CN108611390A - A method of preparing low bitter taste buffalo's milk casein antioxidant peptide powder - Google Patents
A method of preparing low bitter taste buffalo's milk casein antioxidant peptide powder Download PDFInfo
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- CN108611390A CN108611390A CN201810373257.XA CN201810373257A CN108611390A CN 108611390 A CN108611390 A CN 108611390A CN 201810373257 A CN201810373257 A CN 201810373257A CN 108611390 A CN108611390 A CN 108611390A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
Abstract
The invention discloses a kind of method preparing low bitter taste buffalo's milk casein antioxidant peptide powder, this method includes：Fresh buffalo's milk is taken, after degreasing, precipitation and purification process, obtains buffalo milk casein；Buffalo's milk caseic aqueous solution is prepared, it is denatured by boiling, then into buffer solution after addition animal hydrolyzed protein enzyme progress primary enzymolysis, add Prote AX protease and carry out secondary enzymolysis, boil enzyme deactivation after enzymolysis, obtain buffalo's milk casein enzymolysis liquid；The animal hydrolyzed protein enzyme is purchased from Nanning Pang Bo Co., Ltds；For the buffalo's milk casein enzymolysis liquid successively through centrifuging with after hyperfiltration treatment, freeze-drying obtains buffalo's milk casein antioxidant peptide powder.The present invention detaches from fresh buffalo's milk, purifies and obtains buffalo milk casein, and two-stage enzymolysis is carried out to buffalo milk casein using animal hydrolyzed protein enzyme and Prote AX protease, obtain the buffalo's milk casein antioxidant peptide powder that nitrogen recovery is high, molecular weight is small, antioxidant activity is strong and bitter taste is low.
Description
Technical field
The present invention relates to dairy products technical field more particularly to a kind of preparing low bitter taste buffalo's milk casein antioxidant peptide powder
Method.
Background technology
Breast is one of food with the longest history in human diet, and milk and milk products not only provide to human body abundant
Nutrition, and be the important sources of biologically active peptide.Casein is the mixed protein that content is most abundant in lactoprotein, content
Account for 80% or so of newborn total protein.Buffalo is distributed across a kind of Important Economic herding kind of south China, with the means of production
Be constantly progressive, new century buffalo has been gradually transformed into the development trend of " breast use based on, supplemented by meat labour ", and buffalo's milk became already
One new industry.Protein content is up to 4.3% in buffalo's milk, and wherein essential amino acids content is high-quality to prepare more than 30%
Newborn source activity peptide provides raw material guarantee.
In recent years, it is increasingly paid close attention to by people about the peptide fragment with physiological activity contained in bovine casein.It is small
Transhipment, absorption and utilization rate of the molecular peptide in enteron aisle are better than single amino acid, and by its different amino acid range side
Formula can have different physiological activity, such as participate in immunological regulation in vivo, remove free radical, adjust blood pressure balance, antithrombotic
With strengthen bone health etc..Wherein antioxidation active peptides are a studied wide directions, in a variety of breasts and breast
Different anti-oxidant segments is identified in product, contains abundant hydrophobic amino acid and basic amine group in buffalo milk casein
Acid is the quality raw materials for preparing anti-oxidation peptide.
Production problem urgently to be resolved hurrily in the always active peptide product of bitter taste caused by enzymolysis.Monomeric hydrophobic acidic amino acid
Itself does not have bitter taste, but the increase of hydrophobic amino acid ratio obviously can be such that bitter taste increases in small peptide, reduce the receiving of consumer
Degree.The mode that addition adsorbent or embedding medium are often used in industry removes or so that bitter taste is reduced in conjunction with hydrophobic amino acid, but this
A little modes can bring necessary amino acid loss, the problems such as reducing the nutritive value of cow's milk itself, improve finished product.
Notification number is that the application for a patent for invention of CN1546682 discloses a kind of enzymolysis and membrane filtration is integrated continuously produces casein
The technique of biologically active polypeptide, the technique is using casein as raw material, in ultrafiltration, the nanofiltration by enzymatic vessel and different molecular weight cut off
In the multistage enzyme mebrane reactor that film is composed, through at least one albumen enzyme effect, the bioactivity with multiple functions is made
Polypeptide；Wherein, protease is pancreatin, trypsase, pepsin, neutral proteinase, alkali protease, papain, spinach
One kind in trailing plants protease, carboxypeptidase or compounding.
Had to reducing polypeptide products bitter taste by the way of endopeptidase and peptide ending enzyme (aminopeptidase and carboxypeptidase) synergetic hydrolysis
Positive effect.First with the strong endoproteolytic enzyme r e lease small-molecular peptides of hydrolysis ability, followed by peptide ending enzyme cut-out bitter peptides C
The hydrophobic amino acid at end or the basic amino acid of N-terminal discharge single amino acid, to achieve the effect that reduce bitter taste.Double
By the condition difference that different protease are suitble to during enzyme synergetic hydrolysis, constantly regulate soda acid and temperature are needed, in reality
It also needs to increase desalination process in production, does not meet energy-saving and environment-friendly industry trends.
Therefore, it finds hydrolysis ability by force and the matched de- bitter protease of hydrolysis environment is most important for active peptide industry
's.
Invention content
The present invention provides a kind of methods preparing low bitter taste buffalo's milk casein antioxidant peptide powder, are prepared using this method
Not only nitrogen recovery is high for the casein small peptide of acquisition, and molecular weight is low, and antioxidant activity is strong, free to iron atom reduction rate and ABTS
The clearance rate of base is higher, and can significantly reduce the bitter taste of product, is conducive to the popularization of product.
Specific technical solution is as follows：
A method of low bitter taste buffalo's milk casein antioxidant peptide powder is prepared, including：
(1) fresh buffalo's milk is taken, after degreasing, precipitation and purification process, obtains buffalo milk casein；
(2) prepare buffalo's milk caseic aqueous solution, it is denatured by boiling, then into buffer solution be added animal hydrolyzed protein enzyme into
After row primary enzymolysis, adds Prote AX protease and carry out secondary enzymolysis, boil enzyme deactivation after enzymolysis, obtain buffalo cheese
For protein enzymatic hydrolyzate (3) the buffalo's milk casein enzymolysis liquid successively through centrifuging with after hyperfiltration treatment, freeze-drying obtains buffalo's milk
Casein antioxidant peptide powder.
Animal hydrolyzed protein enzyme used in the present invention is purchased from Nanning Pang Bo Co., Ltds, mainly by alkali protease,
Neutral proteinase, papain, excision enzyme and food flavor enzyme compound, and applicable elements are extensive, and restriction enzyme site is more and specific aim
It is not strong, it is energy saving and environmentally friendly without adjusting pH value, thus the step of reducing follow-up desalination in use.
Prote AX protease used in the present invention is purchased from Japanese amano enzyme company, is that one kind being derived from aspergillus oryzae
Aminopeptidase made of (Aspergillus oryzae) fermentation is refined, is suitble to partial neutral condition to use, and exopeptidase vigor is
1400U/g。
Suitable centrifugal condition can improve the purity of gained buffalo milk casein with efficient removal fat.Preferably, step
Suddenly in (1), the temperature of the degreasing is 3~6 DEG C, and rotating speed is 3500~5000r/min, and the time is 10~15min.
Enzymolysis efficiency can be improved in suitable albumen concentration, increases nitrogen recovery, and albumen concentration is too low to increase manufacturing cost,
The too high protease that is unfavorable for of albumen concentration efficiently digests.Preferably, in step (2), the buffalo's milk caseic aqueous solution
In, the mass fraction of buffalo milk casein is 2~5%.
Preferably, in step (2), the denatured by boiling temperature is 90~100 DEG C, and the time is 10~15min.High temperature
The stereochemical structure that protein can be destroyed, exposes peptide chain, is conducive to protease digestion, hence it is evident that improves digesting efficiency.
The additive amount of animal hydrolyzed protein enzyme is to the molecular size range of nitrogen recovery, lactalbumin anti-oxidation peptide and anti-oxidant
Performance has an impact.Preferably, in step (2), in terms of 1g caseins, animal hydrolyzed protein enzyme described in buffer solution adds
Dosage is 0.01~0.03g.
Different animals protease hydrolyzed condition can obtain the enzymolysis liquid of different final state pH, suitable protease additive amount and enzyme
Solution condition can get the enzymolysis liquid that the follow-up Prote AX protease of optimum takes off hard work sequence.Preferably, in step (2), it is described
The temperature of primary enzymolysis is 50~60 DEG C, and the time is 2~4h.The enzymatic hydrolysis condition can efficiently play proteinase activity, improve more
Peptide yield.
The additive amount of Prote AX protease can have a significant effect to the bitter taste of lactalbumin anti-oxidation peptide, meanwhile, Prote
AX has certain endopeptidase activity, can further scinderin matter and polypeptide, influence nitrogen recovery, peptide molecular weight and anti-oxidant
Ability.Preferably, in step (2), in terms of 1g caseins, the additive amount of the protease of Prote AX described in buffer solution is
0.005~0.02g.
Preferably, in step (2), the temperature of the secondary enzymolysis is 40~60 DEG C, and the time is 2~4h.The enzymolysis item
Part can efficiently play proteinase activity, improve polypeptide yield, reduce Gly-His-Lys bitter taste.
Preferably, in step (3), the rotating speed of the centrifugation is 6000~8000r/min, and the time is 10~20min；It can
With isolated polypeptide and insoluble protein as far as possible, polypeptide yield is improved.
Preferably, in step (3), the interception of the ultrafiltration is 6000~10000Da, and pressure is 0.4~0.6MPa.
Ultrafiltration can remove a small number of residual lipids and high molecular weight protein and polypeptide, can effectively remove impurity, obtain limpid pure peptide liquid,
Improve the antioxygenic property of product.
Compared with prior art, the invention has the advantages that：
(1) present invention detaches from fresh buffalo's milk, purifies and obtain buffalo milk casein, and using animal hydrolyzed protein enzyme and
Prote AX protease carries out two-stage enzymolysis to buffalo milk casein, obtains that nitrogen recovery is high, molecular weight is small, antioxidant activity is strong
And the buffalo's milk casein antioxidant peptide powder that bitter taste is low.
(2) the method for the present invention is not necessarily to adjust pH and adjusts temperature repeatedly, only needs a step enzyme deactivation, not only green energy conservation,
And follow-up desalting steps are reduced, it is easy to industrial continuous large-scale production.
(3) the method for the present invention de-bittering effect is excellent, and Gly-His-Lys product sensory is good, stay in grade, can add directly as polypeptide
Agent is added to be applied in other products.
Specific implementation mode
With reference to specific embodiment, the invention will be further described, and what is be exemplified below is only the specific implementation of the present invention
Example, but protection scope of the present invention is not limited only to this.
Iron atom reduction rate, ABTS free radical scavenging activities, nitrogen recovery involved in the following example and molecular weight distribution
Assay method it is as follows：
(1) iron ion reducing power measures：
1. FRAP preparation of reagents：By the hac buffer of 0.1mol/L pH 3.6,10mmol/L TPTZ solution,
The liquor ferri trichloridi of 20mmoL/L presses 10:1:1 preparation.
2. being separately added into 0.1mL candidate polypeptides liquid (1mg/mL) and 1.9mL FRAP reagents in 5mL centrifuge tubes, shake up anti-
It answers after 10min with the light absorption value A under UV spectrophotometer measuring its 593nmj；
3. being separately added into 0.1mL pure water and 1.9mL FRAP reagents in 5mL centrifuge tubes, purple is used after shaking up reaction 10min
Outer spectrophotometer detects the light absorption value A under its 593nmi；
4. iron ion reducing power following formula result of calculation：
(2) ABTS free radical scavenging activities measure：
1. prepared by ABTS free radicals：It is a concentration of that the potassium persulfate solution of 440 a concentration of 140mmoL/L of μ L is added to 25mL
In the ABTS solution of 7mmoL/L, mixing is settled to 100mL after being protected from light 12h.
2. being separately added into 0.1mL pure water and 1.9mL ABTS reaction solutions in 5mL centrifuge tubes, mixing shakes up reaction
The light absorption value A under its 734nm of UV spectrophotometer measuring is used after 10min0；
3. being separately added into 0.1mL candidate polypeptides liquid (1mg/mL) and 1.9mL ABTS reaction solutions in 5mL centrifuge tubes, mix
The light absorption value A under its 734nm of UV spectrophotometer measuring is used after even reaction 10min1；
4. with following formula result of calculation：
(3) nitrogen recovery measures：The nitrogen percentage measured respectively by Kjeldahl's method in buffalo milk casein and Gly-His-Lys contains
Amount is respectively N1And N2。
Nitrogen recovery is calculate by the following formula：
(4) small molecular weight titanium assay：Using GE AKTApure protein purification instrument, it is equipped with Superdex Peptide
10/300GL chromatographic columns, mobile phase are 30% (V/V) acetonitrile solution containing 0.1% (V/V) TFA, flow velocity 0.5mL/min, detection
Wavelength is 215nm.The polypeptide solution of 2mg/mL is prepared, 100 μ L of sample introduction after 0.22 μm of water system filter membrane are crossed.Molecular weight standards are selected
Aprotinin (6511Da), bacitracin (1422Da), reduced glutathione (614Da) and tyrosine (181Da).The molecule of Gly-His-Lys
It is small-molecular peptides that amount, which is distributed within the scope of 200~1500Da, and the ratio of small molecular weight titanium is determined using different molecular weight standard items.
(5) subjective appreciation：Subjective appreciation is carried out using the peptide liquid of Gly-His-Lys and 10mg/mL.Selecting 8 has subjective appreciation base
Assessment officer's (4 male 4 female, 22~25 years old age) of plinth, the bitter taste, color and luster and solubility of Gly-His-Lys and peptide liquid are carried out according to grade form
It judges, for wherein bitterness value using the tanning solution of various concentration as evaluation criteria, 50mmol/L tanning solutions are faint bitter taste,
100mmol/L tanning solutions are apparent bitter taste, and 200mmol/L tanning solutions are strong bitterness.Summarize 8 parts of scorings, items are respectively
It is averaged as product score after removing best result and minimum point.
1 subjective appreciation table of table
Embodiment 1
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 55 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.02g albumen is added by per 1g caseins
The ratio of enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 3h, and not enzyme deactivation after primary enzymolysis continues to add by every 1g caseins
Add the ratio of 0.015g protease to add Prote AX protease, 55 DEG C of constant temperature is kept to digest 3h, after digesting twice, boils
Enzyme deactivation obtains buffalo's milk casein enzymolysis liquid.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.6MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Embodiment 2
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 2% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.01g albumen is added by per 1g caseins
The ratio of enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 2.5h, and not enzyme deactivation after primary enzymolysis continues by every 1g caseins
The ratio for adding 0.01g protease adds Prote AX protease, and 50 DEG C of constant temperature is kept to digest 2.5h, after digesting twice,
Enzyme deactivation is boiled, buffalo's milk casein enzymolysis liquid is obtained.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Embodiment 3
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 5% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 55 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.02g albumen is added by per 1g caseins
The ratio of enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 3.5h, and not enzyme deactivation after primary enzymolysis continues by every 1g caseins
The ratio for adding 0.01g protease adds Prote AX protease, and 55 DEG C of constant temperature is kept to digest 3.5h, after digesting twice,
Enzyme deactivation is boiled, buffalo's milk casein enzymolysis liquid is obtained.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Embodiment 4
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 40 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.015g eggs are added by per 1g caseins
The ratio of white enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 4h, and not enzyme deactivation after primary enzymolysis continues by every 1g caseins
The ratio for adding 0.01g protease adds Prote AX protease, and 40 DEG C of constant temperature is kept to digest 4h, after digesting twice, boils
Enzyme deactivation is boiled, buffalo's milk casein enzymolysis liquid is obtained.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Embodiment 5
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 5% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 60 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.01g albumen is added by per 1g caseins
The ratio of enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 2h, and not enzyme deactivation after primary enzymolysis continues to add by every 1g caseins
Add the ratio of 0.01g protease to add Prote AX protease, 60 DEG C of constant temperature is kept to digest 2h, after digesting twice, boils
Enzyme deactivation obtains buffalo's milk casein enzymolysis liquid.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 1
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：Taking the fresh buffalo's milks of 100g 5000r/min under 4 DEG C of low temperature to centrifuge 10min can divide
From upper-layer fat, degreasing buffalo's milk is obtained；Buffalo's milk pH to 4.6, which is adjusted, with 1mol/L dilute hydrochloric acid precipitates casein, 4000r/min
Supernatant is removed after centrifugation 10min, obtains crude product casein；Continue to use pure water casein three times, each time after washing
4000r/min centrifuges 10min and collects sediment, and pure water bovine casein is obtained after freeze-drying.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.02g albumen is added by per 1g caseins
The ratio of enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 3h, after enzymolysis, boils enzyme deactivation, obtains buffalo's milk casein enzymolysis
Liquid.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 2
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：With 1 preparation method of comparative example.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.02g albumen is added by per 1g caseins
The ratio of enzyme adds Prote AX protease, and 50 DEG C of constant temperature is kept to digest 3h, after digesting twice, boils enzyme deactivation, obtains buffalo
Milk casein enzymolysis liquid.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 3
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：With 1 preparation method of comparative example.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.015g eggs are added by per 1g caseins
The ratio of white enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 3h, and not enzyme deactivation after primary enzymolysis continues by every 1g caseins
The ratio for adding 0.010g protease adds flavor protease, and 50 DEG C of constant temperature is kept to digest 3h, after digesting twice, boils and goes out
Enzyme obtains buffalo's milk casein enzymolysis liquid.Flavor protease used is purchased from Nanning Pang Bo bioengineering Co., Ltd, source
It ferments in aspergillus oryzae, contains endo protease and two kinds of activity of circumscribed peptase.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 4
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：With 1 preparation method of comparative example.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 4% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.015g eggs are added by per 1g caseins
The ratio of white enzyme adds animal hydrolyzed protein enzyme, and constant temperature digests 3h, and not enzyme deactivation after primary enzymolysis is cooled to 40 DEG C, by every
The ratio that 1g caseins add 0.015g protease adds Peptidase R protease, keeps 40 DEG C of constant temperature to digest 3h, twice enzyme
After solution, enzyme deactivation is boiled, obtains buffalo's milk casein enzymolysis liquid.Peptidase R protease used is purchased from Japanese amano enzyme system
Agent company is that one kind aminopeptidase, exopeptidase vigor made of Rhizopus oryzae (Rhizopus oryzae) fermentation refinement are
420U/g。
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 5
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：With 1 preparation method of comparative example.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 37 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.015g eggs are added by per 1g caseins
The ratio of white enzyme adds trypsase, and constant temperature digests 3h, and not enzyme deactivation after primary enzymolysis continues to add by every 1g caseins
The ratio of 0.010g protease adds Prote AX protease, and 37 DEG C of constant temperature is kept to digest 3h, after digesting twice, boils and goes out
Enzyme obtains buffalo's milk casein enzymolysis liquid.Trypsase used is purchased from Nanning Pang Bo bioengineering Co., Ltd, from pig pancreas
Middle extraction purification and obtain.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
Comparative example 6
A method of low bitter taste buffalo's milk casein antioxidant peptide powder being prepared, particular content is as follows：
(1) preparation of buffalo milk casein：With 1 preparation method of comparative example.
(2) protease hydrolyzed：The buffalo's milk casein solution that mass fraction percentage is 3% is prepared with pure water, 95 DEG C are boiled
After boiling pretreatment 10min, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.010g eggs are added by per 1g caseins
The ratio of white enzyme adds neutral proteinase, and constant temperature digests 3h, and not enzyme deactivation after primary enzymolysis continues to add by every 1g caseins
The ratio of 0.010g protease adds Prote AX protease, and 50 DEG C of constant temperature is kept to digest 3h, after digesting twice, boils and goes out
Enzyme obtains buffalo's milk casein enzymolysis liquid.Neutral proteinase used in the present invention is purchased from the limited public affairs of Nanning Pang Bo bioengineering
Department obtains from fermentation of bacillus subtilis.
(3) preparation of Gly-His-Lys：It is thick peptide liquid that enzymolysis liquid 7000r/min centrifugation 15min, which collects supernatant, by the first mistake of thick peptide liquid
Interception is the ultrafiltration membrane of 10kDa, and pressure 0.5MPa sloughs lipid and high molecular weight protein and polypeptide, then using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
To the Gly-His-Lys product that above-described embodiment 1~5 and comparative example 1~6 prepare carries out nitrogen recovery, small-molecular peptides contain
Amount, the measurement and bitter taste of iron atom reduction rate and ABTS free radical scavenging activities, color and luster and deliquescent evaluation, as a result such as table 2
It is shown.
The Comparative result of table 2 different Examples 1 to 5 and reference examples 1~6
Claims (10)
1. a kind of method preparing low bitter taste buffalo's milk casein antioxidant peptide powder, which is characterized in that including：
(1) fresh buffalo's milk is taken, after degreasing, precipitation and purification process, obtains buffalo milk casein；
(2) buffalo's milk caseic aqueous solution is prepared, it is denatured by boiling, then animal hydrolyzed protein enzyme is added into buffer solution and carries out one
After secondary enzymolysis, adds Prote AX protease and carry out secondary enzymolysis, boil enzyme deactivation after enzymolysis, obtain buffalo milk casein
Enzymolysis liquid；The animal hydrolyzed protein enzyme is purchased from Nanning Pang Bo Co., Ltds；
(3) for the buffalo's milk casein enzymolysis liquid successively through centrifuging with after hyperfiltration treatment, freeze-drying obtains buffalo milk casein
Antioxidant peptide powder.
2. the method as described in claim 1, which is characterized in that in step (1), the temperature of the degreasing is 3~6 DEG C, rotating speed
For 3500~5000r/min, the time is 10~15min.
3. the method as described in claim 1, which is characterized in that in step (2), in the buffalo's milk caseic aqueous solution,
The mass fraction of buffalo milk casein is 2~5%.
4. the method as described in claim 1, which is characterized in that in step (2), the denatured by boiling temperature is 90~100
DEG C, the time is 10~15min.
5. the method as described in claim 1, which is characterized in that in step (2), in terms of 1g caseins, described in buffer solution
The additive amount of animal hydrolyzed protein enzyme is 0.01~0.03g.
6. the method as described in claim 1, which is characterized in that in step (2), the temperature of the primary enzymolysis is 50~60
DEG C, the time is 2~4h.
7. the method as described in claim 1, which is characterized in that in step (2), in terms of 1g caseins, described in buffer solution
The additive amount of Prote AX protease is 0.005~0.02g.
8. the method as described in claim 1, which is characterized in that in step (2), the temperature of the secondary enzymolysis is 40~60
DEG C, the time is 2~4h.
9. the method as described in claim 1, which is characterized in that in step (3), the rotating speed of the centrifugation is 6000~8000r/
Min, time are 10~20min.
10. the method as described in claim 1, which is characterized in that in step (3), the interception of the ultrafiltration is 6000~
10000Da, pressure are 0.4~0.7MPa.
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