CN103571905B - 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 with the preparation method of the high-purity casein phosphopeptide that is raw material of casein in cow's milk.
Background technology
Phosphopeptide caseinate (CPP) is raw material with bovine casein, obtained by biotechnology, has the polypeptide that divalent-metal ions such as promoting calcium, iron, zinc absorbs and utilizes, can be used for various nutrition, protective foods.Phosphopeptide caseinate is the casein with pancreatin or trypsin hydrolyzing, make through refining, purifying, 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 (-Se r(P)-in this structure) cluster existence, electronegative under enteron aisle PH weakly alkaline environment, the further effect of digestive ferment can be stoped, phosphopeptide caseinate can not be hydrolyzed further.Phosphopeptide caseinate can be combined with calcium and suppress the formation of calcium phosphate precipitation under the environment of PH7 ~ 8, small intestine lower end, makes the concentration that free ca keeps higher, promotes the Passive intake of calcium.
Although as far back as the fifties, just start the research to phosphopeptide caseinate abroad, the research work of preparation of industrialization is until in recent years just really start, and China is less in 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 about 20%), the product shortcoming such as have bitter taste, face partially yellow.Chinese patent disclosed " a kind of method preparing phosphopeptide caseinate " (201310054681.5), employing adds proteolytic enzyme and carries out enzyme digestion reaction in casein, utilize Ultra filtration membrane to go out 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 (most high product content is about 16%), product do not carry out debitterize.
The present invention is on the basis of traditional phosphopeptide caseinate production technology, with casein in cow's milk for raw material, through explained hereafter phosphopeptide caseinates such as enzymolysis, separation, purifying, debitterize, membrane filtration, dryings, promote the upgrading of phosphopeptide caseinate product, improve 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, be lily, that technique simply can be applicable to actual production, lower-cost phosphopeptide caseinate preparation method without bitter taste, good water solubility, good fluidity, color.
For solving the problem, the method for a kind of phosphopeptide caseinate provided by the present invention, comprises the following steps:
(1) water of casein 6-10 times quality is added, stirring and dissolving under 40 DEG C of-50 DEG C of conditions;
(2) add compound protease and carry out enzymolysis, enzymatic hydrolysis condition is: enzyme concentration is the 0.05%-0.2% of casein quality, temperature of reaction 40 DEG C-50 DEG C, 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, chrymotrypsin and papoid, after compound protease enzymolysis, then the stomach en-of 0.05%-0.1% adding casein quality enzymolysis 1h-2h again under temperature 40 DEG C-50 DEG C, pH value 4.0-5.0;
(3) temperature is adjusted to 80 ~ 90 DEG C, is incubated 20 min-30 min, carries out sterilizing;
(4) adjust pH 4.0-5.0, crosses the high molecular weight protein precipitation filtering non-enzymolysis;
(5) supernatant liquor adjustment pH is 4.0-5.0, uses Zeo-karb to absorb purifying 1h-4h, filters, filtrate adjustment refined solution pH is 6.0-7.0, re-uses anionite-exchange resin and carries out purifying 1h-4h, first wash with water, after discarding water lotion, then be the hydrochloric acid wash-out of 0.5%-1% by concentration;
(6) elutriant utilizes Ultra filtration membrane to go out phosphopeptide caseinate, then uses nanofiltration membrane desalination, obtains the phosphopeptide caseinate liquid being less than 5000D;
(7) through lyophilize, spraying dry, vacuum drying any one, powder is dried to the product obtained.
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: chrymotrypsin=4:3:2:1, or trypsinase: Sumizyme MP: papoid: neutral protease=2:3:1:4, or neutral protease: Sumizyme MP: chrymotrypsin: 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.
The present invention adopts after the compound protease be made up of trypsinase, neutral protease, Sumizyme MP, chrymotrypsin, papoid carries out enzymolysis certain hour, use stomach en-enzymolysis again, and adopt first Zeo-karb to absorb purifying, re-use the purifying process that anionite-exchange resin carries out purifying, effectively can improve yield (>=17%) and the purity (phosphopeptide caseinate content >=90%) of phosphopeptide caseinate, obtained phosphopeptide caseinate is without bitter taste, the aqueous solution clear of good water solubility, 20%CPP, color is fine white powder.The molecular weight of milk-derived 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, 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 casein phosphopeptide, comprises the following steps:
In reactor, add pure water 150L, open and stir 200rpm, be heated to 50 DEG C, in reactor, slowly drop into casein 25Kg, pH is adjusted to 7.8; Drop into trypsinase: neutral protease: Sumizyme MP: chrymotrypsin=4:3:2:1(mass ratio) the compound protease 12.5g that is mixed.After enzymolysis 1h, feed liquid is cooled to 37 DEG C, pH is adjusted to 4.0, add 12.5g stomach en-, continue enzymolysis, after 1h, enzymolysis terminates.Adjust pH4.5 to go out enzyme, temperature is adjusted to 90 DEG C, is incubated 20 min.Use 732 Zeo-karbs to absorb purifying 1h, filter, filtrate adjustment refined solution pH is 7.0, uses 717 anionite-exchange resin to carry out purifying 1h, first washes with water, after discarding water lotion, then use the hydrochloric acid wash-out of 1%.Elutriant adjust pH 6.8, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains the phosphopeptide caseinate liquid being less than 5000D, then use the nanofiltration membrane desalination of 150D.Carry out spraying dry to filtrate, obtain phosphopeptide caseinate finished product 4.52kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, and the aqueous solution clear of good water solubility, 20%CPP.
embodiment 2: a kind of preparation of high-purity casein phosphopeptide, comprises the following steps:
In reactor, add pure water 200L, open and stir 200rpm, be heated to 45 DEG C, in reactor, slowly drop into casein 25Kg, pH is adjusted to 7.5; Drop into trypsinase: neutral protease: papoid: the compound protease 25g that chrymotrypsin=3:2:4:1 is mixed.After enzymolysis 1.5h, feed liquid is cooled to 40 DEG C, pH is adjusted to 5, add 25g stomach en-, continue enzymolysis, after 1h, enzymolysis terminates.Adjust pH4.6, temperature is adjusted to 80 DEG C, is incubated 30 min, go out enzyme.Cross the precipitation such as the high molecular weight protein that filters non-casein hydrolysis, supernatant liquor uses 001 × 7 Zeo-karb to absorb purifying 1h, filter, filtrate adjustment refined solution pH is 6.5, U.S. Amberlite IRA-400 anionite-exchange resin is used to carry out purifying 2h, first wash with water, after discarding water lotion, then use the hydrochloric acid wash-out of 1%.Elutriant adjust pH 6.8, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains the phosphopeptide caseinate liquid being less than 5000D, then use the nanofiltration membrane desalination of 150D.Carry out spraying dry to filtrate, obtain phosphopeptide caseinate finished product 4.75kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, and the aqueous solution clear of good water solubility, 20%CPP.
embodiment 3: a kind of preparation of high-purity casein phosphopeptide, comprises the following steps:
In reactor, add pure water 250L, open and stir 200rpm, be heated to 45 DEG C, in reactor, slowly drop into casein 25Kg, pH is adjusted to 8.5; Drop into trypsinase: Sumizyme MP: papoid: the compound protease 50g that neutral protease=2:3:1:4 is mixed.After enzymolysis 2h, feed liquid is cooled to 37 DEG C, pH is adjusted to 4.5, add 12.5g stomach en-, continue enzymolysis, after 1h, enzymolysis terminates.Adjust pH4.7, temperature is adjusted to 85 DEG C, is incubated 30 min, go out enzyme.Cross the precipitation such as the high molecular weight protein that filters non-casein hydrolysis, supernatant liquor uses ROHM AND HAAS AMBERJET UP6040 Zeo-karb to absorb purifying 1h, filter, filtrate adjustment refined solution pH is 7.0, German Lewatit M500 anionite-exchange resin is used to carry out purifying 2h, first wash with water, after discarding water lotion, then use the hydrochloric acid wash-out of 0.6%.Elutriant adjust pH 5.0, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains the phosphopeptide caseinate liquid being less than 5000D, then use the nanofiltration membrane desalination of 150D.Carry out vacuum-drying to filtrate, obtain phosphopeptide caseinate finished product 4.63kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, and the aqueous solution clear of good water solubility, 20%CPP.
embodiment 4:a preparation for high-purity casein phosphopeptide, comprises the following steps:
In reactor, add pure water 175L, open and stir 200rpm, be heated to 50 DEG C, in reactor, slowly drop into casein 25Kg, pH is adjusted to 8.0; Drop into neutral protease: Sumizyme MP: chrymotrypsin: the compound protease 37.5g that papoid=1:2:3:4 is mixed.After enzymolysis 1.5h, feed liquid is cooled to 37 DEG C, pH is adjusted to 4.5, add 25g stomach en-, continue enzymolysis, after 1h, enzymolysis terminates.Adjust pH4.6, temperature is adjusted to 85 DEG C, is incubated 30 min, go out enzyme.Cross the precipitation such as the high molecular weight protein that filters non-casein hydrolysis, supernatant liquor uses Mitsubishi WK60L Zeo-karb to absorb purifying 2h, filter, filtrate adjustment refined solution pH is 6.8, SA10AX anionite-exchange resin is used to carry out purifying 2h, first wash with water, after discarding water lotion, then use the hydrochloric acid wash-out of 0.8%.Elutriant adjust pH 6.0, adopts the ultra-filtration membrane ultrafiltration of 5000D, obtains the phosphopeptide caseinate liquid being less than 5000D, then use the nanofiltration membrane desalination of 150D.Carry out vacuum-drying to filtrate, obtain phosphopeptide caseinate finished product 4.7kg, CPP content >=90%, molecular weight is 1000D-5000D, without bitter taste, and the aqueous solution clear of good water solubility, 20%CPP.
In the present invention, compound protease and pepsic secondary enzymolysis, effectively can improve the yield of phosphopeptide caseinate.For this reason, the compound protease that adopts with single proteolytic enzyme and embodiment 1-4 of the present invention and Pepsin secondary enzymolysis have made simultaneous test.Result shows, use the single protease hydrolyzed of trypsinase, neutral protease, papoid or chrymotrypsin, the yield of phosphopeptide caseinate is respectively 16 %, 14.5%, 15.3% and 14.8 %, the method of two use 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 the phosphopeptide caseinate of the single protease hydrolyzed of use.
Adopt compound protease enzymolysis, the molecular weight of phosphopeptide caseinate (CPP) can also be made less.Experiment shows, single enzyme is hydrolyzed, and gained phosphopeptide caseinate main molecules amount section is 3000D-8000D, and adopt compound protease (CPP), phosphopeptide caseinate (CPP) yield is main molecules amount section 1000D-5000D.
The present invention's experiment shows, the exchange resin secondarily purified process of anions and canons Zeo-karb independent purification process regulating YIN and YANG ionic cation 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 the purity of independent anionite-exchange resin or the many phosphopeptide caseinate (CPP) of the independent purification process of Zeo-karb.
Claims (4)
1. a high-purity casein phosphopeptide preparation method, is characterized in that
,comprise the following steps:
(1) water of casein 6-10 times quality is added, stirring and dissolving under 40 DEG C of-50 DEG C of conditions;
(2) add compound protease and carry out enzymolysis, enzymatic hydrolysis condition is: enzyme concentration is the 0.05%-0.2% of casein quality, temperature of reaction 40 DEG C-50 DEG C, reaction times 1 h-2h, pH 7.0-9.0, described compound protease is trypsinase, neutral protease, Sumizyme MP and chymotrypsin enzyme mixture, or neutral protease, Sumizyme MP, the mixture of chrymotrypsin and papoid, after compound protease enzymolysis, add the stomach en-of the 0.05%-0.1% of casein quality again temperature 40 DEG C-50 DEG C, enzymolysis 1h-2h again under pH value 4.0-5.0,
(3) temperature is adjusted to 80 ~ 90 DEG C, is incubated 20 min-30 min, carries out sterilizing;
(4) adjust pH 4.0-5.0, crosses the high molecular weight protein precipitation filtering non-enzymolysis;
(5) supernatant liquor adjustment pH is 4.0-5.0, uses Zeo-karb to absorb purifying 1h-4h, filters, filtrate adjustment refined solution pH is 6.0-7.0, re-uses anionite-exchange resin and carries out purifying 1h-4h, first wash with water, after discarding water lotion, then be the hydrochloric acid wash-out of 0.5%-1% by concentration;
(6) elutriant utilizes Ultra filtration membrane to go out phosphopeptide caseinate, then uses nanofiltration membrane desalination, obtains the phosphopeptide caseinate liquid being less than 5000D;
(7) through lyophilize, spraying dry, vacuum drying any one, powder is dried to the product obtained.
2. high-purity casein phosphopeptide preparation method according to claim 1, is characterized in that
,in step (2), in described compound protease, trypsinase, neutral protease, Sumizyme MP and chrymotrypsin, or the quality proportioning of the mixture of neutral protease, Sumizyme MP, chrymotrypsin and papoid is 1 ~ 4:1 ~ 4:1 ~ 4:1 ~ 4:.
3. high-purity casein phosphopeptide preparation method according to claim 2, is characterized in that
,the quality proportioning of compound protease is
:trypsinase: neutral protease: Sumizyme MP: chrymotrypsin=4:3:2:1, or neutral protease: Sumizyme MP: chrymotrypsin: papoid=1:2:3:4.
4. high-purity casein phosphopeptide 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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| CN107557418A (en) * | 2016-06-30 | 2018-01-09 | 天津唐朝食品工业有限公司 | CPP extracting method |
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| CN111528480B (en) * | 2020-05-21 | 2023-07-04 | 南通大学 | Calcium nutrition supplement and preparation method thereof |
| CN117567586A (en) * | 2023-10-25 | 2024-02-20 | 广州菲勒生物科技有限公司 | Preparation method of hydrolyzed casein rich in sialylated peptide and application of hydrolyzed casein in regulating intestinal flora |
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| CN1232649C (en) * | 2003-12-16 | 2005-12-21 | 天津大学 | Process of continuous production of casein bioactive peptide by enzymolysis and filtering membrane concentration |
| CN101768551A (en) * | 2010-02-04 | 2010-07-07 | 南京工业大学 | Multipole immobilized enzyme film reaction device and method of preparing casein phosphopeptides by using reaction device |
| CN102187935B (en) * | 2010-03-19 | 2012-12-19 | 江南大学 | Preparation method of Casein Phosphopeptides (CPPs) and ACE inhibitory peptides |
| CN101812495A (en) * | 2010-04-29 | 2010-08-25 | 广州绿萃生物科技有限公司 | Process for preparing casein phosphopeptide |
| CN101948524A (en) * | 2010-09-02 | 2011-01-19 | 天津大学 | Multi-phosphopeptide-enriched casein hydrolyzate and preparation method thereof |
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