CN101904406B - Preparation method and use of sunflower seed polypeptide - Google Patents
Preparation method and use of sunflower seed polypeptide Download PDFInfo
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Abstract
本发明提供了一种葵花籽多肽的制备方法，步骤包括：将葵花籽分离蛋白加水溶解，采用碱性蛋白酶和复合酸性蛋白酶两阶段酶水解葵花籽分离蛋白制备多肽，在水解过程中，实施超声波间断变频辅助酶解处理；酶解液经微滤、超滤后得到葵花籽多肽。本发明针对不同的蛋白酶采用不同的超声波频率和处理时间，有效提高了葵花籽蛋白的水解度和多肽得率，比传统工艺的生产效率明显提高。制得的产品具有清除自由基、消除亚硝酸盐、阻断亚硝胺合成、抑制红细胞氧化、美容护肤和抗肝损伤等多种保健功能，可用于制备功能食品和化妆品。The invention provides a preparation method of sunflower seed polypeptide, the steps include: dissolving the sunflower seed protein isolate with water, using alkaline protease and compound acid protease to hydrolyze the sunflower seed protein isolate to prepare the polypeptide, and during the hydrolysis process, implementing ultrasonic Intermittent frequency conversion assisted enzymatic hydrolysis treatment; the enzymatic hydrolysis solution was subjected to microfiltration and ultrafiltration to obtain sunflower seed polypeptide. The invention adopts different ultrasonic frequencies and treatment times for different proteases, effectively improves the degree of hydrolysis of sunflower protein and the yield of polypeptides, and significantly improves the production efficiency compared with traditional techniques. The prepared product has a variety of health functions such as scavenging free radicals, eliminating nitrite, blocking nitrosamine synthesis, inhibiting red blood cell oxidation, beautifying skin care and resisting liver damage, and can be used to prepare functional food and cosmetics.
Description
Technical field
The present invention relates to the deep processing of sunflower seeds, specifically is a kind of preparation method and purposes of sunflower seed polypeptide.
Background technology
Modern study shows, much with the plant polypeptide of protease hydrolytic have anti-oxidant, remove functions such as free radical or stopping nitrosation be synthetic, can be used as the raw material of food or health products.For example the disclosed zein of patent CN101284872 (200810064296.8) prepares antioxidation active peptides; The anti-oxidant powder of the disclosed walnut polypeptide of patent CN101228918 (200710017281.1).Some micromolecule polypeptide Transdermal absorption is effective, is applied on the skin, not only can resist the oxidation of cosmetics self; And can remove human free radical; Suppress the degraded of human collagen albumen and elastin laminin, reduce the skin senescence process, thereby play crease-resistant, the flexible effect of maintenance skin.For example U.S.A of Japan can many cosmetics the researcher of Co., Ltd. from the oryzenin of rice, extract a kind of novel polypeptide class material, can effectively suppress wrinkle of skin, phenomenon such as lax, can be used as the functional cosmetics base-material.The antioxidation activity of plant polypeptide and security make one of its direction that becomes functional food and cosmetics exploitation from now on.
Sunflower is one of important oil crops of China, and annual sunflower seeds output is about more than 420 ten thousand tons.Sunflower protein is of high nutritive value, and its amino acid is formed, and except that the content of lysine was low, all the other essential amino acids all reached or are higher than the standard that FAO (Food and Agriculture Organization of the United Nation) is recommended.Protein content is 29%～43% in the sunflower seeds, comprising albumin, globulin, glutelin and alcohol soluble protein etc.For a long time, the grouts after the sunflower seeds oil expression are most as fertilizer or feed, do not obtain further processing and utilization.It is less both at home and abroad to produce the research report of active peptides about sunflower protein, and adopts the single enzymolysis mode mostly, and its degree of hydrolysis is lower; The yield of micromolecule polypeptide is also lower; Adopt alkali protease, flavor protease or 1.398 neutral proteinases that sunflower protein is carried out single enzymolysis research like people such as Ren Jian, degree of hydrolysis only reach 8～12.5% (appoint strong, Zheng Xiqun. neutral proteinase is to the research of sunflower separation proteolysis effect. Chinese oil; 2007,8; Liu Huan appoints strong. and alkali protease is to the research of degreasing sunflower meal enzymatic hydrolysis condition. grain and oil processing, 2008,1; Du Guojun appoints strong. and flavor protease is to the research of degreasing sunflower meal enzymatic hydrolysis condition. grain and oil processing, 2010; 2), adopt in addition papain (Bai Yu. enzyme process prepares the research of sunflower seeds peptide. food with machinery, 2007; 10) or 1.398 bacillus subtilis neutral proteinases (Chen Jie. the research of the enzyme modification of sunflower protein concentrate. agricultural mechanical journal; 2008,5) sunflower protein is hydrolyzed, degree of hydrolysis is merely 4～15.6%.In addition Cheong Kuoc Va adopt alkali protease and pepsin two one-step hydrolysis prepare certain herbaceous plants with big flowers dregs of rice polypeptide (research of the anti-skin photoage effect of certain herbaceous plants with big flowers dregs of rice polypeptide. research and development of natural products, 2010,22), its hydrolysis time is long, energy consumption is bigger, is not suitable for suitability for industrialized production.
Present ultrasonic wave ancillary technique has been used in biological enzymolysis and has separated in the protein process, like the disclosed method of carrying out quick aminosal with one or more enzymes in the protease such as trypsase, chymotrypsin and V8 protease of patent CN101457247 (200810207956.3); Before the combined hydrolysis laver albumen of the disclosed employing pepsin of patent CN101003826 (200610097286.5), trypsase, neutral proteinase or papain and these enzymes; Earlier carry out enzymolysis in the laver solution with sneaking into again behind the ultrasonic pretreatment liquid of protease; Obviously shorten hydrolysis time; But the method for the ultrasound-assisted enzymolysis of this continuous use single-frequency tends to cause problems such as enzyme activity decline, protein action site are destroyed.
Summary of the invention
To the deficiency that prior art exists, the object of the present invention is to provide a kind of time short, energy consumption is lower, and enzymolysis efficiency is high, the preparation method of the sunflower seed polypeptide that the micromolecule polypeptide yield is big and the sunflower seed polypeptide purposes in functional food and cosmetics.
The preparation method of a kind of sunflower seed polypeptide provided by the invention comprises the steps:
1), extract albumen: sunflower seeds dissolves the heavy method of acid with alkali and extracts the sunflower seeds protein isolate after pulverizing, degreasing, and is subsequent use after the vacuum drying;
2), hydrolysis by novo: take by weighing certain herbaceous plants with big flowers seed protein isolate, water-soluble by 4%～8% mass percentage concentration, add alkali protease; Enzyme concentration is 1.5%～2.5% of a protein isolate, mixes, and regulates pH to 8.0～9.5 with NaOH; Constant temperature is 50～60 ℃ in supersonic generator, prior to 20～28KHz supersonic frequency hydrolysis 2-3h, hydrolysis 1-3h under the supersonic frequency of 28～40KHz again; In two stage ultrasonic hydrolysis processes, every at a distance from 5-12min sonicated 1-4min;
3), combination of acidic protease hydrolytic: above-mentioned treatment fluid is regulated pH 2.5～4.5 with HCl, adds combination of acidic protease, and enzyme concentration is 4%～6% of a protein isolate; The control temperature is at 35～45 ℃; Hydrolysis 4～6h under 24～40KHz supersonic frequency, during every at a distance from 6-12min sonicated 1-3min, 5～10 minutes enzymes that go out of boiling water bath; Through micro-filtration, milipore filter separation and purification, obtain molecular weight and account for the product more than 90% at the following polypeptide of 3000Da.
Described combination of acidic protease is any two or three in aspergillus oryzae acid protease, Aspergillus usamii acid protease, mould product acid protease, aspergillus niger acid protease and the pepsin.
In sunflower protein hydrolytic process of the present invention, why to adopt the ultrasonic wave of different frequency to carry out discontinuity treatment, one of which is because of the ultrasonic wave long duration of action, may cause the inactivation of enzyme, causes enzymolysis usefulness to reduce; Its two, adopt lower frequency, long period ultrasonic Treatment in the protease hydrolyzed initial reaction stage, mainly be in order to promote interaction and the contact area between the enzyme-to-substrate; And along with the lengthening of hydrolysis time, the hydrophobicity peptide bond of more difficult hydrolysis can appear, and help the fracture of this type peptide bond with the ultrasonic wave of upper frequency this moment, but the processing time is unsuitable long, the processing time shortened than the enzymolysis initial stage at every turn.In second stage-combination of acidic enzyme hydrolysis process, adopt different ultrasonic frequencies, the acid protease that also will take into account various combination has different ultrasonic frequency accommodation, exceeds certain ultrasonic frequency scope, and enzymolysis activity can descend.
The application of sunflower seed polypeptide in preparation functional food and cosmetics:
In vitro test finds that this sunflower seed polypeptide product is to hydroxy radical (OH), ultra-oxygen anion free radical (O
2) and the clearance rate of DPPH free radical reach 55.37%～98.79%, 55.43%～96.87% and 48.7%～73.56% respectively; Clearance rate to nitrite reaches 68.4%～98.5%; The blocking-up rate synthetic to nitrosamine reaches 52.1%～90.75%; Inhibiting rate to oxidative hemolysis of erythrocyte reaches 43.02%～81.4% in addition, compares with glutathione with Vc, and the antioxidation activity of sunflower seed polypeptide is stronger; Can be used as a kind of efficient natural anti-oxidant, be used for the additive of functional food and cosmetics.
The mouse aging of inducing with the D-galactolipin is a model; Irritate the stomach sunflower seed polypeptide; Can significantly reduce mda content in the mice serum (p＜0.01), and can improve the content (p＜0.01) of hydroxyproline in the skin histology, have the effect of delaying decrepitude of skin, recovery skin elasticity; Can add cosmetics to, play the effect of beauty and skin care; With tetrachloro-methane induction mouse chemical damage is model; Irritate the stomach sunflower seed polypeptide; Can significantly reduce MDA content (p＜0.01) in vigor (p＜0.01) and the serum of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease in the mice serum; Certain herbaceous plants with big flowers seed polypeptide has protective effect for chemical damage, can be used as a kind of safe liver health product.
Compared with prior art, the present invention has following advantage and effect:
1. the present invention adopts acid compound protease to carry out the second stage enzymolysis, and this combination of acidic enzyme hydrolysis mode can obtain more little molecule sunflower seed polypeptide.With compare with two stage enzymolysis sunflower proteins of alkali protease and pepsin combination, little peptide proportion content below the 3000Da is high, the preparation enzymolysis efficiency is high, and anti-oxidation function is stronger.
2. it is different with the characteristic of combination of acidic protease to the present invention is directed to alkali protease, adopts the ultrasonic wave of different frequency to carry out discontinuity treatment respectively, has improved enzymolysis efficiency effectively.Do not add the auxiliary enzymolysis process of ultrasonic wave with tradition and compare, the sunflower protein degree of hydrolysis has improved 15～25%, and polypeptide yield improves 10～20%, and maximum polypeptide yield can reach 96.7%.Simultaneously also can shorten the hydrolysis sunflower protein time 12～30%, be more suitable in suitability for industrialized production.
3. the sunflower seed polypeptide of the present invention's preparation is with a wide range of applications; Can be used for fields such as functional food, cosmetics; Sunflower seed polypeptide has the effect of tangible removing hydroxy radical, ultra-oxygen anion free radical and DPPH free radical; And the antioxidation activity that suppresses oxidative hemolysis of erythrocyte, eliminate the synthetic effect of nitrite and blocking nitrosamine formation in addition, and animal feeding test is found; This sunflower seed polypeptide has delaying decrepitude of skin, improves the content of hydroxyproline in the skin, recovers the cosmetic result of skin elasticity; Can also play the protection liver, various health-care such as prevention chemical damage.
Description of drawings
Fig. 1 is the Superdex G-75 gel chromatography figure of the sunflower seed polypeptide of embodiment 1 preparation
The specific embodiment
Embodiment 1
After sunflower seeds pulverizing, degreasing, dissolve the heavy method of acid with alkali and extract protein isolate, get the 20g protein isolate by being dissolved in the 250mL water, accent pH9.0 adds the 0.3g alkali protease, is 0.3W/cm in ultrasonic power density
2Supersonic generator in 50 ℃ of water-baths.In the supersonic frequency hydrolysis 2h of 24KHz, during every at a distance from 12min sonicated 3min; Back 2h handles in the supersonic frequency of 36KHz, and is every at a distance from 8min sonicated 1min.
Regulate pH to 2.5～3.5,38 ℃ of temperature add 0.9g aspergillus oryzae acid protease and 1.0g pepsin; Mix, earlier with 24KHz frequency hydrolysis 3h, the back is with 36KHz frequency ultrasonic hydrolysis 2h; This stage is every at a distance from 10min sonicated 3min; The enzyme 8min that goes out in the last boiling water bath, this sample degree of hydrolysis is 17.93%, polypeptide yield is 92.19%.The SuperdexG-75 gel chromatography figure of sample sees Fig. 1, total P among the figure
1～P
66 absworption peaks are arranged, and the analysis of SuperdexG-75 gel chromatography obtains the regression equation logMr=-0.1809Ve+6.5617 of relative molecular weight Mr and elution volume Ve, calculate thus learn the polypeptide sample less than the content of peptides of 3000Da more than 90%.Enzymolysis liquid obtains the sunflower seed polypeptide product of molecular weight smaller or equal to 3000Da through the micro-filtration membrane of 0.22 μ m and the milipore filter separation and purification of 3000Da.
Measure through in vitro test, this product reaches 72.8% to the clearance rate of nitrite, and the blocking-up rate synthetic to nitrosamine is 67.9%; Get mouse red blood cell and process red cell suspension, add sunflower seed polypeptide, it reaches 80.23% to the oxidative hemolysis of erythrocyte inhibiting rate.This product with 400,600 and the dosage of 800mg/kg body weight the chemical damage mouse model of tetrachloro-methane induction is irritated stomach; Irritating stomach after 8 weeks; The vigor (p＜0.01) of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease in the remarkable reduction mice serum; And mda content (p＜0.01) in the serum, can play the effect of liver protecting.
Embodiment 2
After sunflower seeds pulverizing, the degreasing, dissolve the heavy method of acid with alkali and extract protein isolate.Getting protein isolate 20g and be dissolved in the 300ml water, transfer pH8.5, add the 0.4g alkali protease, is 0.2～0.47W/cm in power density
2Supersonic generator in 60 ℃ of water-baths, in 20KHz supersonic frequency hydrolysis 3h, during every at a distance from 10min sonicated 3min, again in the supersonic frequency hydrolysis 2h of 28KHz, during every at a distance from 10min sonicated 2min;
Transfer pH4.0 with HCl, add aspergillus oryzae acid protease and each 0.85g of aspergillus niger acid protease, in 28KHz supersonic frequency hydrolysis 2h; Hydrolysis 2h under the supersonic frequency of 32KHz is every at a distance from 6min sonicated 2min in the hydrolytic process again; Enzyme 10min goes out in the last boiling water bath.Protein hydrolysis degree through the hydrolysis of two stages is 17.7%, and polypeptide yield is 90.44%.Enzymolysis liquid obtains the sunflower seed polypeptide product of molecular weight smaller or equal to 3000Da through the micro-filtration membrane of 0.22 μ m and 3000 dalton's (Da) milipore filter separation and purification.
Through measuring, this product its to OH, O
2Reach 83.2%, 78.6% and 70.5% respectively with the clearance rate of DPPH, the clearance rate of nitrite is reached 81.7%, the blocking-up rate synthetic to nitrosamine reaches 89.1%; Get mouse red blood cell and process red cell suspension, add sunflower seed polypeptide, it reaches 76.5% to the oxidative hemolysis of erythrocyte inhibiting rate.Irritate stomach to this product with 400 mouse aging of the D-galactolipin being induced with the dosage of 600mg/kg body weight; Irritating stomach after 8 weeks; Measure that mda content reduces by 30% and 35% respectively with respect to model group in the mice serum, hydroxyproline content you can well imagine height 22% and 58.7% with respect to model components in the skin.
Embodiment 3
After sunflower seeds pulverizing, the degreasing, dissolve the heavy method of acid with alkali and extract protein isolate.Get the 20g protein isolate and be dissolved in the 400mL water, transfer pH8.0, add alkali protease 0.5g; Supersonic frequency in 28KHz in 55 ℃ of supersonic generators is handled 3h; Every during this time at a distance from 10min sonicated 3min, be that the supersonic frequency of 32KHz is handled 1h, every at a distance from 8min sonicated 1min.
Regulate pH 3.0, add aspergillus oryzae acid protease, aspergillus niger acid protease and each 0.8g of pepsin, 36 ℃ of temperature, hydrolysis 5h under 24～32KHz supersonic frequency, during every at a distance from 10min sonicated 2min.Hydrolysis finishes the back boiling water bath enzyme 10min that goes out.This sample degree of hydrolysis is 21%, and polypeptide yield is 96.75%, and enzymolysis liquid obtains the sunflower seed polypeptide product of molecular weight smaller or equal to 3000Da through the micro-filtration membrane of 0.22 μ m and the milipore filter separation and purification of 3000Da.
Through measuring, this product is to OH, O
2Reach 97.2%, 95.6% and 73.5% respectively with the clearance rate of DPPH, have obvious antioxidation activity.Irritate stomach to this product with 400 mouse aging of the D-galactolipin being induced with the dosage of 600mg/kg body weight; Irritating stomach after 8 weeks; Mda content reduces by 34% and 45% respectively with respect to model group in the mensuration mice serum; Hydroxyproline content improves 45% and 60.1% with respect to model group in the skin, can play the effect of delaying decrepitude of skin.
Embodiment 4
After sunflower seeds pulverizing, the degreasing, dissolve the heavy method of acid with alkali and extract protein isolate.Get the 20g protein isolate and be dissolved in the 450mL water, transfer pH8.0, add alkali protease 0.45g; Supersonic frequency in 24KHz in 58 ℃ of supersonic generators is handled 3h; Every during this time at a distance from 8min sonicated 3min, be that the supersonic frequency of 28KHz is handled 1h, every at a distance from 8min sonicated 2min.
Regulate pH 3.5, add Aspergillus usamii acid protease and each 0.9g of aspergillus niger acid protease, 40 ℃ of temperature, in 24KHz supersonic frequency hydrolysis 2h; Hydrolysis 1h under the supersonic frequency of 32KHz is every at a distance from 8min sonicated 2min in the hydrolytic process again; Hydrolysis finishes the back boiling water bath enzyme 10min that goes out.This sample degree of hydrolysis is 18.5%, and polypeptide yield is 91.3%, and enzymolysis liquid obtains the sunflower seed polypeptide product of molecular weight smaller or equal to 3000Da through the micro-filtration membrane of 0.22 μ m and the milipore filter separation and purification of 3000Da.Through measuring, this product reaches 78.4% to the clearance rate of nitrite, and the blocking-up rate synthetic to nitrosamine reaches 81.3%; Get mouse red blood cell and process red cell suspension, add sunflower seed polypeptide, it reaches 77.5% to the oxidative hemolysis of erythrocyte inhibiting rate, has obvious antioxidation activity.This product with 400,600 and the dosage of 800mg/kg body weight the chemical damage mouse model of tetrachloro-methane induction is irritated stomach; Irritating stomach after 8 weeks; The vigor (p＜0.01) of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease in the remarkable reduction mice serum; And mda content (p＜0.01) in the serum, can play the effect of liver protecting.
Embodiment 5
After sunflower seeds pulverizing, the degreasing, dissolve the heavy method of acid with alkali and extract protein isolate.Get the 20g protein isolate and be dissolved in the 500mL water, transfer pH9.0, add alkali protease 0.35g; Supersonic frequency in 20KHz in 60 ℃ of supersonic generators is handled 3h; Every during this time at a distance from 8min sonicated 3min, be that the supersonic frequency of 36KHz is handled 1h, every at a distance from 8min sonicated 1min.
Regulate pH4.0, add the 0.9g mould respectively and produce acid protease and 0.85g pepsin, mix, 38 ℃ of temperature, in 28KHz supersonic frequency hydrolysis 3h; Hydrolysis 1h under the supersonic frequency of 36KHz is every at a distance from 10min sonicated 1min in the hydrolytic process again; Hydrolysis finishes the back boiling water bath enzyme 10min that goes out.This sample degree of hydrolysis is 17.95%, and polypeptide yield is 91.18%, and enzymolysis liquid obtains the sunflower seed polypeptide product of molecular weight smaller or equal to 3000Da through the micro-filtration membrane of 0.22 μ m and the milipore filter separation and purification of 3000Da.This product with 400,600 and the dosage of 800mg/kg body weight the chemical damage mouse model of tetrachloro-methane induction is irritated stomach; Irritating stomach after 8 weeks; The vigor (p＜0.01) of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease in the remarkable reduction mice serum; And mda content (p＜0.01) in the serum, can play the effect of liver protecting.
Claims (4)
1. the preparation method of a sunflower seed polypeptide is characterized in that, comprises the steps:
1), sunflower seeds is through pulverizing, after the degreasing, dissolving the heavy method of acid with alkali and extract the sunflower seeds protein isolate, and is subsequent use after the vacuum drying;
2), take by weighing the sunflower seeds protein isolate, water-soluble by 4%～8% mass percentage concentration, add alkali protease; Enzyme concentration is 1.5%～2.5% of a protein isolate, mixes, and regulates pH to 8.0～9.5 with NaOH; Constant temperature is 50～60 ℃ in supersonic generator, prior to 20～28KHz supersonic frequency hydrolysis 2-3h, hydrolysis 1-3h under the supersonic frequency of 28～40KHz again; In two stage ultrasonic hydrolysis processes, every at a distance from 5-12min sonicated 1-4min;
3), above-mentioned treatment fluid regulates pH 2.5～4.5 with HCl, adds combination of acidic protease, enzyme concentration is 4%～6% of a protein isolate; The control temperature is at 35～45 ℃; Hydrolysis 4～6h under 24～40KHz supersonic frequency, during every at a distance from 6-12min sonicated 1-3min, 5～10 minutes enzymes that go out of boiling water bath; Through micro-filtration, milipore filter separation and purification, obtain the sunflower seed polypeptide product.
2. the preparation method of a kind of sunflower seed polypeptide as claimed in claim 1 is characterized in that described combination of acidic protease is any two or three in aspergillus oryzae acid protease, Aspergillus usamii acid protease, mould product acid protease, aspergillus niger acid protease and the pepsin.
3. the application of the sunflower seed polypeptide that makes of the preparation method of a kind of sunflower seed polypeptide as claimed in claim 1 in the preparation functional food.
4. the application of the sunflower seed polypeptide that makes of the preparation method of a kind of sunflower seed polypeptide as claimed in claim 1 in the preparation cosmetics.
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