CN105211496B - A method of removing peanut meal protein peptides pigment in the way of freeze concentration - Google Patents
A method of removing peanut meal protein peptides pigment in the way of freeze concentration Download PDFInfo
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Abstract
The method that the invention discloses a kind of to remove peanut meal protein peptides pigment in the way of freeze concentration crosses colloid mill and obtains peanut meal slurries method includes the following steps: peanut meal is mixed with water；Protease and cellulase controlled enzymatic hydrolysis is added, centrifuge separation collects supernatant and obtains peanut meal enzymolysis product；Product is concentrated, then stands freezing, peanut meal enzymolysis product natural layering under cryogenic, surface uvea is removed, the defrosting of portion's albumin layer, then the ultrafiltration membrane for being 10000Da by membrane flux are removed, permeate is collected, is spray-dried, obtains the shallower high-quality peanut meal protein peptides of color.The present invention can efficiently remove pigment in peanut protein peptide product using freezing and ultrafiltration synergy is stood, and while efficiently separating purification peanut meal polypeptide, also inexpensively obtain the peanut coat pigment of concentration.
Description
Technical field
The present invention relates to a kind of polypeptide methods of purification, and in particular to a kind of that peanut cake protein is removed in the way of freeze concentration
The method of peptide pigment.
Background technique
Polypeptide refers generally to interconnect the long-chain substance constituted by multiple amino acid peptide bonds, is the centre of protein hydrolysis
Product not only has excellent mechanism of absorption, while also having protein or the unexistent physiological function of amino acid, such as anti-oxidant
With the characteristics such as antihypertensive.
Discovered in recent years, the great theory of nearly all life science such as immune defense, reproduction control, neoplastic lesion, resist
Anti- wait for a long time of declining is directed to related active peptide.In general polypeptide is in a kind of equilibrium state in human body, if after active peptide is reduced,
Disadvantageous changes may occur for the function of human body, it is therefore desirable to supplement the intake of peptide.The preparation method of polypeptide mainly mentions at present
It follows the example of, synthetic method and Hydrolyze method.Direct extraction method is suitable for qualitative research means of the laboratory when carrying out theoretical research more.It closes
It include chemical synthesis, DNA recombination and enzymatic synthesis at method, the method product purity is higher but at high cost, side reaction is more, it is difficult to real
Now it is mass produced.Hydrolyze method is divided into chemical hydrolysis and biological enzyme hydrolysis method, and biologic enzymolysis method is mild because having working condition,
Hydrolytic process is controllable, and the high and safe advantage of peptide yield has gradually replaced traditional chemical method, becomes and prepares the common of polypeptide
Method.
On diet, protein is primarily present in lean meat, eggs, beans and fish.And albumen powder is as important nutrition
Its global marketing volume of replenishers has broken through 18,000,000,000 dollars of high pointes in 2010, occupies an important position in diet nutritional agent.
In China, protein belongs to substance in short supply, and main protein raw material still relies on import, raw if fish meal about 70% needs import
Produce dregs of beans soybean about 70% need import, constitute the basic unit of protein --- 50% or more synthesizing amino acid need into
Mouthful.
Compared to other nutrients such as carbohydrate, grease, not only demand is huge but also source is relatively fewer for protein, extracts
Technique is relative complex, therefore its production cost is generally higher.For source, protein can be divided into animal protein and plant egg
White, animal protein is mainly derived from the meat, egg, milk of fowl, poultry, fish and insect etc.；Vegetable protein is mainly derived from beans, cereal
In class plant.Generally, vegetable protein and animal protein are primarily present amino acid composition and quantitative difference, vegetable protein are general
Store-through results in the unbalanced nutrition of vegetable protein in limiting amino acid.But vegetable protein has from a wealth of sources, extraction
The advantages such as method is simple, can make up the disadvantage of vegetable protein, therefore reasonable development by artificially adding limiting amino acid
Vegetable protein can be effectively relieved protein supply falls short of demand situation, reduce protein production cost.
For example, common lactalbumin and casein belong to animal protein on the market, 0.7% He is only accounted in milk
2.8%, it is the precious albumen that separation and Extraction comes out from milk；Vegetable protein is mainly soybean protein, peanut protein, corn egg
White and mucedin etc., from a wealth of sources and protein content can be up to 30~50%, mainly extract by the way that alkali soluble acid is heavy, technique phase
To being simple and efficient.
Peanut is one of most important four big oil crops in the whole world, and the peanut of China 50-60% is for extracting oil, institute after oil expression
Obtaining peanut meal is the nutrient base material rich in protein, and protein content is up to 40-50%, and amino acid composition is more reasonable, approaches
The recommendation of FAO (Food and Agriculture Organization of the United Nation) (FAO) and the World Health Organization (WHO).China is used for the shelled peanut of oil expression substantially not at present
Clothing is removed, and contains more pigment in peanut coat, so the peanut meal after oil expression is in yellowish-brown, is caused raw using such peanut meal
The polypeptide that output is come generally is in dark brown, utilizes and develops to limit it.
Summary of the invention
In order to overcome existing peanut camellia meal protein polypeptide color generally partially deep defect, the purpose of the present invention is to provide one
The method that kind removes peanut meal protein peptides pigment in the way of freeze concentration, this method use the joint of protease and cellulase
Effect release peanut meal polypeptide, efficiently prepares that color is shallower, peanut protein of high-quality in conjunction with freezing and ultrafiltration means
Peptide product.
The purpose of the invention is achieved by the following technical solution:
A method of removing peanut meal protein peptides pigment in the way of freeze concentration, comprising the following steps:
(1) preparation of peanut meal enzymolysis product: taking 1 part of peanut meal and 5-10 parts of deionized waters to be stirred by weight, and
It crosses colloid mill and obtains peanut meal slurries；Then protease and cellulase is added, is digested under conditions of 50~60 DEG C, when
When degree of hydrolysis reaches 20-25%, enzyme deactivation, centrifuge separation collects supernatant and obtains peanut meal enzymolysis product；
(2) removing of protein peptides pigment: vacuum concentration peanut meal enzymolysis product to solid content is 30-50%, low
Freezing is stood under the conditions of temperature, peanut meal enzymolysis product natural layering removes surface uvea, removes the defrosting of portion's albumin layer, then lead to
The ultrafiltration membrane that membrane flux is 10000Da is crossed, permeate is collected, is spray-dried, obtains the shallower high-quality peanut meal of color
Albumen peptide product；
Step (1) peanut meal is that high temperature squeezing of the fat content no more than 10% and/or solvent mention the band clothing after oil
Peanut cake/the dregs of rice；
Protease described in step (1) is one kind below:
A, be neutral proteinase, one in papain, compound protease, Alcalase 2.4L or alkali protease
Kind or more, enzymatic hydrolysis system initial pH value is 6.0~8.0；
It B, is pepsin, enzymatic hydrolysis system initial pH value is 2.0~4.0；
C, the protease is fermented by aspergillus oryzae and is generated；
Protease total addition level is the 0.5~1.5% of peanut meal quality；
The cellulase is food-grade cellulose element enzyme or composite plant hydrolase, and additive amount is peanut meal quality
1.0-2.0%；
Low temperature described in step (2) is -18 DEG C or less；The freezing is freezing 24 hours or more；
Step (2) the ultrafiltration flow is no more than 70mL/min, and inlet pressure is no more than 10psi, and outlet pressure is no more than
7psi；
Centrifugation of the present invention is centrifuged using room temperature, and generally 5000-8000r/min keeps 15-30min.
The present invention has the following advantages and effects with respect to the prior art:
(1) present invention is by modern biotechnology zymolysis technique degradation peanut cake protein, by hydrolase specific choice and water
Controlled-enzymatic Hydrolysis is realized in the control of solution, improves raw material availability, reduces production cost, utmostly discharges the polypeptide in peanut meal
Component.
(2) present invention can efficiently be removed in peanut protein peptide product using freezing and ultrafiltration synergy is stood
Pigment is also cheap to obtain the peanut coat pigment being concentrated while efficiently separating purification peanut meal polypeptide.
(3) present invention process is easy to operate, production cost is low, high without any pollution, protein recovery, and gained albumen is more
Peptide matters can be widely applied in field of food.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Related test index measuring method in embodiment is as follows:
The measurement of crude protein:
Kjeldahl nitrogen determination/GB5009.5-85.
The measurement of free ammonia nitrogen content:
0.5g is taken using distilled water as blank control using the free ammonia nitrogen content of formaldehyde potentiometric determination sample
Sample is added distilled water to 80g, is titrated to pH 8.2 with the NaOH of 0.1mol/L, 10mL formaldehyde is added, continues to be titrated to pH
9.2, it obtains blank control group and the NaOH volume V of formaldehyde post consumption is added in sample0And Vi.The free ammonia nitrogen content of product are as follows:
Free ammonia nitrogen content=(Vi-V0)×0.7
The measurement of peptide content:
2ml enzymolysis liquid (protein content about 20mg/ml) is taken, the TCA solution of isometric 10% is added, it is quiet after mixing
30min is set, is then centrifuged 10min under the conditions of 4000r/min, supernatant is taken to measure total nitrogen content and free ammonia-nitrogen content respectively.
Then peptide content are as follows:
Peptide content=(the molten protein content-free amino acid of acid)/total protein content × 100% (GB/T22492-2008)
The measurement of general flavone:
The protein sample solution of 1mg/ml is prepared with the NaOH solution that concentration is 0.1mol/L, sufficiently oscillation mixes,
10000g is centrifuged 15min, measures absorbance (total polyphenols) at 328nm.Polyphenol content is single with " g rutin equivalent/100 samples "
Position, by converting by the standard curve of standard specimen of rutin.
The measurement of ORAC:
ORAC reaction carries out in 75mmol/L phosphate buffer (pH=7.4) environment, FL (fluorescein sodium salt), AAPH
(free-radical generating agent), standard antioxidant Trolox and sample to be tested use the buffer solution and dilution.It is specific to survey
Fixed operation are as follows: add buffer solution 20 μ l and FL (70nmol/L) after being separately added into 20 μ l of sample to be tested in each micropore of 96 orifice plates
20 μ L at 37 DEG C after preset 15min, are added AAPH (12.8mmol/L) 140 μ L rapidly with multichannel pipettor in each hole and open
Dynamic reaction, and microwell plate is placed in fluorescence analyser at 37 DEG C and is connected with excitation wavelength 485nm, launch wavelength 528nm
Continuous measurement, every 2min measure primary each hole fluorescence intensity, measure 108min, and fluorescence intensity is denoted as f respectively0, f1, f2…f54.It will note
The absolute fluorescence intensity data f of each micropore different time points of recordiWith initial fluorescent intensity f0It compares, it is strong to be converted to relative fluorescence
Degree, and fluorescence quenching area under the curve (AUC sample) value is counted according to the following formula.
The measurement of value of chromatism:
2mL enzymolysis liquid is quantitatively pipetted for measuring L*, a*, b* value does blank control with water.Measurement is averaged three times.
Relevant parameter is calculated as follows:
Total color difference △ E=(△ L*2+△a*2+△b*2)1/2
Saturation degree C*=(a*2+b*2)1/2
The three primary colors values of standard white plate used: Hongyuan psychrometric colour specification (X0)=75.11；Green primary color values (Y0)=
79.19；Blue primary values (Z0)=85.02)
The measurement of amino acid:
It is analyzed using high performance liquid chromatography (HPLC).Testing conditions are as follows: U.S. Waters HPLC, PICO, TAG amino acid
Analytical column, temperature are 38 DEG C, flow velocity 1mL/min, Detection wavelength 254nm, and sample is hydrolyzed for 24 hours (110 DEG C) with the hydrochloric acid of 6M.
The analysis of tryptophan is then that will be analyzed again with HPLC after sample alkaline hydrolysis.
Embodiment 1
A method of removing peanut meal protein peptides pigment in the way of freeze concentration, comprising the following steps:
(1) preparation of peanut meal enzymolysis product: taking 1 part of peanut meal and 5 parts of deionized waters to be stirred by weight, and crosses glue
Body grinds to obtain peanut meal slurries；PH value in slurries is adjusted to 6.5, the neutral protein of peanut meal quality 0.5% is then added
Enzyme and 1.0% cellulase, digested under conditions of 50 DEG C, when degree of hydrolysis reaches 20%, enzyme deactivation, centrifuge separation,
It collects clear liquid and obtains peanut meal enzymolysis product；
(2) removing of peanut protein peptide pigment: vacuum concentration peanut meal enzymolysis product is to solid content 30%, -18
Freezing 24 hours or more are stood at DEG C, surface uvea is removed after freezing, remove the defrosting of portion's albumin layer, then be by membrane flux
The ultrafiltration membrane of 10000Da collects permeate, is spray-dried, obtains peanut protein peptide.
Embodiment 2
A method of removing peanut meal protein peptides pigment in the way of freeze concentration, comprising the following steps:
(1) preparation of peanut meal enzymolysis product: 1 part of peanut meal and 10 parts of deionized waters is taken to be stirred by weight, and mistake
Colloid mill obtains peanut meal slurries；PH value in slurries is adjusted to 8.0, the alkaline egg of peanut meal quality 1.5% is then added
White enzyme and 2.0% cellulase, digested under conditions of 60 DEG C, when degree of hydrolysis reaches 25%, enzyme deactivation, centrifugation point
From collection clear liquid obtains peanut meal enzymolysis product；
(2) removing of peanut protein peptide pigment: vacuum concentration peanut meal enzymolysis product is to solid content 50%, -36
Freezing 24 hours or more are stood at DEG C, surface uvea is removed after freezing, remove the defrosting of portion's albumin layer, then be by membrane flux
The ultrafiltration membrane of 10000Da collects permeate, is spray-dried, obtains peanut protein peptide.
Embodiment 3
A method of removing peanut meal protein peptides pigment in the way of freeze concentration, comprising the following steps:
(1) preparation of peanut meal enzymolysis product: taking 1 part of peanut meal and 8 parts of deionized waters to be stirred by weight, and crosses glue
Body grinds to obtain peanut meal slurries；PH in slurries is adjusted to 3.0, then be added peanut meal quality 1.0% pepsin and
1.5% composite plant hydrolase is digested under conditions of 55 DEG C, when degree of hydrolysis reaches 22%, enzyme deactivation, and centrifugation point
From collection clear liquid obtains peanut meal enzymolysis product；
(2) removing of peanut protein peptide pigment: vacuum concentration peanut meal enzymolysis product solid content is to 40%, -18
Freezing 24 hours or more are stood at DEG C, surface uvea is removed after freezing, remove the defrosting of portion's albumin layer, then be by membrane flux
The ultrafiltration membrane of 10000Da collects permeate, is spray-dried, obtains peanut protein peptide.
Comparative example 1
A kind of preparation of peanut protein peptide, comprising the following steps:
It takes 1 part of peanut meal and 8 parts of deionized waters to be stirred by weight, and crosses colloid mill and obtain peanut meal slurries；It will slurry
PH in liquid is adjusted to 3.0, and the acid protease of peanut meal quality 1.0% and 1.5% composite plant hydrolase is then added,
It is digested under conditions of 55 DEG C, when degree of hydrolysis reaches 22%, enzyme deactivation, centrifuge separation collects clear liquid and is concentrated in vacuo solid
Then object content is spray-dried to 40%, obtains peanut protein peptide.
The basic index of 1 embodiment and comparative example product of table
The color of 2 embodiment and comparative example product of table changes
The total amino acid composition single amino acids of 3 embodiment and comparative example product of table account for total amino acid ratio %
By table 1 it can be found that enzymatic hydrolysis gained albumen peptide content it is higher, sample protein content (butt) reach 88% with
On, wherein small-molecular peptides content reaches nearly 50%.Sample (embodiment 1,2,3) is decolourized compared with no bleaching sample (comparative example 1),
Free ammonia-nitrogen content and peptide content are slightly decreased, and Flavonoid substances content sharply declines and ORAC value also significantly reduces.This
Illustrate that the freeze concentration that this patent uses is handled for the pigment removal significant effect in protein peptides, but has substantially no effect on protein peptides
Nutriment.Since the pigment composition that peanut meal raw material is brought into is mainly flavone compound (from peanut coat), freezing
This kind of flavone compound can follow ice crystal generation to migrate in the process, surface gradually be concentrated on, using general physics hand
These coloring matters are removed if section (such as excision), to achieve the purpose that efficiently to remove protein peptides pigment.In addition, will removing
Pigment collect, can be used for Western-style ham, cake, in sausage product, can not only play the role of coloring, this even more can be improved
The antioxidant activity of class product.
A kind of balancing method of color is CIE Lab chrominance space, which can reflect human eye to the true of color
Feel.In Lab difference space, L* value is bigger to illustrate that object color is brighter；A* value shows as red if positive value, and negative value is green
Color；B* value positive value is yellow, and negative value is blue；Total color difference Δ E (Total color difference) is indicated and object of reference water
The value of chromatism compared, value is smaller to show that color is more shallow；C* value is saturation degree (Chroma), and the bigger expression full color of value is strong
It is strong, it is worth the sparse dimness of smaller expression color.
By table 2, it can be found that freeze concentration is decolourized, preceding peanut protein peptide is mainly at bronzing, compared with clear；And it decolourizes
Later, peanut protein peptide is then mainly at light yellow, brightness height, solution clear.Illustrate that this patent imitates the decoloration of object
Fruit highly significant.
By table 3 it can be found that peanut meal is hydrolyzed using the method for this patent and decolorization peanut meal enzymatic hydrolysis produce
The amino acid of object forms the influence of not conspicuousness, the delicate flavour ammonia before and after decolorization in peanut meal enzymolysis product amino acid composition
Base acid (Asp+Glu) is up to 32% or more, it is necessary to which amino acid content (Lys+Trp+Thr+Ile+Leu+Phe+Met+Val) is also all
30% or so, nutriment of this this patent during separating peanut protein peptide pigment in peanut protein peptide is demonstrated again that
Lose less, the application field of resulting light color peanut protein peptide is further expanded, first is that a kind of good polypeptide nutrition
Replenishers product.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (6)
1. a kind of method for removing peanut meal protein peptides pigment in the way of freeze concentration, it is characterised in that the following steps are included:
(1) preparation of peanut meal enzymolysis product: taking 1 part of peanut meal and 5-10 parts of deionized waters to be stirred by weight, and crosses glue
Body grinds to obtain peanut meal slurries；Then protease and cellulase is added, is digested under conditions of 50~60 DEG C, works as hydrolysis
When degree reaches 20-25%, enzyme deactivation, centrifuge separation collects supernatant and obtains peanut meal enzymolysis product；
(2) removing of protein peptides pigment: vacuum concentration peanut meal enzymolysis product to solid content is 30-50%, in cryogenic conditions
Lower standing freezing, peanut meal enzymolysis product natural layering remove surface uvea, remove the defrosting of portion's albumin layer, then logical by film
Amount is the ultrafiltration membrane of 10000 Da, collects permeate, is spray-dried, obtains the shallower high-quality peanut cake protein of color
Peptide.
2. the method according to claim 1 for being removed peanut meal protein peptides pigment in the way of freeze concentration, feature are existed
In: protease described in step (1) is one kind below:
A, one or more of for neutral proteinase, papain or alkali protease, enzymatic hydrolysis system initial pH value is 6.0~
8.0；
It B, is pepsin, enzymatic hydrolysis system initial pH value is 2.0~4.0；
C, the protease is fermented by aspergillus oryzae and is generated；
Protease total addition level is the 0.5~1.5% of peanut meal quality.
3. the method according to claim 1 for being removed peanut meal protein peptides pigment in the way of freeze concentration, feature are existed
In: cellulase described in step (1) is composite plant hydrolase, and additive amount is the 1.0-2.0% of peanut meal quality.
4. the method according to claim 1 for being removed peanut meal protein peptides pigment in the way of freeze concentration, feature are existed
In: low temperature described in step (2) is -18 DEG C or less.
5. the method according to claim 1 for being removed peanut meal protein peptides pigment in the way of freeze concentration, feature are existed
In: freezing described in step (2) is freezing 24 hours or more.
6. the method according to claim 1 for being removed peanut meal protein peptides pigment in the way of freeze concentration, feature are existed
The band wrapped peanut dregs of rice after: step (1) peanut meal is high temperature squeezing of the fat content no more than 10% and/or solvent mentions oil.
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| CN102250998A (en) * | 2011-05-27 | 2011-11-23 | 山东省花生研究所 | Preparation method for peanut bioactive peptide |
| CN102363799A (en) * | 2011-09-30 | 2012-02-29 | 华南理工大学 | Preparation method for aromatic-amino-acid-enriched antioxidant |
| CN103392902A (en) * | 2013-07-11 | 2013-11-20 | 华南理工大学 | Method for preparing strong antioxidative peptide by using peanut meal |
| CN103865972A (en) * | 2014-03-20 | 2014-06-18 | 中国农业科学院农产品加工研究所 | Peanut peptide preparation method for enzymatically decomposing thermal-crushed peanut meal step by step with double neutral protease |
| CN104342475A (en) * | 2014-10-16 | 2015-02-11 | 青岛博之源生物技术有限公司 | Method for preparing glucosaccharase-inhibiting active peptide from hot-extraction peanut pulp |
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| JP4220739B2 (en) * | 2002-07-31 | 2009-02-04 | 小林香料株式会社 | Separation method of zein |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250998A (en) * | 2011-05-27 | 2011-11-23 | 山东省花生研究所 | Preparation method for peanut bioactive peptide |
| CN102363799A (en) * | 2011-09-30 | 2012-02-29 | 华南理工大学 | Preparation method for aromatic-amino-acid-enriched antioxidant |
| CN103392902A (en) * | 2013-07-11 | 2013-11-20 | 华南理工大学 | Method for preparing strong antioxidative peptide by using peanut meal |
| CN103865972A (en) * | 2014-03-20 | 2014-06-18 | 中国农业科学院农产品加工研究所 | Peanut peptide preparation method for enzymatically decomposing thermal-crushed peanut meal step by step with double neutral protease |
| CN104342475A (en) * | 2014-10-16 | 2015-02-11 | 青岛博之源生物技术有限公司 | Method for preparing glucosaccharase-inhibiting active peptide from hot-extraction peanut pulp |
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