CN100580088C - Enzyme method for hydrolyzing casein and synchronous preparation of phosphopeptide and non-phosphopeptide - Google Patents
Enzyme method for hydrolyzing casein and synchronous preparation of phosphopeptide and non-phosphopeptide Download PDFInfo
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- CN100580088C CN100580088C CN200710019381A CN200710019381A CN100580088C CN 100580088 C CN100580088 C CN 100580088C CN 200710019381 A CN200710019381 A CN 200710019381A CN 200710019381 A CN200710019381 A CN 200710019381A CN 100580088 C CN100580088 C CN 100580088C
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Abstract
The invention relates to a enzymatical hydrolyzed casein and the method for preparing casein phosphopeptide (CPP) and casein non phosphopeptide (CNPP), belonging to dairy further processing and functional health care food field. The invention takes casein as raw material, takes single alkali prolease Alcalase 2.4L for controlling hydrolysis, takes nitrobenzene-sulfonic acid for pH value monitoring, preparing CPP and CNPP from casein hydrolysate with ethanol- calcium separation method. The nitrogen/ phosphorus molar ratio in CPP is 20- 25: 1, the productivity is above 35%; the productivity of CNPP reaches 45%; the determined amine aicd squence of two ACE inhibiting active peptide and one antioxidant active peptide in CNPP product is Tyr-Leu,Glu-Asp-Val-Pro and His-Ser. The invention is characterized by good enzymolysis effect, low price, rational process route, high raw material vailability ratio, and wide application of CPP and CNPP in functional food ingredient or health care food.
Description
Technical field
A kind of enzymatic hydrolysis casein prepares the method for phospho-peptide (CPP) and non-phosphopeptide (CNPP) simultaneously, belongs to milk-product development of resources and functional health-care food technical field.
Background technology
In recent years, the malnourished problem of human calcium has been subjected to paying close attention to widely.The whole world, especially developing country, calcium deficiency is universal phenomenon already, that wherein the most serious is pregnant woman, children and the elderly.The recommended amounts of China normal people calcium is 800mg/d, but the nutrition survey result shows that calcium intake only is 400mg/d per capita, and the actual intake of children and pregnant woman is less than half of answering intake.The sickness rate of China's senile osteoporosis disease reaches 30%-50%, and the children rachitis sickness rate is up to 20%-50%.There are some researches show at present, take in competent calcium in the meals and can reduce hypertensive sickness rate.Therefore, calcium is all very important to the crowd of different ages and sex.The factor that influences human calcium's absorption is a lot, except that human body self has the malabsorption problem, diet formula that the more important thing is China is based on vegetable food, and animal food is the meals type simply having adequate food and clothing of assisting, and this meals type can cause human body to take in hypocalcia.People must the attention science replenish the calcium, and CPP is the bridge that undisputed science is replenished the calcium.In addition, CPP can and promote them to be absorbed by the body in conjunction with other metallic element equally, and this has increased the interest of people to the CPP research and development more.
Hypertension is " the noiseless killer " that have a strong impact on HUMAN HEALTH of a kind of high incidence, high complication, high disability rate.Statistics shows that developed country's hypertension morbidity is more than 20%, and other national sickness rate is also 10%～20%.The essential hypertension mortality ratio in whole world every year reaches 1,200 ten thousand.For hypertensive treatment, have following mistake at present: (1) is not enough to hypertensive harm understanding, does not do treatment control; (2) only hypotensive merely, the various Hazard Factor of not complex therapy; (3) carry out pharmacological agent once discovery hypertension, and do not do non-drug therapy.Studies show that the blood pressure lowering peptide that comes from food proteins can overcome all side effects that occur in the pharmacological agent fully, it only plays hypotensive effect to the hyperpietic, and the normotensive is not but had hypotensive effect, and it also has immunoregulation effect simultaneously.Further the natural decrease blood pressure peptide manufacture of exploitation is trend of the times, and it will become the important component part in the non-drug therapy.
Be rich in multiple biologically active peptides in the casein, wherein CPP is studied and develops the earliest a kind of, has the effect in conjunction with mineral substance (as calcium).CNPP is as a kind of natural existence and have potential anti-oxidant activity simultaneously and the active material of inhibition ACE, and diseases such as hypertension are played prevention effect, and anosis crowd is edible can functions of prevention and health care, the edible effect of playing non-drug therapy of ill crowd.Therefore, adopting casein is that the feedstock production biologically active peptides has become current research focus.
At present, the investigator has reported the separation and purification of the ace inhibitory peptide of Casein in Milk, but the structure of its functional component is not illustrated; The investigator also utilizes the enzymolysis and the membrane filtration integrated technique that comprise pancreatin, Sumizyme MP to prepare the casein biologically active polypeptides continuously, but its used enzyme kind is many, price is expensive (CN1546682); Be raw material with the bovine casein, adopted protease hydrolysis, ion exchange resin desalination to produce the ace inhibitory peptide of being made up of six peptides to ten peptide (CN1552891) by the investigator in addition, but its operational path is complicated, lyophilize makes cost high; Also have the investigator to adopt the compound protease casein hydrolysis to prepare phosphopeptide caseinate (CPP), but do not utilize CNPP.
Therefore, suitable protease hydrolysis casein prepares CPP and CNPP to adopt price, fully improves raw material availability, has both had very big economic benefit, also has the certain social benefit.And select suitable desalination mode to reduce salt in the polypeptide products, the utilization and extention of CPP and CNPP product is had great importance.Simultaneously, also be necessary to identify its structure, be convenient to further investigation for the important polypeptide that has functional property in the CNPP product.
Summary of the invention
The purpose of this invention is to provide the method that a kind of enzymatic hydrolysis casein prepares phosphopeptide caseinate (CPP) and casein non-phosphopeptide (CNPP) simultaneously, is a kind of important channel of milk-product deep processing, also is the preparation method of biologically active peptides simultaneously.
Technical scheme of the present invention is: be raw material with the casein, adopt single Sumizyme MP Alcalase 2.4L to carry out control hydrolysis, adopt trinitro-benzene-sulfonic acid method monitoring DH value, adopt ethanol-calcium precipitation method separation of C PP and CNPP from casein hydrolyzate then, prepare phosphopeptide caseinate and casein non-phosphopeptide simultaneously, the part of CPP adopts vacuum-drying to obtain the CPP powder-product, and yield is more than 35%.CNPP partly adopts nanofiltration to concentrate, and reclaims ethanol, adopts the macroporous adsorbent resin desalination, adopts spraying drying to obtain the CNPP powder-product then, and yield is more than 45%.Two kinds of ACE inhibition bioactive peptides and a kind of antioxidation active peptides in the CNPP product are identified, obtained its aminoacid sequence.Technology is:
(1) dissolving: under 55～65 ℃ of conditions, it is in 8.0～9.0 the alkaline solution that casein is dissolved in pH, is made into the casein solution of mass concentration 16%～20% (m/m).
(2) enzymolysis: add Alcalase 2.4L in the caseic ratio of 1～1.5mL enzyme liquid/100g, stir 2～4h under 55～65 ℃ temperature, sampling adopts trinitro-benzene-sulfonic acid (TNBS) method monitoring DH value to reach 15～20, be warming up to 80～85 ℃ then, the insulation 15min enzyme that goes out.
(3) ethanol-calcium deposit: add the calcium chloride of 1.2% (calcium chloride quality/hydrolyzed solution volume), add food grade ethanol again, stir 30min to ethanol final concentration 70%～80%, standing over night, the time, temperature was below 30 ℃ more than 6 hours; The method of standing demix also can be substituted by the centrifugal 20min of 3000rpm.
(4) acquisition of CPP product: the siphon supernatant liquor, the sinking part is centrifugal, and (3000rpm 15min), precipitates part and obtains the CPP powder-product 45 ℃ of following vacuum-dryings, and CPP product moisture content merges supernatant liquor below 4.5%, is the CNPP part.
(5) nanofiltration concentrates CNPP and reclaims ethanol: is that the nanofiltration membrane of 300D is carried out nanofiltration to the casein non-phosphopeptide part of (4) to hold back relative molecular mass, obtains casein non-phosphopeptide nanofiltration concentrated solution and reclaims ethanol.
(6) macroporous adsorbent resin desalination: the casein non-phosphopeptide that concentrates gained 60～150mg/ml concentration with sample on the flow velocity of 1 bed volume per hour to the DA201-C macroporous adsorptive resins, washing back 70% alcohol desorption.
(7) spraying drying makes the CNPP powder: turnover wind-warm syndrome degree is respectively 160～180 ℃ and 70～80 ℃, and spraying drying obtains the CNPP powder.
(8) structure of ACE inhibition bioactive peptide and antioxidation active peptides is identified among the CNPP: utilize Size Exclusion Chromatograph SEC Sephadex G-25, ion-exchange chromatography DTF-02, macroporous adsorbent resin chromatography DA201-C and half preparation type C18RP-HPLC to separate from CNPP successively and obtain two kinds of inhibition ACE bioactive peptides and a kind of antioxidation active peptides.Adopt SE-HPLC, LC/MS and amino acid analysis to prove that the aminoacid sequence of these three kinds of peptides is respectively Tyr-Leu, Glu-Asp-Val-Pro and His-Ser again.
The nitrogen of gained phosphopeptide caseinate/phosphorus mol ratio is 20～25: 1, contains in the raw material casein 95% phosphorus, and the phosphopeptide caseinate yield reaches 35%, after the desalination in the phosphopeptide caseinate product ash oontent below 6%.
The ratio of desalinization of gained casein non-phosphopeptide reaches 98%, and the demineralising process casein non-phosphopeptide rate of recovery reaches 90%, and the product yield reaches 45%, after the desalination in the casein non-phosphopeptide product ash oontent below 6%.
Beneficial effect of the present invention: (1) adopts single proteolytic enzyme that casein is hydrolyzed, and has set up casease hydrolysis process efficiently, and raw material availability reaches more than 90%, obtains CPP and CNPP two class biologically active peptidess simultaneously, realizes low-cost, high production; (2) adopt TNBS method monitoring DH value, set up feasible degree of hydrolysis monitoring method, be convenient to control productive rate and the N/P mol ratio of CPP in the product, reduced the ash content in the product; (3) adopt macroporous adsorbent resin and membrane separation technique to remove salinity in the product, after the desalination in CNPP and the CPP product ash oontent below 6%; (4) structure of important component among the CNPP is identified, for structure activity relationship and Study on mechanism lay the foundation.Utilize the preparation method of CPP provided by the invention and CNPP, improved caseic utilization ratio, on economy and preparative-scale, embody its advantage.The structure of critical function composition is clear and definite in the product simultaneously, helps its utilization and extention, and the purposes of CPP and CNPP is functional food ingredient or protective foods.
Description of drawings
The preparation technology of Fig. 1 CPP and CNPP.
The structure of ace inhibitory peptide and anti-oxidation peptide among Fig. 2 amino acid analysis and the mass spectrum evaluation CNPP: (a) Tyr-Leu; (b) Glu-Asp-Val-Pro; (c) His-Ser.
Embodiment
Method of the present invention is not to be defined in this embodiment.
In 10L interlayer reactor, carry out enzyme reaction.5000ml water is added in the reactor, be warming up to 55 ℃, take by weighing the 1000g casein then, slowly add in the entry under stirring.Regulate pH value to 8.0 with 2mol/L NaOH, under 55 ℃ of conditions casein is all dissolved, add 11.5mL Alcalase 2.4L, sampling is monitored DH with TNBS.Add the anhydrous CaCl of 60g
2, be stirred to dissolving fully, under whipped state, slowly add edible ethanol to ultimate density then and reach 70% (v/v), place 30min, it is centrifugal that (3000rpm, 20min), precipitation makes CPP product 354g through vacuum-drying (40 ℃).Supernatant liquor advances nanofiltration device (nanofiltration membrane hold back relative molecular mass be 300D) and concentrates, see through liquid and be used to reclaim ethanol, concentrate DA201-C macroporous adsorbent resin chromatography post on (concentration is 60mg/ml) liquid, use 70% alcohol desorption after the washing desalination, flow velocity is bed volume per hour, 3 hours washing time, desorption time 5 hours.Spraying drying, 160 ℃ of inlet temperature, 80 ℃ of air outlet temperatures make CNPP product 486g.
The CNPP sample is made into 30% solution, separate from CNPP by Size Exclusion Chromatograph SEC Sephadex G-25, DTF-02 ion-exchange chromatography, macroporous adsorbent resin chromatography DA201-C and half preparation type C18RP-HPLC and to obtain two kinds of ACE and suppress bioactive peptides, adopt SE-HPLC, LC/MS and amino acid analysis that the inhibition ACE bioactive peptide among the CNPP is carried out primary structure and identify that really its aminoacid sequence is respectively Tyr-Leu and Glu-Asp-Val-Pro.
In 25L interlayer reactor, carry out enzyme reaction.15000ml water is added in the reactor, be warming up to 65 ℃, take by weighing the 3750g casein then, slowly add in the entry under stirring.Regulate pH value to 8.5 with 2mol/L NaOH, under 65 ℃ of conditions casein is all dissolved, add 55mL Alcalase 2.4L, sampling is monitored DH with TNBS.Add the anhydrous CaCl of 180g
2, be stirred to dissolving fully, under whipped state, slowly add edible ethanol to ultimate density then and reach 80% (v/v), standing over night, it is centrifugal behind the siphon supernatant liquid that (3000rpm, 15min), precipitation makes CPP product 1480g through vacuum-drying (45 ℃).Supernatant liquor advances nanofiltration device (nanofiltration membrane hold back relative molecular mass be 300D) and concentrates, see through liquid and be used to reclaim ethanol, concentrate DA201-C macroporous adsorbent resin chromatography post on (concentration is 150mg/ml) liquid, use 70% alcohol desorption after the washing desalination, flow velocity is bed volume per hour, 3 hours washing time, desorption time 5 hours.Spraying drying, 180 ℃ of inlet temperature, 70 ℃ of air outlet temperatures make CNPF product 1690g.
The CNPP sample ligand is made 25% solution, separate from CNPP by Size Exclusion Chromatograph SEC Sephadex G-25, DTF-02 ion-exchange chromatography, macroporous adsorbent resin chromatography DA201-C and half preparation type C18RP-HPLC and to obtain two kinds of ACE and suppress bioactive peptides, adopt SE-HPLC, LC/MS and amino acid analysis that the inhibition ACE bioactive peptide among the CNPP is carried out primary structure and identify that really its aminoacid sequence is respectively His-Ser.
Claims (3)
1, a kind of enzymatic hydrolysis casein prepares the method for phosphopeptide caseinate and casein non-phosphopeptide simultaneously, it is characterized in that: be raw material with the casein, adopt single Sumizyme MP Alcalase 2.4L to carry out control hydrolysis, adopt trinitro-benzene-sulfonic acid method monitoring DH value, adopt ethanol-calcium partition method to prepare phosphopeptide caseinate and casein non-phosphopeptide simultaneously then from casein hydrolyzate, technology is:
(1) dissolving: under 55～65 ℃, casein is dissolved in the alkaline solution of pH 8.0～9.0, is made into the casein solution of mass concentration 16%～20%;
(2) enzymolysis: add Alcalase 2.4L in the caseic ratio of 1～1.5mL enzyme liquid/100g, stir 2～4h down at 55～65 ℃, sampling adopts trinitro-benzene-sulfonic acid method monitoring DH value to reach 15～20, and the enzyme that goes out then heats up;
(3) ethanol-calcium deposit: add calcium chloride quality/hydrolyzed solution volume than 1.2% calcium chloride, add food grade ethanol again to ethanol final concentration 70%～80%, stirring 30min, 30 ℃ of following standing over night; Or the centrifugal 20min layering of 3000rpm;
(4) acquisition of phosphopeptide caseinate product: siphon goes out supernatant liquid, and precipitation part 3000rpm, the 15min of bottom are centrifugal, and the precipitation part obtains the phosphopeptide caseinate powder-product in vacuum-drying below 45 ℃, and product moisture content is below 4.5%; Supernatant liquor is merged, be casein non-phosphopeptide part;
(5) nanofiltration of casein non-phosphopeptide concentrates with ethanol and reclaims: is that the nanofiltration membrane of 300D is carried out nanofiltration to the casein non-phosphopeptide part of (4) to hold back relative molecular mass, obtains casein non-phosphopeptide nanofiltration concentrated solution and reclaims ethanol;
(6) macroporous adsorbent resin desalination: the casein non-phosphopeptide that concentrates gained 60～150mg/ml concentration with sample on the flow velocity of 1 bed volume per hour to the DA201-C macroporous adsorptive resins, washing back 70% alcohol desorption;
(7) spraying drying: turnover wind-warm syndrome degree is respectively 160～180 ℃ and 70～80 ℃, and spraying drying obtains casein non-phosphopeptide powder;
(8) structure of ACE inhibition bioactive peptide and antioxidation active peptides is identified in the casein non-phosphopeptide: utilize Size Exclusion Chromatograph SEC Sephadex G-25, ion-exchange chromatography DTF-02, macroporous adsorbent resin chromatography DA201-C and half preparation type C18RP-HPLC to separate from the casein non-phosphopeptide successively and obtain two kinds of inhibition ACE bioactive peptides and a kind of antioxidation active peptides; Adopt SE-HPLC, LC/MS and amino acid analysis to prove that the aminoacid sequence of these three kinds of peptides is respectively Tyr-Leu, Glu-Asp-Val-Pro and His-Ser again.
2, method according to claim 1, nitrogen/phosphorus the mol ratio that it is characterized in that phosphopeptide caseinate is 20～25: 1, contain in the raw material casein 95% phosphorus, the phosphopeptide caseinate yield reaches 35%, after the desalination in the phosphopeptide caseinate product ash oontent below 6%.
3, method according to claim 1, the ratio of desalinization that it is characterized in that the casein non-phosphopeptide reaches 98%, the demineralising process casein non-phosphopeptide rate of recovery reaches 90%, and the product yield reaches 45%, after the desalination in the casein non-phosphopeptide product ash oontent below 6%.
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| JP5515031B2 (en) * | 2010-02-23 | 2014-06-11 | ビーンスターク・スノー株式会社 | Peptide composition for inducing oral immune tolerance and method for preparing the same |
| CN101824457B (en) * | 2010-03-03 | 2012-07-04 | 大连工业大学 | Method for preparing casein antioxidant peptide by chymosin |
| CN102187935B (en) * | 2010-03-19 | 2012-12-19 | 江南大学 | Preparation method of Casein Phosphopeptides (CPPs) and ACE inhibitory peptides |
| CN103483455B (en) * | 2013-09-03 | 2015-03-25 | 哈尔滨商业大学 | Preparation method of high-emulsibility CNPPs (casein non-phosphopeptides)-soybean polypeptide assembly protein |
| CN105254714B (en) * | 2015-10-16 | 2019-09-10 | 中国农业大学 | A kind of anti-oxidation peptide and application from casein |
| CN110483622B (en) * | 2019-08-26 | 2021-06-11 | 无限极（中国）有限公司 | Casein phosphopeptide and preparation method and application thereof |
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Non-Patent Citations (8)
| Title |
|---|
| Alcalase水解酪蛋白特性的研究. 赵利.中国乳品工业，第33卷第4期. 2005 |
| Alcalase水解酪蛋白特性的研究. 赵利.中国乳品工业，第33卷第4期. 2005 * |
| Characterization of phosphopeptides prepared usingalcalase:Determination of enzyme specificity. Adamson NJ.Enzyme and Microbial Technology,Vol.19 No.3. 1996 |
| Characterization of phosphopeptides prepared usingalcalase:Determination of enzyme specificity. Adamson NJ.Enzyme and Microbial Technology,Vol.19 No.3. 1996 * |
| 酪蛋白磷酸肽的分离纯化及分子结构鉴定. 冯凤琴.无锡轻工大学学报，第18卷第4期. 1999 |
| 酪蛋白磷酸肽的分离纯化及分子结构鉴定. 冯凤琴.无锡轻工大学学报，第18卷第4期. 1999 * |
| 酪蛋白磷酸肽的制备及性质. 杨曜中.华东理工大学学报，第25卷第6期. 1999 |
| 酪蛋白磷酸肽的制备及性质. 杨曜中.华东理工大学学报，第25卷第6期. 1999 * |
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