weight of oligopeptides has following Gaussian distribution:
9. a Halth-care composition, it contains the Brassica campestris L pollen Oligopeptide Compositions any one of claim 6-8.
10. a make-up composition, it contains the Brassica campestris L pollen Oligopeptide Compositions any one of claim 6-8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106072575A (en) * | 2016-07-07 | 2016-11-09 | 无锡博优特生物科技有限公司 | A kind of nourishing tonic for sport and containing its sport nutrition |
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| CN101058600A (en) * | 2007-04-28 | 2007-10-24 | 中国农业科学院蜜蜂研究所 | Oxidation resistance zymolyte rich in pollen active peptide |
| CN103039709A (en) * | 2013-01-11 | 2013-04-17 | 江西旺大动物科技有限公司 | Preparation method of wall-breaking and enzymolysis bee pollen |
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| CN101058600A (en) * | 2007-04-28 | 2007-10-24 | 中国农业科学院蜜蜂研究所 | Oxidation resistance zymolyte rich in pollen active peptide |
| CN103039709A (en) * | 2013-01-11 | 2013-04-17 | 江西旺大动物科技有限公司 | Preparation method of wall-breaking and enzymolysis bee pollen |
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| CN106072575A (en) * | 2016-07-07 | 2016-11-09 | 无锡博优特生物科技有限公司 | A kind of nourishing tonic for sport and containing its sport nutrition |
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