CN102533918A - Method for preparing rapeseed active polypeptides - Google Patents
Method for preparing rapeseed active polypeptides Download PDFInfo
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- CN102533918A CN102533918A CN2012100156538A CN201210015653A CN102533918A CN 102533918 A CN102533918 A CN 102533918A CN 2012100156538 A CN2012100156538 A CN 2012100156538A CN 201210015653 A CN201210015653 A CN 201210015653A CN 102533918 A CN102533918 A CN 102533918A
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Abstract
The invention provides a method for preparing rapeseed active polypeptides. The method comprises the following steps of: grinding dehusked, defatted and dried rapeseeds, leaching with water, centrifuging, precipitating the resulting supernatant with acids, dialyzing in water, hydrolyzing the resulting rapeseed albumin with two enzymes, decolorizing, and removing foreign matters, to obtain rapeseed active polypeptides. The method has the advantages of simple production process, mild reaction condition and no strict requirement for production equipment, and is suitable for industrial production.
Description
Technical field
The invention belongs to food processing technology field, relate to a kind of preparation method and application of vegetable seed active polypeptide.
Background technology
Rape is one of important oil crops, and China's yield of rape ranks first in the world.Generally speaking, Semen Brassicae campestris can about 3506 rapeseed oils and 65% left and right sides rapeseed meal through processing.Be rich in crude protein, crude fat, Yelkin TTS, multivitamin, amino acid etc. in the rapeseed meal, digestible protein content can reach 27.8% in its protein.In addition, also contain various trace elements such as vitamin B group such as higher choline, vitamin H, folic acid, nicotinic acid and VitB1, vitamin G and Ca, P, Fe, Se, Cu, Zn, Mn in the rapeseed meal.The crude protein content of rapeseed meal is fine full price vegetable-protein generally at 35%-45%, it is developed and utilized have important economic implications and social effect.But China is quite limited to the utilization of rapeseed meal at present, only adds in the feed Ingredient of ruminating animal and freshwater fish as fertilizer or in a small amount of ratio, and this has caused certain wasting of resources.
Protein is the main agent and the basic substance of all vital movements.All vital movements are all relevant with protein, so the research of protein involved all is the focus of the research of biological technical field all the time.And in recent years, increasing research shows that peptide has more outstanding character than protein.Modern biological metabolism is discovered: the oligopeptide specific ionization amino acid by a plurality of amino acid be combined intos is digested and assimilated more easily, wherein is absorbed as preferably the fastest with dipeptides, tripeptides especially.Amino acid than material protein and same composition; The enzymolysis product polypeptide can show different physiological roles such as physical function characteristic such as good retention ability, gelation, surfactivity and anti-oxidant, reducing blood-fat, adjusting immunizing power, and these are material proteins with to form total free aminoacids not available.Therefore, rapeseed meal is used for processing high value added product, has significant social meaning and economic implications.
Proteinic method for hydrolysis generally comprises soda acid edman degradation Edman and enzymolysis process.The acid-base method hydrolyzed animal protein is to utilize protein different solubility under different pH, under extreme conditions makes earlier most protein dissolvings, removes nonsoluble, transfers pH to the recovery of isoelectric point of protein deposition again.Characteristics such as chemical degradation method is simple to operate with it, cost is low, protein yield height become the main means of early protein hydrolysis; But along with the research to proteolysis is goed deep into; Gradually find that there are many defectives in the chemical method protein hydrolysate; As: (1) too extremely violent reaction conditions brings very big pollution to environment, has also increased the weight of the cost that subsequent environments is administered simultaneously; (2) adjusting of soda acid can cause the hydrolysate complicated component, and salinity is too high, and use range is very limited, and peptide series products now is mostly as food, healthcare products, makeup etc.; (3) fierce reaction process can make amino acid whose configuration change D-amino acid into by L-amino acid, and the amino acid whose functional L-amino acid that can not show a candle to of D-; (4) acid-base method is difficult to control reaction process, can not realize the location hydrolysis of title product.Take all factors into consideration, seldom adopted the chemical method protein hydrolysate in recent years, the substitute is zymyhydrolyzed protein matter.
Adopt biological process hydrolysing foodstuff albumen to have more outstanding advantage than chemical acid alkali facture, as proteolytic enzyme can be under the condition of gentleness the hydrolysing foodstuff proteinoid, can not cause big environmental pollution; Proteolytic enzyme protolysate matter can not cause the variation of amino acid configuration yet, better preserve the original effect of amino acid; Protease hydrolysis can realize locating hydrolysis, is easy to realize the control of hydrolysis process, thereby makes the applicability of peptide class and functional change wide, satisfies the demand in market.But enzymolysis protein matter is not a simple technological process, and it relates to a lot of steps.The first step is exactly the suitable enzyme of screening; Because enzyme not only influences the speed of reaction, the yield of product; But also directly have influence on local flavor, physicochemical property and the physiologically active of product; Select according to not only relevantly, also much relations are arranged with substrate and processing condition with the specificity of the action site of enzyme self.
On the whole; Rape seed protein amino acid is formed rationally; Near the amino acid compositional model of Food and Argriculture OrganizationFAO (WHO) and the World Health Organization (FAO) recommendation, but the high temperature milling process can cause protein denaturation, in addition; From rapeseed meal, separate the vegetable seed crude protein that obtains and more or less have some ANFs, make its development and use receive certain limitation.Therefore, how effectively to utilize rape seed protein to become the problem of needing solution badly.
Summary of the invention
In view of the deficiency of prior art, order of the present invention is to provide a kind of preparation method of vegetable seed active polypeptide, thereby further realizes improving the added value of rapeseed meal.
In order to realize the object of the invention, the contriver has finally obtained following technical scheme through a large amount of experimental studies:
A kind of preparation method of vegetable seed active polypeptide comprises the steps:
(1) gets shelling degreaser drying rapeseed meal, pulverized 60 mesh sieves and be placed in the extractor, add 8-12 and doubly measure zero(ppm) water; 40 ± 2 ℃ of following stirring and leaching 0.8-1.5h, 5500-6500rpm, 4 ℃ of centrifugal 20min, filtering supernatant obtains deposition; Carry out identical lixiviate operation 2-3 time again by above-mentioned steps, merge the supernatant of gained, with 1mol/L salt Acid precipitation supernatant; Transfer pH to 4.0 back 5500-6500rpm, 4 ℃ of centrifugal 10min to obtain deposition; Deposition is placed the zero(ppm) water 16-48h that dialyses, and whenever changes zero(ppm) water respectively at a distance from 8h, then after the vacuum lyophilization the vegetable seed white protein;
(2) vegetable seed white protein adding distil water is mixed with the protein solution that massfraction is 5-8%, by the Flavourzyme proteolytic enzyme of every gram vegetable seed white protein interpolation 4000U, 55 ± 2 ℃ of holding temperatures, pH7.0; Hydrolysis 2.5-3h presses the Alcalase proteolytic enzyme that every gram vegetable seed white protein adds 2800U, 52 ± 2 ℃ of holding temperatures, pH8.0 again; Hydrolysis 3.5-4h, hydrolysis is rapidly heated to 95 ℃ after finishing, and keeps 10～15min; After hydrolyzed solution is cooled to normal temperature; The centrifugal 15min of 12000rpm gets supernatant, gets rough polypeptide liquid;
(3) in rough polypeptide liquid, press 2.5% of vegetable seed white protein quality and add gac, at 55 ~ 60 ℃ of following stirring reaction 60min, the centrifugal 15min of 15000rpm gets supernatant, gets rough decolouring polypeptide liquid;
(4) with rough decolouring polypeptide liquid earlier after filter paper filtering, the 100nm micro-filtrate membrane filtration, the 500Da nf membrane is filtered, nanofiltration liquid is-45 ℃ of vacuum lyophilizations, the vegetable seed active polypeptide.
Discover through animal experiment, utilize the vegetable seed active polypeptide of method for preparing to have significant effect in the learning and memory ability to that improves the mouse aging model.Therefore, second purpose of the present invention is to provide a kind of new use of product, the application of vegetable seed active polypeptide in healthcare products, food or the medicine of preparation memory that promptly utilizes aforesaid method to obtain.
The vegetable seed active polypeptide of the present invention's preparation is that rape seed protein is through the proteolytic enzyme effect; The protein hydrolystate that after special processing, obtains again; The little peptide mixt of low molecule by many molecular chain length do not wait is formed, and also contains compositions such as small-amount free amino acid, carbohydrate and inorganic salt.
The preparation method of the vegetable seed active polypeptide that the present invention relates to has following advantage and obvious improvement: (1) screening has obtained ideal compound protease and consumption thereof; Obtained the active polypeptide of better flavor; Realized comprehensive utilization, further improved the added value of rapeseed meal waste material.(2) production technique is simple, and reaction conditions is gentle, to production unit require lowly, be fit to industrialized production.(3) the vegetable seed active polypeptide that utilizes this method to prepare can significantly improve the elderly's learning and memory ability.(4) utilize the vegetable seed active polypeptide of this method preparation not only to have good solubility in acid, low vigorous degree, anti-gel formation property; Digest and assimilate fast in vivo; Characteristics such as protein utilization rate height, and have low antigenicity, can not produce anaphylaxis; Also have good physicochemical property and physiologically active, can be used as the additive of sports nutrition agent, diet food, old people food.
Embodiment
Below be the concrete preparation example that the present invention prepares the vegetable seed active polypeptide, technical scheme of the present invention is done further the description, but protection scope of the present invention be not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
The preparation of embodiment 1 vegetable seed active polypeptide
(1) gets shelling degreaser drying rapeseed meal (crude protein content 38.5%), pulverized 60 mesh sieves and be placed in the extractor, add 10 times of amount zero(ppm) water; 40 ± 2 ℃ of following stirring and leaching 1h, 6000rpm, 4 ℃ of centrifugal 20min, filtering supernatant obtains deposition; Carry out identical lixiviate operation 3 times again by above-mentioned steps, merge the supernatant of gained, with 1mol/L salt Acid precipitation supernatant; Transfer pH to 4.0 back 6000rpm, 4 ℃ of centrifugal 10min to obtain deposition; Deposition is placed the zero(ppm) water 24h that dialyses, and whenever changes zero(ppm) water respectively at a distance from 8h, then after the vacuum lyophilization the vegetable seed white protein;
(2) vegetable seed white protein adding distil water being mixed with massfraction is 6% protein solution, adds the Flavourzyme proteolytic enzyme of 4000U, 55 ± 2 ℃ of holding temperatures, pH7.0 by every gram vegetable seed white protein; Hydrolysis 2.5h, the 2800U Alcalase proteolytic enzyme that adds by every gram vegetable seed white protein again, 52 ± 2 ℃ of holding temperatures, pH8.0; Hydrolysis 3.5h, hydrolysis is rapidly heated to 95 ℃ after finishing, and keeps 10min; After hydrolyzed solution is cooled to normal temperature; The centrifugal 15min of 12000rpm gets supernatant, gets rough polypeptide liquid;
(3) in rough polypeptide liquid, press 2.5% of vegetable seed white protein quality and add gac, at 58 ℃ of following stirring reaction 60min, the centrifugal 15min of 15000rpm gets supernatant, gets rough decolouring polypeptide liquid;
(4) with rough decolouring polypeptide liquid earlier after filter paper filtering, the 100nm micro-filtrate membrane filtration, the 500Da nf membrane is filtered, nanofiltration liquid must not have the vegetable seed active polypeptide of bitter taste, raciness-45 ℃ of vacuum lyophilizations.
Embodiment 2 vegetable seed active polypeptide promote the mouse memory experimental study
Six week healthy mices in age (body weight 22g-25g, male and female half and half), test mice is divided into 3 groups at random, 15 every group, i.e. young control, aging model group, bioactive peptide treatment group.Wherein, 0.8% D-galactose solution 0.5ml/20g (200mg/kg) of the nape portion subcutaneous injection saline water dilution of aging model group, bioactive peptide treatment group mouse, 1 time/d; Continuous 6 weeks; Cause aging model, young control subcutaneous injection equivalent saline water, the vegetable seed active polypeptide of bioactive peptide treatment group embodiment of the invention of 9 fillings stomach in morning every day 1 preparation in modeling; Dosage is the 0.8g/kg body weight; Active polypeptide dilutes with saline water, and in continuous 6 weeks, young control and aging model group are irritated stomach and given equal-volume saline water.
After each is organized mouse and handled for 6 weeks, carry out water maze test, the labyrinth is the double-deck opaque synthetic glass case of 80cm * 50cm * 20cm, in establish starting point, goal platform and four cecums, be the safety zone near the platform.Depth of water 12cm in labyrinth during test, water temperature keeps (25 ± 1) ℃, and all space indicates remain unchanged in entire test around the labyrinth.Official test previous day helps every mouse in the labyrinth, to be familiar with water surrounding 15min, like mouse no show platform in 60s, can help formula ground that mouse is rushed to platform, and make mouse on platform, stop 20s.Beginning official test in the 2nd day.Mouse is put into water towards wall in starting point, and the record mouse is put into the number of times (errors number) that water is reached home the platform time (omnidistance time of trip) and got into cecum, and statistics is seen table 1.
Can find out that according to test-results model group and youth group is compared, the omnidistance time of water maze test trip obviously prolong (
P＜0.01), errors number significantly increase (
P＜0.01); Though and bioactive peptide treatment group mouse does not reach the level of young group, than these two achievements of model group all be significantly improved (
P＜0.05), the vegetable seed active polypeptide of this explanation the present invention preparation can improve the learning and memory ability of mouse aging.
Table 1 vegetable seed active polypeptide is to the influence of mouse water maze test achievement (n=15, X ± s)
| Group | Sample size | The omnidistance time (s) of trip | Errors number |
| Young group | 15 | 60.43±17.61 | 5.48±2.05 |
| Model group | 15 | 118.57±26.10 ** | 19.58±3.64 ** |
| The bioactive peptide group | 15 | 70.64±20.39 # | 7.86±4.28 ## |
Organize comparison with the youth, *
P＜0.05, * *
P＜0.01;
Compare # with model group
P＜0.05, ##
P＜0.01.
Claims (2)
1. the preparation method of a vegetable seed active polypeptide comprises the steps:
(1) gets shelling degreaser drying rapeseed meal, pulverized 60 mesh sieves and be placed in the extractor, add 8-12 and doubly measure zero(ppm) water; 40 ± 2 ℃ of following stirring and leaching 0.8-1.5h, 5500-6500rpm, 4 ℃ of centrifugal 20min, filtering supernatant obtains deposition; Carry out identical lixiviate operation 2-3 time again by above-mentioned steps, merge the supernatant of gained, with 1mol/L salt Acid precipitation supernatant; Transfer pH to 4.0 back 5500-6500rpm, 4 ℃ of centrifugal 10min to obtain deposition; Deposition is placed the zero(ppm) water 16-48h that dialyses, and whenever changes zero(ppm) water respectively at a distance from 8h, then after the vacuum lyophilization the vegetable seed white protein;
(2) vegetable seed white protein adding distil water is mixed with the protein solution that massfraction is 5-8%, by the Flavourzyme proteolytic enzyme of every gram vegetable seed white protein interpolation 4000U, 55 ± 2 ℃ of holding temperatures, pH7.0; Hydrolysis 2.5-3h presses the Alcalase proteolytic enzyme that every gram vegetable seed white protein adds 2800U, 52 ± 2 ℃ of holding temperatures, pH8.0 again; Hydrolysis 3.5-4h, hydrolysis is rapidly heated to 95 ℃ after finishing, and keeps 10～15min; After hydrolyzed solution is cooled to normal temperature; The centrifugal 15min of 12000rpm gets supernatant, gets rough polypeptide liquid;
(3) in rough polypeptide liquid, press 2.5% of vegetable seed white protein quality and add gac, at 55 ~ 60 ℃ of following stirring reaction 60min, the centrifugal 15min of 15000rpm gets supernatant, gets rough decolouring polypeptide liquid;
(4) with rough decolouring polypeptide liquid earlier after filter paper filtering, the 100nm micro-filtrate membrane filtration, the 500Da nf membrane is filtered, nanofiltration liquid is-45 ℃ of vacuum lyophilizations, the vegetable seed active polypeptide.
2. the application of vegetable seed active polypeptide in healthcare products, food or the medicine of preparation memory of method preparation according to claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103740796A (en) * | 2014-01-14 | 2014-04-23 | 天津替代医学科技有限公司 | Method for extracting polypeptides from skimmed protein milk supernatant |
| CN105907826A (en) * | 2016-06-15 | 2016-08-31 | 山东博奥克生物科技有限公司 | Clean preparation method for plant polypeptide/protein |
| CN107259559A (en) * | 2017-05-02 | 2017-10-20 | 江苏大学 | Rapeseed peptides and fruit of Chinese magnoliavine water extract and the method for cooperateing with improvement to sleep |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740796A (en) * | 2014-01-14 | 2014-04-23 | 天津替代医学科技有限公司 | Method for extracting polypeptides from skimmed protein milk supernatant |
| CN103740796B (en) * | 2014-01-14 | 2015-07-22 | 天津替代医学科技有限公司 | Method for extracting polypeptides from skimmed protein milk supernatant |
| CN105907826A (en) * | 2016-06-15 | 2016-08-31 | 山东博奥克生物科技有限公司 | Clean preparation method for plant polypeptide/protein |
| CN105907826B (en) * | 2016-06-15 | 2020-01-10 | 山东博奥克生物科技有限公司 | Clean preparation method of plant polypeptide/protein |
| CN107259559A (en) * | 2017-05-02 | 2017-10-20 | 江苏大学 | Rapeseed peptides and fruit of Chinese magnoliavine water extract and the method for cooperateing with improvement to sleep |
| CN107259559B (en) * | 2017-05-02 | 2020-12-18 | 江苏大学 | Rapeseed polypeptide and schisandra chinensis aqueous extract and method for synergistically improving sleep |
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