CN107557418A - CPP extracting method - Google Patents
CPP extracting method Download PDFInfo
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- CN107557418A CN107557418A CN201610514870.XA CN201610514870A CN107557418A CN 107557418 A CN107557418 A CN 107557418A CN 201610514870 A CN201610514870 A CN 201610514870A CN 107557418 A CN107557418 A CN 107557418A
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Abstract
The invention discloses a kind of CPP extracting method, comprise the following steps：Step (1)：Add water in enzymatic vessel, stirred under the conditions of 42~48 DEG C and 45~55r/min, then casein sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；Step (2)：Add compound protease to be digested, obtain CPP solution；The compound protease is at least three kinds in neutral proteinase, chrymotrypsin, flavor protease and papain；Enzymatic hydrolysis condition is：The amount for adding compound protease is the 0.5~5% of casein sodium phosphate quality, pH value is 6.8~7.2, reaction temperature is 42~48 DEG C, mixing speed is 45~55r/min, the reaction time is 4~6h；Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered CPP successively.This invention simplifies preparation technology, the preparation difficulty and production cost of industrialization production are reduced, and product CPP has higher calcium binding capacity.
Description
Technical field
The present invention relates to biologically active peptide technical field, more specifically, it relates to a kind of CPP extraction side
Method.
Background technology
In recent years, the polypeptide of existing many tool bioactivity is identified out through protein digestibility, and biologically active peptide is
One of focus of recent domestic Animal nutrition research.CPP (Casein Phosphope Ptides, CPPs)
It is using milk casein as raw material, by single or compound protease hydrolysis, then is obtained after being isolated and purified to hydrolysate
Natural physiological active peptide containing phosphoserine cluster.CPPs can be with the divalence such as calcium, iron, zinc, selenium in the small intestine condition of animal
Mineral ion combine, prevent to precipitate, strengthen enteral dissolvable mine material concentration, so as to promote intestinal mucosa to calcium, iron,
The absorption and utilization of zinc, selenium, especially calcium, are described as " mineral carrier ".CPPs is the activity of currently the only promotion calcium uptake
Peptide, while also played an important role improving immunity of organisms, improving reproductive performance etc..The country such as Japan, Germany, U.S. is
CPP is set to functional food, the research of China in this respect is also increasingly by scientist and food worker
Extensive concern.
Although early in the fifties, foreign countries have begun to the research to CPP, the research of preparation of industrialization
Work until in recent years just really start, and China CPP content low (highest CPPs contents are mostly 20% or so),
Product has the shortcomings that bitter taste, color are partially yellow.
Existing application publication number is a kind of CN103114118A patent document " method for preparing CPP ", is adopted
Protease is added in casein and carries out enzyme digestion reaction, goes out enzymolysis product CPP, product using Ultra filtration membrane
CPP obtains after NF membrane desalting processing.This method is simple to operate, and equipment requirement is low, high income, product quality
It is good, and reaction temperature is low, can realize industrialization production, but it uses single protease hydrolyzed, CPP yield and
Content relatively low (highest product content is 16% or so), product do not carry out de- hardship, and do not indicate specific casein phosphorus
Sour peptide components and structure, also hold calcium activity without whether the product that checking finally gives has.
Existing Authorization Notice No. is a kind of CN103571905B patent document " high-purity casein phosphopeptide preparation side
Method " (documents 1), it is using milk-derived casein as raw material, mainly by complex enzyme zymohydrolysis, separation, purifying, de- bitter, film mistake
The technique productions CPP such as filter, dry.
Existing application publication number is CN105085651A patent document " a kind of CPP monomer and its preparation side
Method " (documents 2), its using casein in cow's milk as raw material, mainly by trypsin digestion, enzyme deactivation, refined filtration, alcohol precipitation, from
The technique such as the heart, excessively sephadex column, medium pressure column chromatography, reverse phase silica gel purifying, dry prepares CPP monomer.
The complex process of CPP is prepared in documents 1 and documents 2, especially purification step adds
The preparation difficulty and production cost of industrialization production.
The content of the invention
In view of the deficienciess of the prior art, it is an object of the invention to provide a kind of CPP extracting method,
Simplify preparation technology, reduce the preparation difficulty and production cost of industrialization production, and product CPP have compared with
High calcium binding capacity.
To achieve the above object, the invention provides following technical scheme：
A kind of CPP extracting method, comprises the following steps：
Step (1)：Add water in enzymatic vessel, stirred under the conditions of 42~48 DEG C and 45~55r/min, then casein phosphoric acid
Sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The compound protease is neutrality
At least three kinds in protease, chrymotrypsin, flavor protease and papain；Enzymatic hydrolysis condition is：Add compound protein
The amount of enzyme is the 0.5~5% of casein sodium phosphate quality, pH value is 6.8~7.2, reaction temperature is 42~48 DEG C, mixing speed
It is 4~6h for 45~55r/min, reaction time；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide.
Preferably, in step (1), the mass ratio for adding casein sodium phosphate and water is 1 to the present invention：1.5~3.
Preferably, in step (1), the mass ratio for adding casein sodium phosphate and water is 1 to the present invention：2.
Preferably, in step (2), the amount for adding compound protease is casein sodium phosphate and water quality to the present invention
0.7~1.8%.
Preferably, in step (2), the reaction temperature of enzymolysis is 45 DEG C to the present invention.
Preferably, in step (2), the compound protease is chrymotrypsin, flavor protease and pawpaw to the present invention
Protease, the quality proportioning of the compound protease is chrymotrypsin：Flavor protease：Papain=1.5~3：1.5
~3：1.5~3.
Preferably, in step (2), the compound protease is neutral proteinase, flavor protease and pawpaw to the present invention
Protease, the quality proportioning of the compound protease is neutral proteinase：Flavor protease：Papain=1.5~3：1.5
~3：1.5~3.
Preferably, in step (2), the compound protease is neutral proteinase, chrymotrypsin, pawpaw egg to the present invention
White enzyme, the quality proportioning of the compound protease is neutral proteinase：Chrymotrypsin：Papain=1.5~3：1.5~
3：1.5~3.
Preferably, in step (2), the compound protease is neutral proteinase, chrymotrypsin and flavor to the present invention
Protease, the quality proportioning of the compound protease is neutral proteinase：Chrymotrypsin：Flavor protease=1.5~3：1.5
~3：1.5~3.
Preferably, in step (2), the compound protease is neutral proteinase, chrymotrypsin, flavor egg to the present invention
White enzyme and papain, the quality proportioning of the compound protease is neutral proteinase：Chrymotrypsin：Flavor protease：
Papain=1.5~3：1.5~3：1.5~3：1.5~3.
Compared with prior art, the present invention has the advantages that：
(1) present invention its using casein sodium phosphate as raw material, mainly by the technique productions casein phosphorus such as complex enzyme zymohydrolysis, drying
Sour peptide, but preparation technology is a simplified, reduce the preparation difficulty and production cost of industrialization production, and product casein phosphoric acid
Peptide has higher calcium binding capacity.
(2) the product CPP of present invention gained is 25%CPPs water without bitter taste, good water solubility, mass fraction
Solution clear, color are fine white powder, and molecular weight is small, and phosphoserine residue is more, and calcium binding capacity is high.
(3) junket of the present invention is detected by physiologically active inside protein phosphatase polypeptide, equally has higher calcium absorptivity.
Embodiment
The embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
Casein sodium phosphate in the present invention is purchased by Fonterra Co-operative Group (Auckland, NZL)
.
The present invention, equipment is dish-style high-speed centrifuge used by step (3) high speed centrifuges, and rotating speed is
7500r/min。
Embodiment one：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 42 DEG C and 50r/min, then 33.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For chrymotrypsin：Flavor protease：Papain=1.5：3：2；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 0.5%, pH value 6.8, reaction temperature be 42 DEG C, mixing speed 50r/min, reaction time be
6h；Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment two：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 45 DEG C and 45r/min, then 66.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For chrymotrypsin：Flavor protease：Papain=3：2：1.5；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 2.5%, pH value 7, reaction temperature be 45 DEG C, mixing speed 45r/min, reaction time 5h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment three：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 48 DEG C and 55r/min, then 50Kg casein phosphorus
Sour sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For chrymotrypsin：Flavor protease：Papain=2：1.5：3；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 5%, pH value 7.2, reaction temperature be 48 DEG C, mixing speed 55r/min, reaction time 4h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Example IV：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 42 DEG C and 50r/min, then 33.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Flavor protease：Papain=1.5：3：2；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 0.5%, pH value 6.8, reaction temperature be 42 DEG C, mixing speed 50r/min, reaction time be
6h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment five：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 45 DEG C and 45r/min, then 66.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Flavor protease：Papain=3：2：1.5；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 2.5%, pH value 7, reaction temperature be 45 DEG C, mixing speed 45r/min, reaction time 5h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment six：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 48 DEG C and 55r/min, then 50Kg casein phosphorus
Sour sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Flavor protease：Papain=2：1.5：3；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 5%, pH value 7.2, reaction temperature be 48 DEG C, mixing speed 55r/min, reaction time 4h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment seven：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 42 DEG C and 50r/min, then 33.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Papain=1.5：3：2；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 0.5%, pH value 6.8, reaction temperature be 42 DEG C, mixing speed 50r/min, reaction time be
6h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment eight：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 45 DEG C and 45r/min, then 66.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Papain=3：2：1.5；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 2.5%, pH value 7, reaction temperature be 45 DEG C, mixing speed 45r/min, reaction time 5h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment nine：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 48 DEG C and 55r/min, then 50Kg casein phosphorus
Sour sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Papain=2：1.5：3；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 5%, pH value 7.2, reaction temperature be 48 DEG C, mixing speed 55r/min, reaction time 4h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment ten：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 45 DEG C and 50r/min, then 33.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease=1.5：3：2；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 0.5%, pH value 6.8, reaction temperature be 45 DEG C, mixing speed 50r/min, reaction time be
6h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment 11：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 48 DEG C and 45r/min, then 66.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease=3：2：1.5；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 2.5%, pH value 7, reaction temperature be 48 DEG C, mixing speed 45r/min, reaction time 5h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment 12：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 42 DEG C and 55r/min, then 50Kg casein phosphorus
Sour sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease=2：1.5：3；Enzymatic hydrolysis condition is：The amount for adding compound protease is junket
Protein phosphatase sodium quality 5%, pH value 7.2, reaction temperature be 42 DEG C, mixing speed 55r/min, reaction time 4h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment 13：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 42 DEG C and 50r/min, then 33.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease：Papain=1.5：3：2：1.5；Enzymatic hydrolysis condition is：Add multiple
The amount of hop protein enzyme is the 0.5% of casein sodium phosphate quality, pH value 6.8, reaction temperature are 42 DEG C, mixing speed 50r/
Min, reaction time 6h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment 14：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 45 DEG C and 45r/min, then 66.5Kg caseins
Sodium phosphate is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease：Papain=2：1.5：3：2；Enzymatic hydrolysis condition is：Add compound
The amount of protease is the 2.5% of casein sodium phosphate quality, pH value 7, reaction temperature are 45 DEG C, mixing speed 45r/min,
Reaction time is 5h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment 15：CPP extracting method, comprises the following steps：
Step (1)：100Kg water is added in enzymatic vessel, stirred under the conditions of 48 DEG C and 55r/min, then 50Kg casein phosphorus
Sour sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The quality proportioning of compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease：Papain=3:2:1.5:3；Enzymatic hydrolysis condition is：Add compound
The amount of protease is the 5% of casein sodium phosphate quality, pH value 7.2, reaction temperature are 48 DEG C, mixing speed 55r/min,
Reaction time is 4h；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide, CPPs content >=85%, molecular weight are 1000~1750Da, no bitter taste, good water solubility, mass fraction 25%CPPs
Aqueous solution clear.
Embodiment two, five, eight, ten and 14 is chosen, and makes comparative example one and two, gained CPP is carried out
Performance evaluation.
Comparative example one：Use Authorization Notice No. for the embodiment three in CN103571905B documents 1 as a comparison
Example one.
Add pure water 250L into reactor, open stirring 200rpm, be heated to 45 DEG C, casein is slowly put into reactor
25Kg, pH is adjusted to 8.5；Put into trypsase:Alkali protease:Papain:Neutral proteinase=2:3:1:4 mixing systems
Into compound protease 50g.After digesting 2h, feed liquid is cooled to 37 DEG C, pH is adjusted to 4.5, adds 12.5g pepsins, after
It is continuous to digest, digest and terminate after 1h.PH=4.7 is adjusted, by temperature adjustment to 85 DEG C, is incubated 30min, enzyme deactivation.It is filtered to remove and does not digest
The high molecular weight proteins such as casein are precipitated, and supernatant is absorbed pure using ROHM AND HAAS AMBERJET UP6040 cationic ion-exchange resins
Change 1h, filtering, filtrate adjustment refined solution pH is 7.0, and purifying 2h is carried out using German Lewatit M500 anion exchange resin,
First be washed with water, after discarding water lotion, then with 0.6% hydrochloric acid elute.Eluent adjusts pH value 5.0, is surpassed using 5000D milipore filter
Filter, yield less than 5000D CPP liquid, then the NF membrane desalination with 150D.Filtrate is dried in vacuo, obtained
CPP finished product 4.63Kg, CPP content >=90%, molecular weight 1000D-5000D, no bitter taste, good water solubility, 20%
CPP aqueous solution clear.
Comparative example two：Use application publication number for the embodiment one in CN105085651A documents 2 as a comparison
Example two.
(1) add pure water 4000mL into beaker, open stirring 200rpm, casein 1kg is slowly put into beaker, at 40 DEG C
Under the conditions of stirring and dissolving.Add trypsase to be digested, enzymatic hydrolysis condition is：Enzyme concentration is the 0.1% of casein quality, is reacted
40 DEG C of temperature, reaction time 1h, pH=7.0.By temperature adjustment to 80 DEG C, 30min is incubated, carries out enzyme deactivation sterilizing.
(2) pH value 4.0 is adjusted, is filtered to remove high molecular weight protein.Filtrate adjusts pH value 5.0, crosses the refined filtration dress of 0.2 μm of specification
Put, further remove macromolecular substances.Refined filtration liquid adjusts pH value 6.0, crosses 300Da NF membranes, removes some small molecules and salt,
Concentrated after reverse osmosis membrane, obtain concentrate.
(3) concentrate regulation pH value 5.0,5 DEG C of temperature, ethanol is added, regulation concentration of alcohol to 40%, stands 6h, from
The heart or filtering, collect precipitation.
(4) G25 sephadex columns are crossed after precipitation is dissolved with water, are eluted with water, respectively 50mL before collection eluent, 50~
100mL, 100~200mL, freezed after concentration.Powder dissolved QuikSep-100 medium pressure column chromatography column systems, middle compression leg self-chambering,
Middle pressure elution requirement：Flow velocity 20mL/min；Detection wavelength：215nm；Sample size：5mL；Mobile phase：A pumps ultra-pure water (contains 0.1%
(v/v) TFA), B pumps acetonitrile (contains 0.1% (v/v) TFA), and B pumps initial concentration is 10%；Type of elution：Binary gradient elutes；Wash
De- gradient：0.01min~35min, 10%~20%；35.01min~125min, 20~27%；125.01min~140min,
27%~90%；140.01min~155min, 90%~90%, 155.01min~165min, 90%~10%.Repeat
Sample, CPP eluting peak is collected, be concentrated in vacuo removing organic solvent after collecting eluting peak, freeze-drying, obtain preliminary
The CPP of purifying.Powder is dissolved with water, adjusts pH to 5.0, adds ethanol, adjusts concentration of alcohol 50%, centrifugation
Take precipitation.
(5) reverse phase silica gel purifies.Precipitation dissolving carries out reverse phase silica gel purifying, HPLC system again：LC-8A Shimadzus prepare efficient
Liquid chromatograph；Chromatographic column：HW-0121；Chromatographic condition：Flow velocity 10mL/min；Detection wavelength：215nm；Sample size：1mL；Stream
Dynamic phase：A pumps ultra-pure water (contains 0.1% (v/v) TFA), and B pumps acetonitrile (contains 0.1% (v/v) TFA), and B pumps initial concentration is 5%；Wash
Off-square formula：Binary gradient elutes；Gradient：0.01min~25min, 5%~18%；25.01min~85min, 18~
30%；85.01min~95min, 30%~70%；95.01min~120min, 70%~70%, 120.01min~
150min, 70%~5%.Sample introduction is repeated, collects CPP eluting peak.Vacuum concentration removing is organic after collecting eluting peak
Solvent, freeze-drying, the CPP purified.
(6) reverse phase silica gel purifies again.HPLC system：LC-15C Shimadzu high performance liquid chromatographs；Chromatographic column：C18,AQ-C18,5 μm, 4.6 × 250mm；Chromatographic condition：Flow velocity：1mL/min；Detection wavelength：215nm；Sample size：20
μL；Mobile phase：A pumps ultra-pure water (contains 0.1% (v/v) TFA), and B pumps acetonitrile (contains 0.1% (v/v) TFA), and B pump initial concentrations are
10%；Type of elution：Binary gradient elutes；Gradient 0~15min, 10%~20%；15~50min, 20%~27%；
50.01min~60min, 90%；60.01min~70min, 10%.Sample introduction is repeated, collects CPP eluting peak.Receive
It is concentrated in vacuo after collection CPP eluting peak, removes organic solvent, freeze-drying, obtain CPP monomer.
1st, the iii vitro chemical analysis of CPP
With the legal nitrogen of kjeldahl determination；Determine phosphorus with Subbarow2Fiske methods；Relative molecular mass is surveyed with gel filtration
(SephadexG250, post specification are 100cm × 1.5cm), sample with 0.025mol/L KCl-0.2mol/L HAc solution with
0.2mL/min speed elution；CPPs external calcium binding capacity is surveyed with pH titrations, i.e., is added in the reactor in right amount
NaH2PO4And CPP, NaH2PO4Ultimate density control in 0.008mol/L, CPPs ultimate density 0.1g/L.Reactor is placed in
25 DEG C of water-baths, add the CaCl that ultimate density is 0.008mol/L2, reaction system is adjusted to pH with 0.1mol/L NaOH immediately
=7.2, and 0.1mol/L NaOH are constantly added dropwise, reaction solution pH is maintained at 7.2, since the timing adjusting pH, pH be adjusted to 7.2
2min is about needed, 0.1mol/L NaOH consumption is continuously recorded since 2min.Reaction equation is：
Ca(H2PO4)2→CaHPO4+H+
3CaHPO4→Ca3(PO4)2↓+H PO4 2++2H+
Mole N/P ratio of the CPP of table 1.1
| (N/CPPs)/(g·g-1) | (P/CPPs)/(g·g-1) | (N/P)/(mol·mol-1) | |
| Embodiment two | 0.012 | 0.0046 | 5.78 |
| Embodiment five | 0.084 | 0.0279 | 6.67 |
| Embodiment eight | 0.022 | 0.0069 | 7.01 |
| Embodiment ten | 0.023 | 0.0093 | 5.48 |
| Embodiment 14 | 0.040 | 0.0131 | 6.83 |
| Comparative example one | 0.143 | 0.0089 | 35.58 |
| Comparative example two | 0.029 | 0.0095 | 6.79 |
The relative molecular mass of the CPP of table 1.2
| Relative molecular mass | |
| Embodiment two | 1466 |
| Embodiment five | 1593 |
| Embodiment eight | 1653 |
| Embodiment ten | 1278 |
| Embodiment 14 | 1387 |
| Comparative example one | 3763 |
| Comparative example two | 1350 |
The external calcium binding capacity of the CPP of table 1.3
The phosphoserine residue that the numerical value of mole N/P ratio is smaller to show CPP is more, and relative molecular mass represents
The molecular size of CPP.By embodiment two, five, eight, ten, 14 and comparative example it can be seen from table 1.1~1.3
Mole N/P ratio, relative molecular mass and the external calcium binding capacity of two gained CPPs are more or less the same, i.e., by embodiment
2nd, five, eight, ten, 14 and the gained CPP of comparative example two phosphoserine residue quantity and molecular size difference
Less, and with similar external calcium binding capacity.But to be far superior to the preparation of comparative example two by the preparation technology of the present invention
Technique, this invention simplifies preparation technology, reduce the preparation difficulty and production cost of industrialization production, and product casein phosphorus
Sour peptide has higher calcium binding capacity, but the preparation technology of comparative example two is applied only for preparing in a small amount, it is impossible to realizes industrialization
Production.
By mole N/P ratio of the CPP of the gained of embodiment two, five, eight, ten, 14, relative molecular mass and
External calcium binding capacity will be far superior to the CPP obtained by comparative example one, and this invention simplifies preparation technology, drop
The low preparation difficulty and production cost of industrialization production.
2nd, physiologically active detects inside CPP
40 mouse are randomly divided into 8 groups, each group mouse is raised precedently two weeks, ad lib basal feed, drinking public water supply.So
Afterwards the casein that quantitive feeding is obtained by embodiment two, five, eight, ten and 14 and comparative example one and two is distinguished daily for first 7 groups
Phosphoeptide, the 8th group is blank control.Start to collect mouse excrement after CPP sample 15d is fed with, continuously collect 5d, together
The food ration and excretion of mouse in Shi Jilu 5d.With atomic absorption spectrum measuring instrument to the calcium content in basal feed and mouse excrement
It is measured and (is shown in Table 2), calcium absorptivity=(intake calcium-excretion calcium)/intake calcium.
The calcium absorptivity of each group mouse of the feeding CPP of table 2
| Take in calcium/g | Drain calcium/g | Absorb calcium/g | Calcium absorptivity/% | |
| Embodiment two | 0.66 | 0.30 | 0.36 | 54.5 |
| Embodiment five | 0.69 | 0.32 | 0.37 | 53.6 |
| Embodiment eight | 0.62 | 0.30 | 0.32 | 51.6 |
| Embodiment ten | 0.62 | 0.27 | 0.35 | 56.0 |
| Embodiment 14 | 0.74 | 0.35 | 0.39 | 52.7 |
| Comparative example one | 0.69 | 0.45 | 0.24 | 34.8 |
| Comparative example two | 0.76 | 0.36 | 0.40 | 52.6 |
| Blank control | 0.74 | 0.52 | 0.22 | 29.7 |
As can be seen from Table 2, had by embodiment two, five, eight, ten, 14 and the gained CPP of comparative example two close
Calcium absorptivity, to be far longer than by right by the calcium absorptivity of the CPP of the gained of embodiment two, five, eight, ten, 14
The calcium absorptivity for the CPP that ratio one obtains, this experimental result and above-mentioned experimental result match.
Therefore, this invention simplifies preparation technology, the preparation difficulty and production cost of industrialization production, and product are reduced
CPP has higher calcium binding capacity.
Described above is only the preferred embodiment of the present invention, still, during the present invention is not limited to the above-described embodiments
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, these
Simple variant belongs to the protection domain of the present invention.
Claims (10)
1. a kind of CPP extracting method, it is characterised in that comprise the following steps：
Step (1)：Add water in enzymatic vessel, stirred under the conditions of 42~48 DEG C and 45~55r/min, then casein phosphoric acid
Sodium is added in enzymatic vessel, obtains casein sodium phosphate suspension；
Step (2)：Add compound protease to be digested, obtain CPP solution；The compound protease is neutrality
At least three kinds in protease, chrymotrypsin, flavor protease and papain；Enzymatic hydrolysis condition is：Add compound protein
The amount of enzyme is the 0.5~5% of casein sodium phosphate quality, pH value is 6.8~7.2, reaction temperature is 42~48 DEG C, mixing speed
It is 4~6h for 45~55r/min, reaction time；
Step (3)：CPP solution by being centrifuged at a high speed and being spray-dried, obtains powdered casein successively
Phosphoeptide.
2. CPP extracting method according to claim 1, it is characterised in that in step (1), add casein
The mass ratio of sodium phosphate and water is 1：1.5~3.
3. CPP extracting method according to claim 1, it is characterised in that in step (1), add casein
The mass ratio of sodium phosphate and water is 1：2.
4. CPP extracting method according to claim 1, it is characterised in that in step (2), add compound egg
The amount of white enzyme is the 0.7~1.8% of casein sodium phosphate and water quality.
5. CPP extracting method according to claim 1, it is characterised in that in step (2), the reaction of enzymolysis
Temperature is 45 DEG C.
6. CPP extracting method according to claim 1, it is characterised in that in step (2), the compound egg
White enzyme is chrymotrypsin, flavor protease and papain, and the quality proportioning of the compound protease is chrymotrypsin：
Flavor protease：Papain=1.5~3：1.5~3：1.5~3.
7. CPP extracting method according to claim 1, it is characterised in that in step (2), the compound egg
White enzyme is neutral proteinase, flavor protease and papain, and the quality proportioning of the compound protease is neutral proteinase：
Flavor protease：Papain=1.5~3：1.5~3：1.5~3.
8. CPP extracting method according to claim 1, it is characterised in that in step (2), the compound egg
White enzyme is neutral proteinase, chrymotrypsin, papain, and the quality proportioning of the compound protease is neutral proteinase：
Chrymotrypsin：Papain=1.5~3：1.5~3：1.5~3.
9. CPP extracting method according to claim 1, it is characterised in that in step (2), the compound egg
White enzyme is neutral proteinase, chrymotrypsin and flavor protease, and the quality proportioning of the compound protease is neutral proteinase：
Chrymotrypsin：Flavor protease=1.5~3：1.5~3：1.5~3.
10. CPP extracting method according to claim 1, it is characterised in that described compound in step (2)
Protease is neutral proteinase, chrymotrypsin, flavor protease and papain, the quality proportioning of the compound protease
For neutral proteinase：Chrymotrypsin：Flavor protease：Papain=1.5~3：1.5~3：1.5~3：1.5~3.
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| CN110483622A (en) * | 2019-08-26 | 2019-11-22 | 无限极（中国）有限公司 | Casein phosphopeptide and preparation method thereof, application |
| CN114958951A (en) * | 2022-07-07 | 2022-08-30 | 卫仕宠物营养科学研究院(江苏)有限公司 | Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110483622A (en) * | 2019-08-26 | 2019-11-22 | 无限极（中国）有限公司 | Casein phosphopeptide and preparation method thereof, application |
| 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 |
| CN114958951A (en) * | 2022-07-07 | 2022-08-30 | 卫仕宠物营养科学研究院(江苏)有限公司 | Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats |
| CN114958951B (en) * | 2022-07-07 | 2024-04-09 | 卫仕宠物营养科学研究院(江苏)有限公司 | Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats |
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