CN103571905A - Preparation method of high-purity casein phosphopeptide - Google Patents
Preparation method of high-purity casein phosphopeptide Download PDFInfo
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Abstract
The invention discloses a preparation method of high-purity casein phosphopeptide. The method comprises the following steps: adding water to dissolve casein; carrying out secondary enzymolysis by using compound protease and pepsase prepared from any four of trypsase, neutral protease, alkaline protease, curd protease and nematolyt; carrying out secondary purification treatment through cation exchange resin and anion exchange resin after sterilizing and filtering; washing hydrolysate by water; eluting by hydrochloric acid of which the concentration is 0.5-1%; desalting and drying by a nanofiltration membrane. By adopting the preparation method, the yield (greater than or equal to 17%) and the purity (greater than or equal to 90%) of the casein phosphopeptide can be effectively improved; the prepared casein phosphopeptide is bitter in taste, and good in water-solubility; a water solution containing 20% of casting polypropylene (CPP) is clear and transparent; the casein phosphopeptide is powder of which the color is pure white. The molecular weight of the milk casein phosphopeptide prepared by the method mainly is between 1000 and 5000; absorption of a human body is facilitated; the preparation method disclosed by the invention is a preparation method which is low in cost, simple in process, and applicable to industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of take the high purity phosphopeptide caseinate that casein is raw material in cow's milk.
Background technology
Phosphopeptide caseinate (CPP) is to take bovine casein as raw material, by biotechnology, makes, and has the polypeptide that divalent-metal ions such as promoting calcium, iron, zinc absorbs and utilizes, and can be used for various nutrition, protective foods.Phosphopeptide caseinate is the casein with pancreatin or trypsin hydrolyzing, through refining, purifying, make, its core texture is :-Se r(P)-Se r(P)-Se r(P)-G lu-G lu-(Se r: Serine, G lu: L-glutamic acid, P: phosphate).Phosphoserine residue in this structure (Se r(P)-) cluster existence, electronegative under enteron aisle PH weakly alkaline environment, can stop the further effect of digestive ferment, phosphopeptide caseinate can be further hydrolyzed.Phosphopeptide caseinate can be combined with calcium and be suppressed the formation of calcium phosphate precipitation under the environment of PH7～8, small intestine lower end, makes free ca keep higher concentration, promotes the Passive intake of calcium.
Although as far back as the fifties, abroad just started the research to phosphopeptide caseinate, the research work of preparation of industrialization is until in recent years just really to start ，Er China less aspect the research of casein phosphoric acid skin.Tradition phosphopeptide caseinate production technique, with low content of technology, phosphopeptide caseinate content low (the highest CPP content mostly is 20% left and right), product have the shortcomings such as bitter taste, face is partially yellow.Chinese patent disclosed " a kind of method of preparing phosphopeptide caseinate " (201310054681.5), employing adds proteolytic enzyme to carry out enzyme digestion reaction in casein, utilize ultra-filtration membrane to isolate enzymolysis product phosphopeptide caseinate, product phosphopeptide caseinate is after nanofiltration membrane desalting treatment and get final product.The method is simple to operate, and equipment requirements is low, and yield is high, good product quality, and temperature of reaction is low, can realize industrialization and produce, but it adopts single protease hydrolyzed, phosphopeptide caseinate obtaining ratio and content lower (high product content is 16% left and right), product do not carry out debitterize.
The present invention is on the basis of traditional phosphopeptide caseinate production technology, the casein in cow's milk of take is raw material, through enzymolysis, separation, purifying, debitterize, membrane filtration, the explained hereafter phosphopeptide caseinate such as dry, promote the upgrading of phosphopeptide caseinate product to have improved added value of product.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of purity high, without bitter taste, good water solubility, good fluidity, color, be preparation method lily, that technique simply can be applicable to actual production, lower-cost phosphopeptide caseinate.
For addressing the above problem, the method for a kind of phosphopeptide caseinate provided by the present invention, comprises the following steps:
(1) add the water of casein 6-10 times quality, stirring and dissolving under 40 ℃ of-50 ℃ of conditions;
(2) add compound protease to carry out enzymolysis, enzymatic hydrolysis condition is: enzyme concentration is the 0.05%-0.2% of casein quality, 40 ℃-50 ℃ of temperature of reaction, reaction times 1 h-2h, pH 7.0-9.0, described compound protease is the mixture of any four kinds in trypsinase, neutral protease, Sumizyme MP, curdled milk proteolytic enzyme and papoid, after compound protease enzymolysis, then the stomach en-of 0.05%-0.1% that adds casein quality enzymolysis 1h-2h again under 40 ℃-50 ℃ of temperature, pH value 4.0-5.0;
(3) temperature is adjusted to 80 ~ 90 ℃, is incubated 20 min-30 min, carry out sterilizing;
(4) adjust pH 4.0-5.0, removes by filter the not high molecular weight protein precipitation of enzymolysis;
(5) supernatant liquor adjustment pH is 4.0-5.0, uses Zeo-karb to absorb purifying 1h-4h, filters, it is 6.0-7.0 that filtrate is adjusted refined solution pH, re-uses anionite-exchange resin and carries out purifying 1h-4h, first washes with water, discard after water lotion, then the hydrochloric acid wash-out that is 0.5%-1% by concentration;
(6) elutriant utilizes ultra-filtration membrane to isolate phosphopeptide caseinate, then uses nanofiltration membrane desalination, obtains being less than the phosphopeptide caseinate liquid of 5000D;
(7) through lyophilize, spraying dry, vacuum drying any one, the product obtaining is dried to powder.
In step (2), in described compound protease, the quality proportioning of any four kinds of proteolytic enzyme is 1～4:1～4:1～4:1～4.What optimize is, trypsinase: neutral protease: Sumizyme MP: curdled milk proteolytic enzyme=4:3:2:1, or trypsinase: Sumizyme MP: papoid: neutral protease=2:3:1:4, or neutral protease: Sumizyme MP: curdled milk proteolytic enzyme: papoid=1:2:3:4.
In step (6), described ultra-filtration membrane molecular weight cut-off is 5000D, and nanofiltration membrane molecular weight cut-off is 150D.
After the compound protease that the present invention's employing is made by trypsinase, neutral protease, Sumizyme MP, curdled milk proteolytic enzyme, papoid carries out enzymolysis certain hour, use again stomach en-enzymolysis, and adopt first Zeo-karb to absorb purifying, re-use the purifying process that anionite-exchange resin carries out purifying, can effectively improve yield (>=17%) and the purity (phosphopeptide caseinate content >=90%) of phosphopeptide caseinate, prepared phosphopeptide caseinate is without bitter taste, the aqueous solution clear of good water solubility, 20%CPP, color is pure white powder.The molecular weight of newborn source property phosphopeptide caseinate prepared by the present invention mainly, between 1000-5000, is conducive to absorption of human body, is that a kind of cost is low, and the simple preparation method of technique is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of gained phosphopeptide caseinate (CPP) before anion-cation exchange resin purification process;
Fig. 2 is the high-efficient liquid phase chromatogram of Zeo-karb purification process gained phosphopeptide caseinate (CPP);
Fig. 3 is the high-efficient liquid phase chromatogram of anionite-exchange resin purification process gained phosphopeptide caseinate (CPP);
Fig. 4 is first Zeo-karb purification process, then the high-efficient liquid phase chromatogram of anionite-exchange resin purification process gained phosphopeptide caseinate (CPP).
Embodiment
embodiment 1: a kind of preparation of high purity phosphopeptide caseinate, comprises the following steps:
In reactor, add pure water 150L, open and stir 200rpm, be heated to 50 ℃, in reactor, slowly drop into casein 25Kg, pH is adjusted to 7.8; Drop into trypsinase: neutral protease: Sumizyme MP: compound protease 12.5g curdled milk proteolytic enzyme=4:3:2:1(mass ratio) being mixed.After enzymolysis 1h, feed liquid is cooled to 37 ℃, pH is adjusted to 4.0, add 12.5g stomach en-, continue enzymolysis, after 1h, enzymolysis finishes.Adjust the pH4.5 enzyme that goes out, temperature is adjusted to 90 ℃, be incubated 20 min.Use 732 Zeo-karbs to absorb purifying 1h, filter, it is 7.0 that filtrate is adjusted refined solution pH, uses 717 anionite-exchange resin to carry out purifying 1h, first washes with water, discards after water lotion, then uses 1% hydrochloric acid wash-out.Elutriant adjust pH 6.8, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains being less than the phosphopeptide caseinate liquid of 5000D, then uses the nanofiltration membrane desalination of 150D.Filtrate is sprayed dry, obtained phosphopeptide caseinate finished product 4.52kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, the aqueous solution clear of good water solubility, 20%CPP.
embodiment 2: a kind of preparation of high purity phosphopeptide caseinate, comprises the following steps:
In reactor, add pure water 200L, open and stir 200rpm, be heated to 45 ℃, in reactor, slowly drop into casein 25Kg, pH is adjusted to 7.5; Drop into trypsinase: neutral protease: papoid: the compound protease 25g that curdled milk proteolytic enzyme=3:2:4:1 is mixed.After enzymolysis 1.5h, feed liquid is cooled to 40 ℃, pH is adjusted to 5, add 25g stomach en-, continue enzymolysis, after 1h, enzymolysis finishes.Adjust pH4.6, temperature is adjusted to 80 ℃, be incubated 30 min, enzyme goes out.Remove by filter not the high molecular weight protein precipitations such as casein hydrolysis, supernatant liquor is used 001 * 7 Zeo-karb to absorb purifying 1h, filter, it is 6.5 that filtrate is adjusted refined solution pH, use U.S. Amberlite IRA-400 anionite-exchange resin to carry out purifying 2h, first wash with water, discard after water lotion, then use 1% hydrochloric acid wash-out.Elutriant adjust pH 6.8, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains being less than the phosphopeptide caseinate liquid of 5000D, then uses the nanofiltration membrane desalination of 150D.Filtrate is sprayed dry, obtained phosphopeptide caseinate finished product 4.75kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, the aqueous solution clear of good water solubility, 20%CPP.
embodiment 3: a kind of preparation of high purity phosphopeptide caseinate, comprises the following steps:
In reactor, add pure water 250L, open and stir 200rpm, be heated to 45 ℃, in reactor, slowly drop into casein 25Kg, pH is adjusted to 8.5; Drop into trypsinase: the compound protease 50g that Sumizyme MP: papoid: neutral protease=2:3:1:4 is mixed.After enzymolysis 2h, feed liquid is cooled to 37 ℃, pH is adjusted to 4.5, add 12.5g stomach en-, continue enzymolysis, after 1h, enzymolysis finishes.Adjust pH4.7, temperature is adjusted to 85 ℃, be incubated 30 min, enzyme goes out.Remove by filter not the high molecular weight protein precipitations such as casein hydrolysis, supernatant liquor is used ROHM AND HAAS AMBERJET UP6040 Zeo-karb to absorb purifying 1h, filter, it is 7.0 that filtrate is adjusted refined solution pH, use German Lewatit M500 anionite-exchange resin to carry out purifying 2h, first wash with water, discard after water lotion, then use 0.6% hydrochloric acid wash-out.Elutriant adjust pH 5.0, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains being less than the phosphopeptide caseinate liquid of 5000D, then uses the nanofiltration membrane desalination of 150D.Filtrate is carried out to vacuum-drying, obtain phosphopeptide caseinate finished product 4.63kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, the aqueous solution clear of good water solubility, 20%CPP.
embodiment 4:a preparation for high purity phosphopeptide caseinate, comprises the following steps:
In reactor, add pure water 175L, open and stir 200rpm, be heated to 50 ℃, in reactor, slowly drop into casein 25Kg, pH is adjusted to 8.0; Drop into neutral protease: Sumizyme MP: the compound protease 37.5g that curdled milk proteolytic enzyme: papoid=1:2:3:4 is mixed.After enzymolysis 1.5h, feed liquid is cooled to 37 ℃, pH is adjusted to 4.5, add 25g stomach en-, continue enzymolysis, after 1h, enzymolysis finishes.Adjust pH4.6, temperature is adjusted to 85 ℃, be incubated 30 min, enzyme goes out.Remove by filter not the high molecular weight protein precipitations such as casein hydrolysis, supernatant liquor is used the WK60L of Mitsubishi Zeo-karb to absorb purifying 2h, filter, it is 6.8 that filtrate is adjusted refined solution pH, use SA10AX anionite-exchange resin to carry out purifying 2h, first wash with water, discard after water lotion, then use 0.8% hydrochloric acid wash-out.Elutriant adjust pH 6.0, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains being less than the phosphopeptide caseinate liquid of 5000D, then uses the nanofiltration membrane desalination of 150D.Filtrate is carried out to vacuum-drying, obtain phosphopeptide caseinate finished product 4.7kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, the aqueous solution clear of good water solubility, 20%CPP.
In the present invention, compound protease and pepsic secondary enzymolysis, can effectively improve the yield of phosphopeptide caseinate.For this reason, the present invention adopts with single proteolytic enzyme and embodiment 1-4 compound protease and Pepsin secondary enzymolysis have been made simultaneous test.Result shows, use trypsinase, neutral protease, papoid or the single protease hydrolyzed of curdled milk proteolytic enzyme, the yield of phosphopeptide caseinate is respectively 16 %, 14.5%, 15.3% and 14.8 %, two use the method for embodiment of the present invention 1-4, the yield of phosphopeptide caseinate is respectively 18.08%, 18.0%, 18.52%, 18.88%, all far away higher than the yield of using the phosphopeptide caseinate of single protease hydrolyzed.
Adopt compound protease enzymolysis, can also make the molecular weight of phosphopeptide caseinate (CPP) less.Experiment shows, single enzyme hydrolysis, and gained phosphopeptide caseinate main molecules amount section is 3000D-8000D, adopts compound protease (CPP), phosphopeptide caseinate (CPP) yield is main molecules amount section 1000D-5000D.
The present invention tests and shows, the cation exchange resin secondarily purified processing of the independent purification process regulating YIN and YANG of yin, yang ion Zeo-karb ion exists notable difference.For this reason, the present invention has done contrast experiment, and it the results are shown in Figure 1, shown in Fig. 2, Fig. 3, Fig. 4.Realization shows, adopt first Zeo-karb purification process, the purity of anionite-exchange resin purification process gained phosphopeptide caseinate (CPP) again, apparently higher than separately by the purity of anionite-exchange resin or the many phosphopeptide caseinate (CPP) of the independent purification process of Zeo-karb.
Claims (4)
1. a high purity phosphopeptide caseinate preparation method, is characterized in that
,comprise the following steps:
(1) add the water of casein 6-10 times quality, stirring and dissolving under 40 ℃ of-50 ℃ of conditions;
(2) add compound protease to carry out enzymolysis, enzymatic hydrolysis condition is: enzyme concentration is the 0.05%-0.2% of casein quality, 40 ℃-50 ℃ of temperature of reaction, reaction times 1 h-2h, pH 7.0-9.0, described compound protease is the mixture of any four kinds in trypsinase, neutral protease, Sumizyme MP, curdled milk proteolytic enzyme and papoid, after compound protease enzymolysis, then the stomach en-of 0.05%-0.1% that adds casein quality enzymolysis 1h-2h again under 40 ℃-50 ℃ of temperature, pH value 4.0-5.0;
(3) temperature is adjusted to 80 ~ 90 ℃, is incubated 20 min-30 min, carry out sterilizing;
(4) adjust pH 4.0-5.0, removes by filter the not high molecular weight protein precipitation of enzymolysis;
(5) supernatant liquor adjustment pH is 4.0-5.0, uses Zeo-karb to absorb purifying 1h-4h, filters, it is 6.0-7.0 that filtrate is adjusted refined solution pH, re-uses anionite-exchange resin and carries out purifying 1h-4h, first washes with water, discard after water lotion, then the hydrochloric acid wash-out that is 0.5%-1% by concentration;
(6) elutriant utilizes ultra-filtration membrane to isolate phosphopeptide caseinate, then uses nanofiltration membrane desalination, obtains being less than the phosphopeptide caseinate liquid of 5000D;
(7) through lyophilize, spraying dry, vacuum drying any one, the product obtaining is dried to powder.
2. high purity phosphopeptide caseinate preparation method according to claim 1, is characterized in that
,in step (2), in described compound protease, the quality proportioning of any four kinds of proteolytic enzyme is 1～4:1～4:1～4:1～4:.
3. high purity phosphopeptide caseinate preparation method according to claim 2, is characterized in that
,the quality proportioning of compound protease is
:trypsinase: neutral protease: Sumizyme MP: curdled milk proteolytic enzyme=4:3:2:1, or trypsinase: Sumizyme MP: papoid: neutral protease=2:3:1:4, or neutral protease: Sumizyme MP: curdled milk proteolytic enzyme: papoid=1:2:3:4.
4. high purity phosphopeptide caseinate preparation method according to claim 1, is characterized in that
,in step (6), described ultra-filtration membrane molecular weight cut-off is 5000D, and nanofiltration membrane molecular weight cut-off is 150D.
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| CN108531531A (en) * | 2018-04-02 | 2018-09-14 | 临夏州华安生物制品有限责任公司 | A kind of casein phosphopeptide preparation method of beta-cyclodextrin and the de- hardship of chitosan |
| CN110037163A (en) * | 2019-04-24 | 2019-07-23 | 王书敏 | A kind of preparation method of compound plant protein peptide |
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