CN108588156A - A kind of preparation method of buffalo's milk casein antioxidant active peptide - Google Patents
A kind of preparation method of buffalo's milk casein antioxidant active peptide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of buffalo's milk casein antioxidant active peptide, this method includes：Fresh buffalo's milk is taken, after degreasing, precipitation and purification process, obtains buffalo milk casein；Buffalo milk casein is dissolved into buffer solution, it is denatured by boiling, then bacillus stearothermophilus protease is added into buffer solution and is digested, enzyme deactivation is boiled after enzymolysis, obtains buffalo's milk casein enzymolysis liquid；For the buffalo's milk casein enzymolysis liquid successively after centrifugation, ultrafiltration and nanofiltration processing, freeze-drying obtains buffalo's milk casein antioxidant active peptide powder.The present invention detaches from fresh buffalo's milk, purifies and obtains buffalo milk casein, and use bacillus stearothermophilus protease hydrolyzed buffalo milk casein, after further isolating and purifying, the buffalo's milk casein antioxidant active peptide powder that nitrogen recovery is high, molecular weight is small, antioxidant activity is strong is obtained.
Description
Technical field
The present invention relates to dairy products technical field more particularly to a kind of preparation sides of buffalo's milk casein antioxidant active peptide
Method.
Background technology
Newborn source biologically active peptide refers to naturally occurring in a variety of source lactoproteins or is easily degraded by proteases process at it
In the obtained polypeptide with different physiological activity.As a variety of newborn source peptides with different bioactivity are constantly found, breast
Albumen has become the new hot spot of physiology educational circles, nutrition educational circles and the research of food educational circles.
Newborn source activity peptide has anti-oxidant, adjusting immunity of organisms, antihypertensive, antitumor, adjusting metabolic syndrome etc.
Different physiological roles, wherein antioxidation active peptides are a studied wide directions.Although synthetized oxidation preventive agent has
Efficient and at low cost advantage, but its potential side effect is also controversial, therefore modern consumer is more favored in selection naturally
Antioxidant.
Casein is one of major product of dairy industry, is the main composition of cheese, and content accounts for newborn total protein content
80% or so.Preparation and structural research to antioxidation active peptides in bovine casein comparative maturity, such as：Publication number
Disclose the preparation method of yak milk casein anti-oxidation peptide for the application for a patent for invention of CN102787156A, this method include with
Lower step：(1) yak milk casein dissolves：Yak milk casein 20g~40g is weighed, water 500mL is added, adjusts reaction temperature 30
DEG C~60 DEG C, pH7.0~9.0, stirring makes yak milk casein dissolve；(2) add hydrolysis by novo：Draw 250 μ L~
500 μ L enzyme solutions, 30 DEG C~60 DEG C of maintaining reaction temperature, pH7.0~9.0 are reacted 1~4 hour；(3) alkali protease is inactivated, is risen
Temperature maintains 15~30 minutes to 80 DEG C~100 DEG C；(4) casein is precipitated：With 5mol/L salt acid for adjusting pH to 4.1~4.6, make
Casein is precipitated at its isoelectric point；(5) it centrifuges：4000g~10000g centrifugal force centrifuges 5~30 minutes, is centrifuged off junket egg
White precipitation, stays supernatant；(6) ultrafiltration：Centrifuged supernatant is taken, the ultrafiltration membrane of interception 6000Da is crossed, collects permeate, is removed
High molecular weight protein and polypeptide；(7) it is spray-dried：The ultrafiltration permeate in step (6) is taken, spray drying is carried out using spray dryer,
Obtain yak milk casein anti-oxidation peptide.
However, the releasing research at present about buffalo lactoprotein moderate resistance oxidation activity peptide is also fewer.Buffalo's milk is the world
The second largest breast, yield accounts for about the 13% of global milk production, and China is buffalo milk production big country.China's buffalo's milk and other
The breast of mammal is compared, and protein content is up to 4.3%, is 1.5 times of common Holstein cow breast, wherein essential amino acids content
Up to 30.29%, this provides raw material guarantee to prepare guaranteed milk source activity peptide.Therefore, research and utilization buffalo lactoprotein, research and development
Preparing buffalo's milk protein antioxidant peptide has important economic implications.
Invention content
The present invention provides a kind of preparation methods of buffalo's milk casein antioxidant active peptide, are prepared using this method
Casein small peptide not only nitrogen recovery is high, molecular weight is low, and antioxidant activity is also strong, free to DPPH free radicals and ABTS
The clearance rate of base is higher.
Specific technical solution is as follows：
A kind of preparation method of buffalo's milk casein antioxidant active peptide, including：
(1) fresh buffalo's milk is taken, after degreasing, precipitation and purification process, obtains buffalo milk casein；
(2) buffalo milk casein is dissolved into buffer solution, it is denatured by boiling, then stearothermophilus is added into buffer solution
Bacillus protein enzyme is digested, and enzyme deactivation is boiled after enzymolysis, obtains buffalo's milk casein enzymolysis liquid；
(3) for the buffalo's milk casein enzymolysis liquid successively after centrifugation, ultrafiltration and nanofiltration processing, freeze-drying obtains water
Bovine casein antioxidant activity Gly-His-Lys.
The bacillus stearothermophilus protease is THERMOASE C100 protease, derives from stearothermophilus gemma bar
Bacterium (Bacillus stearothermophilus) Var.Rokko is purchased from amano enzyme preparation Co., Ltd.Experiment discovery,
THERMOASE C100 protease can effectively hydrolyzing hydrophobic amino acid at relatively high temperatures, obtain strong more of oxidation resistance
Peptide.
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.
Preferably, in step (2), the buffer solution is the mixed aqueous solution of sodium dihydrogen phosphate and disodium hydrogen phosphate,
The mass fraction of buffalo milk casein is 2~5% in buffer solution.The phosphate buffer can quick solubilising casein, and it is straight
The step of connecing and adjust within the scope of solution to peak enzymolysis-ability pH, saving subsequent adjustment pH.
Preferably, in step (2), the denatured by boiling temperature is 90~100 DEG C, and the time is 5~10min.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.
Molecular size range and antioxygenic property of the additive amount of protease to nitrogen recovery and casein antioxidant active peptide
Have an impact.Preferably, in step (2), in terms of 1g caseins, bacillus stearothermophilus protease described in buffer solution
Additive amount be 0.005~0.020g.
Preferably, in step (2), the temperature of the enzymolysis is 55~70 DEG C, and the time is 2~4h.The enzymatic hydrolysis condition can
Efficiently to play proteinase activity, polypeptide yield is improved.
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；
The interception of the nanofiltration is 100~200Da, and pressure is 2~2.5MPa.Ultrafiltration can remove a small number of residual lipids and macromolecular
Albumen and polypeptide, nanofiltration can remove small molecule salinity, and two filtration steps can effectively remove impurity, obtain limpid pure peptide
Liquid improves the antioxygenic property of product.
Compared with prior art, the invention has the advantages that：
The present invention detaches from fresh buffalo's milk, purifies and obtains buffalo milk casein, and uses bacillus stearothermophilus egg
White enzyme digests buffalo milk casein, after further isolating and purifying, obtains that nitrogen recovery is high, molecular weight is small, antioxidant activity is strong
Buffalo's milk casein antioxidant active peptide powder.
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.
DPPH free radical scavenging activities, ABTS free radical scavenging activities, nitrogen recovery involved in the following example and molecular weight
The assay method of distribution is as follows：
(1) measurement of DPPH free radical scavenging activities：
1. being separately added into 0.1mL pure water and 1.9mL DPPH ethanol solutions (1mmoL/L) in 5mL centrifuge tubes, shake
After even after 30min is reacted in dark place, with the light absorption value A under UV spectrophotometer measuring its 515nmc；
2. being separately added into 0.1mL candidate polypeptides liquid (2mg/mL) and 1.9mLDPPH ethanol solutions in 5mL centrifuge tubes
(1mmoL/L), after shaking up after 30min is reacted in dark place, with the light absorption value A under UV spectrophotometer measuring its 515nmi；
3. DPPH clearance rates following formula result of calculation：
(2) measurement of ABTS free radical scavenging activities：
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, which is protected from light after 12h, to be settled to 100mL and obtains ABTS radical reaction liquid.
2. being separately added into 0.1mL pure water and 1.9mL ABTS reaction solutions (2mmoL/L) in 5mL centrifuge tubes, mixing is shaken
The light absorption value A under its 734nm of UV spectrophotometer measuring is used after even reaction 10min0；
3. being separately added into 0.1mL candidate polypeptides liquid (2mg/mL) and 1.9mLABTS reaction solutions in 5mL centrifuge tubes, mix
Close the light absorption value A used after shaking up reaction 10min under its 734nm of UV spectrophotometer measuring1；
4. ABTS clearance rates 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 AKTA pure 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.
Embodiment 1
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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：With sodium dihydrogen phosphate/disodium hydrogen phosphate buffer quality of 0.04mol/L pH 7.5
The buffalo's milk casein solution that percentage fractional is 3% is cooled to 60 DEG C, then be placed in magnetic force after 95 DEG C are boiled pretreatment 10min
In constant temperature water bath apparatus, THERMOASE C100 protease is added in the ratio for adding 0.020g protease per 1g caseins, it is permanent
Temperature enzymolysis 4h, is adjusted with acid/base pH value is kept to maintain between 7.0~7.5 in the process.After enzymolysis, enzyme deactivation is boiled, obtains water
Bovine casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 80.6%, and small molecular weight titanium ratio is 82.3%.It is more that Gly-His-Lys are configured to 2mg/mL
Peptide liquid, it is 54.5% that measure its DPPH free radical scavenging activity, which be 63.8%, ABTS free radical scavenging activities,.
Embodiment 2
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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：With sodium dihydrogen phosphate/disodium hydrogen phosphate buffer quality point of 0.04mol/L pH 8
The buffalo's milk casein solution that number percentage is 4% is cooled to 65 DEG C after 95 DEG C are boiled pretreatment 10min, then is placed in magnetic force perseverance
In warm water bath apparatus, THERMOASE C100 protease, constant temperature are added in the ratio for adding 0.015g protease per 1g caseins
3h is digested, is adjusted with acid/base pH value is kept to maintain between 7.5~8.0 in the process.After enzymolysis, enzyme deactivation is boiled, obtains buffalo
Milk casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 77.5%, and small molecular weight titanium ratio is 80.1%.It is more that Gly-His-Lys are configured to 2mg/mL
Peptide liquid, it is 50.3% that measure its DPPH free radical scavenging activity, which be 61.2%, ABTS free radical scavenging activities,.
Embodiment 3
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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：With sodium dihydrogen phosphate/disodium hydrogen phosphate buffer quality of 0.04mol/L pH 7.5
The buffalo's milk casein solution that percentage fractional is 2% is cooled to 55 DEG C, then be placed in magnetic force after 95 DEG C are boiled pretreatment 10min
In constant temperature water bath apparatus, THERMOASE C100 protease is added in the ratio for adding 0.010g protease per 1g caseins, it is permanent
Temperature enzymolysis 2.5h, is adjusted with acid/base pH value is kept to maintain between 7.0~7.5 in the process.After enzymolysis, enzyme deactivation is boiled, is obtained
Buffalo's milk casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 74.8%, and small molecular weight titanium ratio is 80.2%.It is more that Gly-His-Lys are configured to 2mg/mL
Peptide liquid, it is 50.0% that measure its DPPH free radical scavenging activity, which be 59.6%, ABTS free radical scavenging activities,.
Embodiment 4
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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：With sodium dihydrogen phosphate/disodium hydrogen phosphate buffer quality of 0.04mol/L pH 8.0
The buffalo's milk casein solution that percentage fractional is 5% is cooled to 70 DEG C, then be placed in magnetic force after 95 DEG C are boiled pretreatment 15min
In constant temperature water bath apparatus, THERMOASE C100 protease is added in the ratio for adding 0.020g protease per 1g caseins, it is permanent
Temperature enzymolysis 2.5h, is adjusted with acid/base pH value is kept to maintain between 7.5~8.0 in the process.After enzymolysis, enzyme deactivation is boiled, is obtained
Buffalo's milk casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 71.4%, and small molecular weight titanium proportion is 75.6%.Gly-His-Lys are configured to 2mg/
ML polypeptide liquids, it is 48.7% that measure its DPPH free radical scavenging activity, which be 59.8%, ABTS free radical scavenging activities,.
Comparative example 1
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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：With sodium dihydrogen phosphate/disodium hydrogen phosphate buffer quality of 0.04mol/L pH 8.0
The buffalo's milk casein solution that percentage fractional is 3% is cooled to 37 DEG C, then be placed in magnetic force after 95 DEG C are boiled pretreatment 15min
In constant temperature water bath apparatus, trypsase is added in the ratio for adding 0.015g protease per 1g caseins, constant temperature digests 3h, process
Middle adjusted with acid/base keeps pH value to maintain between 7.5~8.0.After enzymolysis, enzyme deactivation is boiled, obtains buffalo's milk casease
Solve liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 74.3%, and small molecular weight titanium proportion is 77.0%.Gly-His-Lys are configured to 2mg/
ML polypeptide liquids, it is 50.2% that measure its DPPH free radical scavenging activity, which be 62.4%, ABTS free radical scavenging activities,.
Comparative example 2
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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
Boiling pretreatment 10min postcoolings are placed in magnetic force constant temperature water bath apparatus to 50 DEG C, and 0.020g albumen is added by per 1g caseins
The ratio of enzyme adds neutral proteinase, and constant temperature digests 3.5h, adjusted in the process with acid/base keep pH value maintain 6.5~7.5 it
Between.After enzymolysis, enzyme deactivation is boiled, obtains buffalo's milk casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 68.4%, small molecular weight titanium proportion is 73.2%.Gly-His-Lys are configured to 2mg/
ML polypeptide liquids, it is 48.7% that measure its DPPH free radical scavenging activity, which be 60.1%, ABTS free radical scavenging activities,.
Comparative example 3
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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, 50 DEG C are cooled to, then be placed in magnetic force constant temperature water bath apparatus, 0.020g eggs are added by per 1g caseins
The ratio of white enzyme adds pancreatin, and constant temperature digests 3.5h, do not adjust pH value in the process.After enzymolysis, enzyme deactivation is boiled, obtains buffalo's milk
Casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 70.2%, small molecular weight titanium proportion is 69.7%.Gly-His-Lys are configured to 2mg/
ML polypeptide liquids, it is 45.3% that measure its DPPH free radical scavenging activity, which be 57.6%, ABTS free radical scavenging activities,.
Comparative example 4
A kind of preparation method of buffalo's milk casein antioxidant active peptide, particular content are 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, 0.010g eggs are added by per 1g caseins
The ratio of white enzyme adds compound protease (alkali protease:Neutral proteinase:Papain:Bromelain=1:1:1:
1), constant temperature digests 3.5h, does not adjust pH value in the process.After enzymolysis, enzyme deactivation is boiled, obtains buffalo's milk casein enzymolysis liquid.
(3) preparation of desalination low molecular weight antioxidant peptide powder：Enzymolysis liquid 7000r/min centrifugations 15min collects supernatant and is
Thick peptide liquid, is first crossed the ultrafiltration membrane that interception is 10kDa by thick peptide liquid, pressure 0.5MPa, slough lipid and high molecular weight protein and
Polypeptide, after 200Da NF membranes, pressure 2MPa sloughs salinity and single amino acid, and nanofiltration recovered liquid is using freeze-drying
Method obtains buffalo's milk casein antioxidant peptide powder.
The present embodiment nitrogen recovery is 65.5%, and small molecular weight titanium proportion is 66.7%.Gly-His-Lys are configured to 2mg/
ML polypeptide liquids, it is 46.9% that measure its DPPH free radical scavenging activity, which be 58.7%, ABTS free radical scavenging activities,.
Claims (9)
1. a kind of preparation method of buffalo's milk casein antioxidant active peptide, 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 milk casein is dissolved into buffer solution, it is denatured by boiling, then stearothermophilus gemma is added into buffer solution
Bacillus protease is digested, and enzyme deactivation is boiled after enzymolysis, obtains buffalo's milk casein enzymolysis liquid；
(3) for the buffalo's milk casein enzymolysis liquid successively after centrifugation, ultrafiltration and nanofiltration processing, freeze-drying obtains buffalo's milk
Casein antioxidant active peptide powder.
2. preparation method as described in claim 1, which is characterized in that the bacillus stearothermophilus protease is
THERMOASE C100 protease derives from bacillus stearothermophilus (Bacillus stearothermophilus)
Var.Rokko。
3. preparation 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 is 3500~5000r/min, and the time is 10~15min.
4. preparation method as described in claim 1, which is characterized in that in step (2), the buffer solution is sodium dihydrogen phosphate
With the mixed aqueous solution of disodium hydrogen phosphate, the mass fraction of buffalo milk casein is 2~5% in buffer solution.
5. preparation method as described in claim 1, which is characterized in that in step (2), the denatured by boiling temperature be 90~
100 DEG C, the time is 5~10min.
6. preparation method as described in claim 1, which is characterized in that in step (2), in terms of 1g caseins, in buffer solution
The additive amount of the bacillus stearothermophilus protease is 0.005~0.020g.
7. preparation method as described in claim 1, which is characterized in that in step (2), the temperature of the enzymolysis is 55~70
DEG C, the time is 2~4h.
8. preparation 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.
9. preparation method as described in claim 1, which is characterized in that in step (3), the interception of the ultrafiltration is 6000~
10000Da, pressure are 0.4~0.6MPa；The interception of the nanofiltration is 100~200Da, and pressure is 2~2.5MPa.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109824774A (en) * | 2019-03-18 | 2019-05-31 | 时代生物科技（深圳）有限公司 | A method of recycling high activity peptide |
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