CN105506046B - Casein hypoglycemic peptide based on yak milk Qula and its preparation method and application - Google Patents
Casein hypoglycemic peptide based on yak milk Qula and its preparation method and application Download PDFInfo
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- CN105506046B CN105506046B CN201610029083.6A CN201610029083A CN105506046B CN 105506046 B CN105506046 B CN 105506046B CN 201610029083 A CN201610029083 A CN 201610029083A CN 105506046 B CN105506046 B CN 105506046B
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Classifications
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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
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a kind of casein hypoglycemic peptides and its preparation method and application based on yak milk Qula for belonging to field of biotechnology.The preparation method is that: it is pre-processed after yak milk Qula is crushed, impregnated using physics auxiliary dissolving technology, then adjusts pH to 4-7, filtered after centrifugation, obtain yak milk Qula casein solution；After successively being hydrolyzed to it with pepsin and trypsase, centrifuging and taking supernatant carries out ultrafiltration, obtains the casein hypoglycemic peptide solution of yak milk Qula；It carries out de- suffering reason after concentration to it using plastein reaction, the casein hypoglycemic Gly-His-Lys of yak milk Qula is obtained after dry.The prices of raw materials that the preparation method uses are cheap, and the hypoglycemic peptide nutritive value of acquisition is high, without side-effects, good absorption effect, good assistant hypoglycemic effect for reducing fat can be played to diabetes and hyperlipidemia, had a good application prospect in preparing food-borne food for reducing blood sugar and blood fat or health care product.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of casein hypoglycemic peptide based on yak milk Qula and
Preparation method and application.
Background technique
In recent years, with the improvement of people ' s living standards and living-pattern preservation, diabetes, obesity, artery are hard
The disease incidence of the metabolic diseases such as change, fatty liver increases year by year.According to International Diabetes Federation (International Diabetes
Federation, IDF) statistics, by 2014, global diabetes number had reached 3.82 hundred million, it is contemplated that by 2035, the whole world will
There is puzzlement of 5.92 hundred million people by diabetes.Currently, China's diabetic's number is up to 0.96 hundred million.Body is chronically at high blood
Sugared state can induce fat metabolic disturbance, lead to dyslipidemia, increase the disease incidence of cardiovascular disease.According to the World Health Organization
(World Health Organization, WHO) announces data and shows, there are about 17,500,000 people to die of angiocarpy for the whole world in 2012
Disease accounts for the 31% of global dead sum.The Chinese cardiovascular disease report of 2014 publications, China's cardiovascular patient number are
2.9 hundred million, cardiovascular disease is occupied first of people's various diseases cause of the death.The medicine of the treatment metabolic diseases such as diabetes and hyperlipidemia at present
There are the adverse reactions such as hepar damnification, lactic acidosis and gastrointestinal discomfort in object, therefore exploitation is natural, pacifies there is an urgent need to seek
Entirely, efficiently with the food source function factor of hypoglycemic biological activity.
Qula is the exclusive newborn resource in China, it is yak milk crude cheese, junket made from spontaneous fermentation after degreasing
Protein content is up to 70% or more.The multiple biological activities sequence that yak milk casein contains utilizes protease hydrolytic technology energy
It enough releases, generates the polypeptide with the biological activities such as blood pressure lowering, anti-oxidant, antibacterial, hypoglycemic.
The dissolution of yak milk casein needs under alkaline pH system, and solubility is very under neutral and faintly acid pH system
It is low.Therefore, in order to sufficiently dissolve yak milk casein, protease is realized to its effectively hydrolyzing, patented technology adds mostly at present
Enter a large amount of lye solubilising caseins, but to will lead to salt content in enzymolysis liquid excessively high for this processing mode, not only influences product
Quality, and intake with high salt can also threaten the health of human body.Also there is the yak milk casein using nanofiltration to preparation
Blood pressure lowering peptide carries out desalination, but this method is cumbersome and time-consuming, low efficiency, is unfavorable for industrialized production.
In addition, a series of molecular weight can be generated after casein hydrolysis in 6000Da hereinafter, and containing hydrophobic amino acid
Polypeptide, this kind of polypeptide have different degrees of bitter taste, influence its application in field of food.Therefore, it is necessary to enzymolysis product
Carry out de- suffering reason.With active carbon adsorption can the trypsin hydrolysis liquid to yak milk casein carry out de- suffering reason, but this
Kind method can lose the functional component in polypeptide and reduce its biological activity；It can also be solved by carrier of cycloheptaamylose
The bitterness problem of hydrophobic peptides in yak milk casein peptide, however, the addition of cycloheptaamylose, not only draws protein peptides system
Exogenous material is entered, but also system can be made to generate and certain be charred peculiar smell.
Summary of the invention
The purpose of the present invention is to provide a kind of casein hypoglycemic peptide and preparation method thereof based on yak milk Qula
And application.
To achieve the goals above, the technical solution adopted by the present invention is as follows:
A kind of preparation method of the casein hypoglycemic peptide based on yak milk Qula, comprising the following steps:
(1) yak milk Qula raw material is crushed to 30-120 mesh, by weight: volume ratio 1g:(10-30ml) 30- is added
In 50 DEG C of water, 1-5h is impregnated, obtains yak milk Qula aqueous solution；It is water-soluble to yak milk Qula using physics auxiliary dissolving technology
After liquid pretreatment, its pH to 4-7 is adjusted, stirs 30-90min；It is then centrifuged for degreasing, removal impurity, is carried out after collecting supernatant
Filtering collects filtrate, obtains yak milk Qula casein solution；
(2) pH to 2-4 for adjusting yak milk Qula casein solution, is added pepsin, carries out under the conditions of 25-65 DEG C
Enzyme digestion reaction, reaction time 1-3h；
(3) the temperature-resistant of enzyme digestion reaction system is kept, the pH to 6-8 of reaction system is adjusted, trypsase is added, is continued
After digesting 1-3h, reaction system is warming up to 75-95 DEG C, 10-20min is kept, inactivates pepsin and trypsase therein
Reaction is terminated, yak milk Qula casein hydrolyzate is obtained；
(4) by centrifuging and taking supernatant after the cooling of yak milk Qula casein hydrolyzate, and supernatant is passed through into the molecule that shuts off
Amount is the ultrafiltration membrane of 3000-10000Da, collects ultrafiltrate, obtains the casein hypoglycemic peptide solution of yak milk Qula；
(5) the casein hypoglycemic peptide solution of yak milk Qula is concentrated into mass concentration is 30%-60%, adjusts temperature
Degree is added protease and carries out plastein reaction to 20-65 DEG C, after reacting 0.5-3h, heats enzyme deactivation, obtains the light yak of bitter taste
Milk casein hypoglycemic peptide solution；
(6) the casein hypoglycemic peptide solution of yak milk Qula is dried, obtains the junket of yak milk Qula
Albumen hypoglycemic Gly-His-Lys.
Physics described in step (1) auxiliary dissolving technology mode are as follows: pressure be 200-600MPa, temperature 35-
15-30min is handled under conditions of 65 DEG C；Or ultrasonic power be 1000-1500W, temperature be 40-60 DEG C under conditions of handle
20-40min；Or microwave power be 100-700W, temperature be 40-100 DEG C under conditions of handle 10-300s.
The additional amount of pepsin described in step (2) is the stomach cardia that 500-1000KU is added in every 100g yak milk Qula
Enzyme；Wherein, 1U is defined as the amount of 1 μ g tyrosine of caseinhydrolysate generation per minute at 40 DEG C.
The additional amount of trypsase described in step (3) is the tryptose that 500-1000KU is added in every 100g yak milk Qula
Enzyme；Wherein, 1U is defined as the amount of 1 μ g tyrosine of caseinhydrolysate generation per minute at 40 DEG C.
Protease described in step (5) is pepsin, trypsase, flavor protease, neutral proteinase or pawpaw
Protease.
The additional amount of protease described in step (5) are as follows: the casein hypoglycemic peptide of every 100g yak milk Qula is added
The protease of 50-1000KU；Wherein, 1U is defined as the amount of 1 μ g tyrosine of caseinhydrolysate generation per minute at 40 DEG C.
The hypoglycemic peptide that a kind of preparation method of casein hypoglycemic peptide based on yak milk Qula obtains.
The hypoglycemic peptide is preparing the application in food-borne food for reducing blood sugar and blood fat or health care product.
The hypoglycemic peptide can be used alone or be used in compounding with natural hypoglycemic active constituent.
The invention has the benefit that the preparation method combines simulation by super-pressure/ultrasonic wave/microwave assisted techniques
Gastro-intestinal digestion hydrolysis pattern effectively increases the degree of hydrolysis of casein, realizes the controlled release of active amino acid sequence, improves
Its stomach and intestine stability；The plastein reaction modification debitterness technology of use solves drop on the basis of lossless polypeptide biological activity
The bitterness problem of sugared lipid-loweringing peptide；And the prices of raw materials that the preparation method uses are cheap, reduce production cost, acquisition
Hypoglycemic peptide nutritive value is high, without side-effects, good absorption effect, can play good auxiliary to diabetes and hyperlipidemia
Hypoglycemic lipid-lowering effect has a good application prospect in preparing food-borne food for reducing blood sugar and blood fat or health care product.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Embodiment 1:
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 40 DEG C of 200mL of water is added and impregnates 3h, it is bent to obtain yak milk
Draw aqueous solution；It is that 300MPa adjusts it after temperature handles yak milk Qula aqueous solution 20min under conditions of being 50 DEG C in pressure
PH to 5 stirs 30min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is filtered after collecting supernatant, is received
Collect filtrate, obtains yak milk Qula casein solution；
2) pH to 2.0 for adjusting yak milk Qula casein solution, is added pepsin 50KU, under 40 DEG C of water bath conditions into
Row enzyme digestion reaction reacts 1h；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 50KU is added, is continued after hydrolyzing 2h, it will
Reaction system is warming up to 85 DEG C, keeps 10min, and enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, measurement is at this time
The degree of hydrolysis of enzymolysis liquid is 22.8%；
3) supernatant will be taken after 4000r/min is centrifuged 15min after the cooling of yak milk Qula casein hydrolyzate, and will be upper
Clear liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 10000Da that shuts off, and obtains the casein drop of 150mL yak milk Qula
Sugared lipid-loweringing peptide solution, wherein the content of hypoglycemic peptide is 7.5g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 20mL, adjusts solution temperature to 50 DEG C, is added
Papain 45KU reacts 1h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measurement produces
The content of ashes of product is less than 2%, bitterness value 2.
Each experimental data is obtained according to following measuring methods in above-mentioned experimental procedure:
(1) measurement (trinitrobenzene sulfonic acid method) of degree of hydrolysis:
It draws the yak milk Qula casein hydrolyzate of 0.5mL enzyme deactivation and utilizes 1% lauryl sodium sulfate after cooling
It is settled to 25mL, is stood.Take the above-mentioned enzymolysis liquid dilution of 0.125mL and 1mL phosphate buffer (pH 8.2,0.2mol/L) and
The 0.1% trinitrobenzene sulfonic acid solution of 1mL mixes, and is protected from light on 50 DEG C of shaking baths.2mL is added after 60min
The HCl solution of 0.1mol/L terminates reaction, and after being stored at room temperature 30min, its absorbance value is measured under 420nm.Using 0-5.0 ×
10-3The L-Leu of mol/L concentration draws standard curve.Degree of hydrolysis is calculated using following formula.
In formula, h: every gram of peptide bond mM number (meq/g protein) being cleaved in hydrolysate is indicated；Htot: it indicates
Total peptide bond mM number in every gram of material protein.
(2) measurement (Method of burning of high temperature) of ash content:
The casein hypoglycemic Gly-His-Lys for weighing 3g yak milk Qula are placed in the crucible of constant weight, and 1mL24% second is added
Sour magnesium solution makes the casein hypoglycemic Gly-His-Lys complete wetting of yak milk Qula.After placing 10min, by moisture in water-bath
After being evaporated, first carbonize the casein hypoglycemic Gly-His-Lys of yak milk Qula sufficiently to smokeless on electric hot plate with small fire heating,
It is subsequently placed in Muffle furnace, in 550 DEG C of ± 25 DEG C of calcination 4h.200 DEG C or so are cooled to, is taken out, is put into drier cooling
30min is weighed.
In formula: X1The content of ash content, g/100g in-sample；
m0The quality of-magnesia (product after magnesium acetate calcination), g；
m1The quality of-crucible and ash content, g；
m2The quality of-crucible, g；
m3The quality of-crucible and sample, g；
(3) measurement (sensory evaluation method) of bitterness value:
Sample to be tested (the casein hypoglycemic Gly-His-Lys of yak milk Qula) is soluble in water with 5% mass concentration, by 1:1
Ratio carries out gradient dilution；Support samples are soluble in water by 5% mass concentration, gradient dilution is carried out in 1:1 ratio；Utilize three
Point experimental method carries out sensory evaluation and is supplied to two support samples and a sample to be tested random number of same dilution
Can subject, detection distinguish their bitterness；Bitterness value is indicated so that the minimum extension rate of bitter taste difference cannot be tasted,
The more big then bitterness value of the extension rate is higher, illustrates that the bitterness of product is heavier.
Embodiment 2:
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 40 DEG C of 200mL of water is added and impregnates 3h, it is bent to obtain yak milk
Draw aqueous solution；It is that 350MPa adjusts it after temperature handles yak milk Qula aqueous solution 20min under conditions of being 50 DEG C in pressure
PH to 5.5 stirs 30min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is filtered after collecting supernatant,
Filtrate is collected, yak milk Qula casein solution is obtained；
2) pH to 3.0 for adjusting yak milk Qula casein solution, is added pepsin 80KU, under 45 DEG C of water bath conditions into
Row enzyme digestion reaction reacts 1h；The pH to 6.0 of enzyme digestion reaction system is adjusted, trypsase 60KU is added, is continued after hydrolyzing 1.5h,
Reaction system is warming up to 85 DEG C, keeps 10min, enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, measure this
When enzymolysis liquid degree of hydrolysis be 20.5%；
3) will yak milk Qula casein hydrolyzate it is cooling after take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate, obtains the casein hypoglycemic of 150mL yak milk Qula by the ultrafiltration membrane that molecular weight is 10000Da that shuts off
Lipid-loweringing peptide solution, wherein the content of hypoglycemic peptide is 7.1g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 15mL, adjusts temperature to 50 DEG C, flavor is added
Protease 55KU reacts 0.5h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measure product
Content of ashes less than 2%, bitterness value 3.The measuring method of each data is the same as embodiment 1.
Embodiment 3:
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 45 DEG C of 200mL of water is added and impregnates 1.5h, obtains yak milk
Qula aqueous solution；It is that 1500W is adjusted after temperature handles yak milk Qula aqueous solution 20min under conditions of being 40 DEG C in ultrasonic power
Its pH to 6.5 is saved, 50min is stirred；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is carried out after collecting supernatant
Filtering collects filtrate, obtains yak milk Qula casein solution；
2) pH to 2.5 for adjusting yak milk Qula casein solution, is added pepsin 100KU, under 40 DEG C of water bath conditions
Enzyme digestion reaction is carried out, 0.5h is reacted；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 80KU is added, continues to hydrolyze 1h
Afterwards, reaction system is warming up to 85 DEG C, keeps 10min, enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, surveys
The degree of hydrolysis of fixed enzymolysis liquid at this time is 21.2%；
3) will yak milk Qula casein hydrolyzate it is cooling after take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 8000Da that shuts off, and obtains the casein hypoglycemic drop of 150mL yak milk Qula
Lipopeptide solution, wherein the content of hypoglycemic peptide is 7.2g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 18mL, adjusts temperature to 40 DEG C, pancreas egg is added
White enzyme 40KU reacts 1.5h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measure product
Content of ashes is less than 2%, bitterness value 5.The measuring method of each data is the same as embodiment 1.
Embodiment 4:
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 45 DEG C of 150mL of water is added and impregnates 2h, it is bent to obtain yak milk
Draw aqueous solution；It is that 1000W is adjusted after temperature handles yak milk Qula aqueous solution 40min under conditions of being 50 DEG C in ultrasonic power
Its pH to 6.0 stirs 60min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, was carried out after collecting supernatant
Filter collects filtrate, obtains yak milk Qula casein solution；
2) pH to 3.5 for adjusting yak milk Qula casein solution, is added pepsin 60KU, under 37 DEG C of water bath conditions into
Row enzyme digestion reaction reacts 2h；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 100KU is added, is continued after hydrolyzing 2h, it will
Reaction system is warming up to 85 DEG C, keeps 10min, and enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, measurement is at this time
The degree of hydrolysis of enzymolysis liquid is 22.5%；
3) will yak milk Qula casein hydrolyzate it is cooling after take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate, obtains the casein hypoglycemic of 100mL yak milk Qula by the ultrafiltration membrane that molecular weight is 10000Da that shuts off
Lipid-loweringing peptide solution, wherein the content of hypoglycemic peptide is 7.2g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 20mL, adjusts temperature to 35 DEG C, stomach egg is added
White enzyme 60KU reacts 1h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measure the ash of product
Divide content less than 2%, bitterness value 6.The measuring method of each data is the same as embodiment 1.
Embodiment 5:
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 45 DEG C of 300mL of water is added and impregnates 2h, it is bent to obtain yak milk
Draw aqueous solution；It is that 400W adjusts it after temperature handles yak milk Qula aqueous solution 100s under conditions of being 80 DEG C in microwave power
PH to 5.5 stirs 90min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is filtered after collecting supernatant,
Filtrate is collected, yak milk Qula casein solution is obtained；
2) pH to 4.0 for adjusting yak milk Qula casein solution, is added pepsin 100KU, under 37 DEG C of water bath conditions
Enzyme digestion reaction is carried out, 1.5h is reacted；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 50KU is added, continues to hydrolyze 3h
Afterwards, reaction system is warming up to 85 DEG C, keeps 10min, enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, surveys
The degree of hydrolysis of fixed enzymolysis liquid at this time is 23.8%；
3) will yak milk Qula casein hydrolyzate it is cooling after take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 6000Da that shuts off, and obtains the casein hypoglycemic drop of 250mL yak milk Qula
Lipopeptide solution, wherein the content of hypoglycemic peptide is 6.8g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 15mL, adjusts temperature to 50 DEG C, flavor is added
Protease 3 5KU reacts 3h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measure product
Content of ashes is less than 2%, bitterness value 4.The measuring method of each data is the same as embodiment 1.
Embodiment 6:
1) the yak milk Qula that 10g is crushed to 80 mesh is weighed, 40 DEG C of 250mL of water is added and impregnates 2h, it is bent to obtain yak milk
Draw aqueous solution；It is that 500W adjusts it after temperature handles yak milk Qula aqueous solution 70s under conditions of being 60 DEG C in microwave power
PH to 6.0 stirs 45min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is filtered after collecting supernatant,
Filtrate is collected, yak milk Qula casein solution is obtained；
2) pH to 4.0 for adjusting yak milk Qula casein solution, is added pepsin 50KU, under 40 DEG C of water bath conditions into
Row enzyme digestion reaction reacts 0.5h；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 80KU is added, is continued after hydrolyzing 2h,
Reaction system is warming up to 85 DEG C, keeps 10min, enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, measure this
When enzymolysis liquid degree of hydrolysis be 21.1%；
3) will yak milk Qula casein hydrolyzate it is cooling after take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 6000Da that shuts off, and obtains the casein hypoglycemic drop of 200mL yak milk Qula
Lipopeptide solution, wherein the content of hypoglycemic peptide is 5.0g；
4) yak milk Qula casein hypoglycemic peptide solution is concentrated into 10mL, adjusts temperature to 50 DEG C, pawpaw is added
Protease 50KU reacts 0.5h, heats enzyme deactivation.Casein hypoglycemic Gly-His-Lys through dry obtained yak milk Qula.Measure product
Content of ashes less than 2%, bitterness value 3.The measuring method of each data is the same as embodiment 1.
Embodiment 7 (comparative example 1):
The yak milk Qula that 10g is crushed to 60 mesh is weighed, 40 DEG C of 200mL of water is added and impregnates 3h, obtains yak milk Qula
Aqueous solution adjusts its pH to 9.0, stirs 30min；Then 20min is centrifuged under conditions of revolving speed is 4000r/min, in collection
It is filtered after clear, collects filtrate, obtain yak milk Qula casein solution.
The pH to 2.0 of yak milk Qula casein solution is adjusted, pepsin 50KU is added, under 37 DEG C of water bath conditions, into
Row hydrolysis reacts 2h；Then the pH to 7.0 for adjusting reaction system, is added trypsase 50KU, continues to hydrolyze 2h, hydrolysis
Being continuously added lye in the process maintains reaction system pH constant；Reaction system is warming up to 85 DEG C, keeps 10min, enzyme deactivation terminates
Reaction obtains yak milk Qula casein hydrolyzate, and enzymolysis liquid is hydrolyzed to 15.4% at this time for measurement.
By yak milk Qula casein hydrolyzate it is cooling after, take supernatant after 4000r/min centrifugation 15min, and by supernatant
Liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 10000Da that shuts off, and solution temperature is adjusted after concentration to 50 DEG C, wood is added
Melon protease 45KU reacts 1h, heats enzyme deactivation.The casein hypoglycemic peptide of yak milk Qula is obtained after drying.Measure product
Content of ashes is more than 5%, bitterness value 3.Measuring method is the same as embodiment 1.
Embodiment 8 (comparative example 2):
1) the yak milk Qula that 10g is crushed to 60 mesh is weighed, 40 DEG C of 200mL of water is added and impregnates 3h, it is bent to obtain yak milk
Draw aqueous solution；It is that 300MPa adjusts it after temperature handles yak milk Qula aqueous solution 25min under conditions of being 60 DEG C in pressure
PH to 5.5 stirs 40min；Then under conditions of revolving speed is 4000r/min, it is centrifuged 20min, is filtered after collecting supernatant,
Filtrate is collected, yak milk Qula casein solution is obtained；
2) pH to 2.0 for adjusting yak milk Qula casein solution, is added pepsin 40KU, under 40 DEG C of water bath conditions into
Row enzyme digestion reaction reacts 1.5h；The pH to 7.0 of enzyme digestion reaction system is adjusted, trypsase 60KU is added, continues to hydrolyze 1.5h
Afterwards, reaction system is warming up to 75 DEG C, keeps 20min, enzyme deactivation terminates reaction, obtains yak milk Qula casein hydrolyzate, surveys
The degree of hydrolysis of fixed enzymolysis liquid at this time is 25.3%；
3) supernatant will be taken after 4000r/min is centrifuged 15min after the cooling of yak milk Qula casein hydrolyzate, and will be upper
Clear liquid collects ultrafiltrate by the ultrafiltration membrane that molecular weight is 10000Da that shuts off, and obtains the casein drop of 150mL yak milk Qula
Sugared lipid-loweringing peptide solution, wherein the content of hypoglycemic peptide is 7.0g；
4) 1.8g cycloheptaamylose is weighed, 100mL water is added, dissolves it sufficiently, obtains carrier solution；Then by yak
The casein hypoglycemic peptide solution of cow's milk Qula is added in carrier solution, after 2h is stirred at room temperature, spray-dried obtained yak
The casein hypoglycemic Gly-His-Lys of cow's milk Qula.The content of ashes of measurement product is less than 2%, bitterness value 15, and with certain
Peculiar smell (being charred taste).Through the foregoing embodiment with comparative example it is found that method of the invention can effectively improve yak milk casein
Degree of hydrolysis, significantly reduce polypeptide products in content of ashes and bitterness value.Method of the invention is not necessarily to subsequent cumbersome desalination
Link is conducive to industrialized production, and it is good that product special flavour is made.
Embodiment 9: the effect measuring of the casein hypoglycemic peptide of yak milk Qula
1. experimental material and method
1.1 experimental material
1) the casein hypoglycemic peptide of yak milk Qula
The casein hypoglycemic peptide of yak milk Qula made from embodiment 1.
2) experimental animal and feed
C57BL/6 mouse, male, 4 week old, are purchased from Beijing Vital River Experimental Animals Technology Co., Ltd. by 40；It is high
Rouge feed and normal diet are purchased from U.S. Research Diets company.
3) main agents
Glucose testing kit, total cholesterol kit, triglyceride reagent box, high-density lipoprotein cholesterol kit and
Low density lipoprotein cholesterol kit builds up Bioengineering Research Institute purchased from Nanjing.
1.2 experimental method
1) foundation of obese model
40 C57BL/6 mouse to be grouped after basal feed adaptable fed 1 week at random according to weight.32
C57BL/6 mouse continuously feeds high lipid food 12 weeks, establishes obesity C57BL/6 mouse model.8 C57BL/6 mouse feedings are just
Normal feed, as Normal group.
2) experimental design
By 32 C57BL/6 obesity mices, it is divided into 4 groups, every group 8, it is bent is set to obese model control group, yak milk
Casein hypoglycemic peptide low dose group, the casein hypoglycemic peptide middle dose group of yak milk Qula and the yak milk Qula of drawing
Casein hypoglycemic peptide high dose group.Specific experiment animal packet and dosage are as described in Table 1.
The grouping of 1 experimental animal of table and given low
| Group | Feed for nursing | Stomach-filling substance | Dosage (mg/kg b.w.) |
| Normal group | Chow diet | Distilled water | / |
| Obese model control group | High lipid food | Distilled water | / |
| Yak milk casein peptide low dose group | High lipid food | Yak milk casein peptide | 50 |
| Yak milk casein peptide middle dose group | High lipid food | Yak milk casein peptide | 100 |
| Yak milk casein peptide high dose group | High lipid food | Yak milk casein peptide | 200 |
3) feeding manner
Every group of 8 C57BL/6 mouse, are divided into 2 mouse cage raisings, and 5 groups of experiments are carried out simultaneously, fed according to the design of table 1
Different feeds, normal water supply.
4) administration mode
Rat body weight is measured before weekly administration, according to different groups of other different dosing dosage, is dissolved in the distillation of same volume
In water, it is administered in a manner of stomach-filling.Daily timing gastric infusion is primary.Test continuous 6 weeks, during experiment mouse free water and
It ingests.
5) index determining
After experiment, fasting 12h measures weight.Broken end takes blood after anesthesia, collects serum, measures fasting blood in serum
Sugar, triglycerides (TC), total cholesterol (TG), high-density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol
(LDL-c)。
2. experimental result and analysis
Different disposal group C57BL/6 mouse weight and blood items biochemical indicator, as described in Table 2.
2 different disposal group C57BL/6 mouse weight of table and blood items biochemical indicator
Note: numerical value is indicated in the form of mean+SD；Different letters represent significant difference, p < 0.05.
Before experiment, hyperlipidemia model control group and each dosage group mouse weight be all remarkably higher than Normal group (p <
0.05) Mice model of obesity can be successfully established by, illustrating that high lipid diet is continuously fed mouse 12 weeks.After experiment, with hyperlipidemia model
Group is compared, and each dosage group mouse weight is remarkably decreased (p < 0.05), moreover, dose-dependent effect is presented.
Compared with hyperlipidemia model group, the fasting blood-glucose of middle dose group and high dose group mouse is substantially less than hyperlipidemia model group (p
<0.05), difference is not significant (p>0.05) between Normal group.
Each dosage group mouse TG, TC and LDL-c content is substantially less than hyperlipidemia model group (p < 0.05), poor between each dosage group
Different not significant (p > 0.05).
Each dosage group mouse HDL-c content is significantly higher than hyperlipidemia model group (p < 0.05), middle dose group, high dose group with just
Difference is not significant (p > 0.05) between normal control group.
3. experiment conclusion
The casein hypoglycemic peptide of the yak milk Qula of 100mg/kg b.w. dosage, being capable of effective obesity controlling
The increase of C57BL/6 mouse weight reduces TG, TC and LDL-c content in its fasting blood-glucose and blood, increases in its blood
HDL-c content.The casein hypoglycemic peptide of yak milk Qula of the present invention is taken for a long time to hyperglycemia and hyperlipemia
Disease has certain auxiliary curative effect.
The foregoing is merely presently preferred embodiments of the present invention, and embodiments of the present invention are not by the limit of above-described embodiment
System, other are any without departing from made changes, modifications, substitutions, combinations, simplifications under spirit of the invention and principle, should be
Equivalent substitute mode, is included within the scope of the present invention.
Claims (5)
1. a kind of preparation method of the casein hypoglycemic peptide based on yak milk Qula, which comprises the following steps:
(1) yak milk Qula raw material is crushed to 30-120 mesh, by weight: volume ratio 1g:(10-30ml) it is added 30-50 DEG C
Water in, impregnate 1-5 h, obtain yak milk Qula aqueous solution；Using physics auxiliary dissolving technology to yak milk Qula aqueous solution
After pretreatment, its pH to 4-7 is adjusted, stirs 30-90 min；It is then centrifuged for degreasing, removal impurity, is carried out after collecting supernatant
Filtering collects filtrate, obtains yak milk Qula casein solution；
The mode of the described physics auxiliary dissolving technology are as follows: in pressure be 200-600 MPa, under conditions of temperature is 35-65 DEG C
Handle 15-30 min；Or ultrasonic power be 1000-1500 W, temperature be 40-60 DEG C under conditions of handle 20-40 min；
Or microwave power be 100-700 W, temperature be 40-100 DEG C under conditions of handle 10-300 s；
(2) pH to 2-4 for adjusting yak milk Qula casein solution, is added pepsin, enzyme is carried out under the conditions of 25-65 DEG C
Solution reaction, reaction time are 1-3 h；
(3) the temperature-resistant of enzyme digestion reaction system is kept, the pH to 6-8 of reaction system is adjusted, trypsase is added, continues enzyme
After solving 1-3 h, reaction system is warming up to 75-95 DEG C, 10-20 min is kept, inactivates pepsin and trypsase therein
Reaction is terminated, yak milk Qula casein hydrolyzate is obtained；
(4) centrifuging and taking supernatant after cooling down yak milk Qula casein hydrolyzate, and be by the molecular weight that shuts off by supernatant
The ultrafiltration membrane of 3000-10000 Da collects ultrafiltrate, obtains the casein hypoglycemic peptide solution of yak milk Qula；
(5) the casein hypoglycemic peptide solution of yak milk Qula is concentrated into mass concentration is 30%-60%, is adjusted the temperature to
20-65 DEG C, protease is added and carries out plastein reaction, after reacting 0.5-3 h, heats enzyme deactivation, obtain the light yak milk of bitter taste
Casein hypoglycemic peptide solution；The protease is pepsin, trypsase, flavor protease, neutral proteinase or wood
Melon protease；
The protease additional amount are as follows: the casein hypoglycemic peptide of every 100 g yak milk Qula is added 50-1000 KU's
Protease；
(6) the casein hypoglycemic peptide solution of yak milk Qula is dried, obtains the casein of yak milk Qula
Hypoglycemic Gly-His-Lys.
2. a kind of preparation method of casein hypoglycemic peptide based on yak milk Qula according to claim 1, special
Sign is, the additional amount of pepsin described in step (2) are as follows: the stomach egg of 500-1000 KU is added in every 100 g yak milk Qula
White enzyme.
3. a kind of preparation method of casein hypoglycemic peptide based on yak milk Qula according to claim 1, special
Sign is, the additional amount of trypsase described in step (3) are as follows: the pancreas egg of 500-1000 KU is added in every 100 g yak milk Qula
White enzyme.
4. a kind of described in any item preparation method systems of the casein hypoglycemic peptide based on yak milk Qula of claim 1-3
Standby hypoglycemic peptide.
5. hypoglycemic peptide as claimed in claim 4 is preparing the application in food-borne food for reducing blood sugar and blood fat or health care product.
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| CN101736067A (en) * | 2010-01-08 | 2010-06-16 | 中国农业大学 | Lactoalbumin antihypertensive peptide, preparation method and application thereof |
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| CN101736067A (en) * | 2010-01-08 | 2010-06-16 | 中国农业大学 | Lactoalbumin antihypertensive peptide, preparation method and application thereof |
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| CN103053788A (en) * | 2013-01-28 | 2013-04-24 | 甘肃农业大学 | Method for preparing enzymatic cross-linked casein by taking yak milk casein as raw material |
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