CN103461643A - Method for extracting bran polypeptide and bran estrogen through wet ball milling in combination with enzymic method - Google Patents
Method for extracting bran polypeptide and bran estrogen through wet ball milling in combination with enzymic method Download PDFInfo
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- CN103461643A CN103461643A CN2013104011814A CN201310401181A CN103461643A CN 103461643 A CN103461643 A CN 103461643A CN 2013104011814 A CN2013104011814 A CN 2013104011814A CN 201310401181 A CN201310401181 A CN 201310401181A CN 103461643 A CN103461643 A CN 103461643A
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Abstract
The invention discloses a method for extracting bran polypeptide and bran estrogen through wet ball milling in combination with an enzymic method. The method comprises the following steps of: (1) ball milling and grinding: taking bran, adding water which is 4-6 times of the material in weight and adding 20 U/g of cellulase and 10 U/g of amylase, and performing wet grinding on the wheat bran for 1-3 hours by using a ball mill under the condition that a rotating speed is in the range from 250 to 400 rpm to obtain an ultrafine powder suspension; (2) extraction of polypeptide; and (3) extraction of estrogen. The method for extracting the bran polypeptide and the bran estrogen through wet ball milling in combination with the enzymic method provided by the invention has the advantages that the raw material is pretreated through wet ball milling in combination with the enzymic method so that compact crystalline structure in the wheat bran particle can be broken to promote dissolving-out of active ingredients such as protein, starch and phytoestrogen, and the conversion rate of the polypeptide in bran polypeptide enzymolysis and the dissolving-out quantity of the estrogen in alcohol extraction process can be effectively increased.
Description
Technical field
The invention belongs to food processing technology field, relate to a kind of wet ball grinding associating Enzymatic Extraction wheat bran polypeptide and the estrogenic method of wheat bran.
Background technology
Research in recent years is found, in the enzymolysis product of food protein, exist some to there is the small peptide than high bioactivity, as antithrombotic peptide, blood pressure lowering peptide, anti-oxidation peptide, immunity promote peptide etc., the exploitation that the research of these biologically active peptides is new functionalized food provides new approach, becomes one of noticeable research direction in current Food Science field.Protein digestion is the process of a complexity, dezymotizes outside the characteristic of itself, also is subject to the impact of the many factors such as concentration of substrate, pH value, temperature, and the change of any one condition all may produce different products, affects the conversion ratio of protein.At present, the domestic research for biologically active peptide concentrates on separation, purifying and functional study, but polypeptide yield is all lower, and there is the serious waste problem in material protein.
Phytoestrogen refers to that plant contains is similar to animal estrogen (estradiol, progesterone etc.) bioactivator, this class material shows estrogenic antagonist in the high estrogen environment, can reduce and the relevant disease that cause too high with estrogen such as breast cancer, fibroid, play estrogen agonist in the low estrogen environment, can prevent and treat as diseases relevant with decrease in estrogen such as osteoporosis, climacteric metancholias.Be rich in estrogen in wheat bran, have the effects such as anticancer, anti-cancer, the estrogen in wheat bran mainly concentrates in the wheat bran cell, and bioavailability is extremely low.
Be rich in phytoestrogen and albumen in wheat bran, and be utilized mainly as feed and fertilizer at present, added value is extremely low.The present invention selects wheat bran to extract wheat bran albumen and phytoestrogen as raw material, by wet ball grinding, combines each wheat bran biologically active polypeptide of enzymolysis system and estrogenic mixed solution, and the method mild condition, pollution-free, efficiency is high.
Summary of the invention
The purpose of this invention is to provide a kind of wet ball grinding associating Enzymatic Extraction wheat bran polypeptide and the estrogenic method of wheat bran, its method mild condition, pollution-free, efficiency is high.
To achieve these goals, the present invention proposes following technical scheme:
A kind of method of wet ball grinding associating Enzymatic Extraction wheat bran polypeptide comprises the following steps:
(1) ball mill grinding: get a certain amount of wheat bran, with the wheat bran weighing scale, add weight of material 4-6 water doubly, and add cellulase 20U/g, amylase 10U/g, adopting ball mill is under the 250-400rpm condition, wheat bran to be carried out to wet pulverizing 1-3h to obtain the superfine powder suspension at rotating speed;
(2) extraction of polypeptide: suspension is separated with ball mill, and to add water to solid-liquid ratio be 1:9, adjust pH to 9.5, with the wheat bran weighing scale, add alkali protease 50U/g, enzymolysis 1h under 55 ℃ of conditions, enzymolysis liquid centrifugal 20min under the 4000rpm condition obtains polypeptide solution.
Preferably, in described step (1), a certain amount of wheat bran is placed in to the agate tank.
Preferably, the ball mill in described step (1) is planetary ball mill.
Preferably, in described step (2), utilize sodium hydroxide solution to adjust pH to 9.5.
The present invention also protects the polypeptide solution that adopts above-mentioned method to extract.
The present invention provides a kind of wet ball grinding associating Enzymatic Extraction wheat bran estrogenic method in addition, after above-mentioned steps (1) and step (2), further comprising the steps of:
(3) estrogenic extraction: get the filter residue that filter (2), the amount that is 1:2 according to solid-liquid ratio adds the ethanol extract that volume fraction is 30%-60%, 60-80 ℃ of backflow 1h, under the 4000rpm condition, centrifugal 20min obtains the estrogen extract, residue repeats to extract once according to above-mentioned steps, merge the supernatant extracted for 2 times, obtain the estrogen extract.
The present invention further protects the estrogen extract that adopts said method to extract.
The present invention adopts following raw material: cellulase (2000U/g), amylase (2000U/g), three kinds of enzymes of alkali protease (20,000 U/g) are bought in Zhaodong Sun Shine Enzyme Co., Ltd..
Beneficial effect of the present invention is: the invention provides wet ball grinding associating Enzymatic Extraction wheat bran polypeptide and the estrogenic method of wheat bran, raw material adopts wet ball grinding associating enzyme process to carry out pretreatment, can break crystalline texture fine and close in the wheat bran particle, promote the stripping of the active ingredients such as albumen, starch, phytoestrogen, can effectively improve estrogenic stripping quantity in the conversion ratio of polypeptide in the wheat bran polypeptide enzymolysis and alcohol extracting process.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
The specific embodiment
Below by embodiment, technical scheme of the present invention is described further, but can not be interpreted as limitation of the present invention.
Embodiment 1
(1) ball mill grinding: get the wheat bran of 500 grams as in the agate tank, add water 2000mL, and add cellulase 5g, amylase 2 .5g, adopting planetary ball mill (XQM-20L, test apparatus research institute is analysed by Nanjing section) is under the 400rpm condition, wheat bran to be carried out to wet pulverizing 3h to obtain the superfine powder suspension at rotating speed;
(2) extraction of polypeptide: suspension is separated with spherical tank, utilize 2500ml water to rinse spherical tank, merge 2 times suspension, utilize sodium hydroxide solution to adjust pH to 9.5, add alkali protease 1.25g, enzymolysis 1h under 55 ℃ of conditions, enzymolysis liquid centrifugal 20min under the 4000rpm condition obtains polypeptide solution.Adopt biuret method to detect the content of polypeptide in solution, the conversion ratio that calculates wheat bran polypeptide is 87%, and adopting the kit method to detect progesterone content in solution is that 0.63ng/mL, estradiol content are 0.19pg/mL.The step of wheat bran polypeptide separation and purification is: rough polypeptide solution ultra-filtration and separation, molecular cut off is the following polypeptide of 10KDa, through vacuum freeze drying, must obtain wheat bran polypeptide.The active component that polypeptide solution is collected after gel chromatography, ion-exchange chromatography and RT-HPLC is carried out cellulose acetate electrophoresis, and electrophoresis is a single band, illustrates that the active peptides purity of collecting is higher, and its molecular weight of mass spectroscopy is 6523.2Da.
(3) estrogenic extraction: get the filter residue that filter (2), add the ethanol extract 1000mL that volume fraction is 60%, 80 ℃ of backflow 1h, under the 4000rpm condition, centrifugal 20min obtains the estrogen extract, residue repeats to extract once according to above-mentioned steps, merge the supernatant extracted for 2 times, obtain the estrogen extract, adopting the kit method to detect progesterone content in solution is that 3.2ng/mL, estradiol content are 2.5pg/mL.After ultrafiltration in step (2) being removed to the estrogen supernatant merging of solution and extraction, utilize the macroporous resin adsorption post to separate, adopt water-miscible solvent to carry out gradient elution, obtain the concentrate that contains Conjugated Estrogen; By the Conjugated Estrogen concentrate extracted, through anti-phase C18 post enriching and purifying, carry out loading, water-miscible solvent gradient elution, to collect component concentrated, obtains the estrogen mixture of purifying.
Detection method: free amino acid adopts formol titration; Protein adopts Kjeldahls method.Polypeptide adopts biuret method to detect.
Polypeptide conversion ratio computing formula: conversion ratio=(T2-T1)/(P1-P2).
In formula: T2 is content of peptides after enzymolysis; T1 is content of peptides before enzymolysis; P1 is total protein content before enzymolysis; P2 is acid-soluble protein content before enzymolysis.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: in the situation that do not break away from principle of the present invention and aim can be carried out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.
Claims (7)
1. the method for a wet ball grinding associating Enzymatic Extraction wheat bran polypeptide, is characterized in that, comprises the following steps:
(1) ball mill grinding: get a certain amount of wheat bran, with the wheat bran weighing scale, add weight of material 4-6 water doubly, and add cellulase 20U/g, amylase 10U/g, adopting ball mill is under the 250-400rpm condition, wheat bran to be carried out to wet pulverizing 1-3h to obtain the superfine powder suspension at rotating speed;
(2) extraction of polypeptide: suspension is separated with ball mill, and to add water to solid-liquid ratio be 1:9, adjust pH to 9.5, with the wheat bran weighing scale, add alkali protease 50U/g, enzymolysis 1h under 55 ℃ of conditions, enzymolysis liquid centrifugal 20min under the 4000rpm condition obtains polypeptide solution.
2. method according to claim 1, is characterized in that, described step is placed in the agate tank by a certain amount of wheat bran in (1).
3. method according to claim 1, is characterized in that, the ball mill in described step (1) is planetary ball mill.
4. method according to claim 1, is characterized in that, in described step (2), utilizes sodium hydroxide solution to adjust pH to 9.5.
5. the polypeptide solution that the described method of claim 1-4 is extracted.
6. the estrogenic method of wet ball grinding associating Enzymatic Extraction wheat bran, is characterized in that, after the described step of claim 1-4 any one (1) and step (2), further comprising the steps of:
(3) estrogenic extraction: get the filter residue that filter (2), the amount that is 1:2 according to solid-liquid ratio adds the ethanol extract that volume fraction is 30%-60%, 60-80 ℃ of backflow 1h, under the 4000rpm condition, centrifugal 20min obtains the estrogen extract, residue repeats to extract once according to above-mentioned steps, merge the supernatant extracted for 2 times, obtain the estrogen extract.
7. the estrogen extract that method claimed in claim 6 is extracted.
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Application publication date: 20131225 |