CN105506046A - Yak milk Qula based casein glucose-lowering lipid-lowering peptide as well as preparation method and application thereof - Google Patents
Yak milk Qula based casein glucose-lowering lipid-lowering peptide as well as preparation method and application thereof Download PDFInfo
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Classifications
-
- 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
Abstract
The invention discloses a yak milk Qula based casein glucose-lowering lipid-lowering peptide as well as a preparation method and application thereof, belonging to the field of biotechnology. The preparation method comprises: crushing and soaking yak milk Qula, then carrying out pretreatment by adopting a physical assisted dissolution technology, then adjusting the pH value to 4-7, and carrying out filtration after centrifugation to obtain a yak milk Qula casein solution; sequentially hydrolyzing the yak milk Qula casein solution by using pepsin and trypsin, carrying out centrifugation, and taking a supernatant to carry out ultrafiltration so as to obtain a casein glucose-lowering lipid-lowering peptide solution of the yak milk Qula; and carrying out bitterness removal treatment on the casein glucose-lowering lipid-lowering peptide solution by using plastein reaction after concentration, and drying to obtain yak milk Qula casein glucose-lowering lipid-lowering peptide powder. The raw materials adopted by the preparation method are cheap, and the obtained glucose-lowering lipid-lowering peptide is high in nutritive value, has no side effects, is good in absorption effect, can achieve a good auxiliary glucose-lowering lipid-lowering effect on diabetes and hyperlipemia, and has a good application prospect in the preparation of food-borne glucose-lowering lipid-lowering foods or health care products.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of based on bent casein hypoglycemic peptide drawn of yak milk and its preparation method and application.
Background technology
In recent years, along with raising and the living-pattern preservation of people's living standard, the sickness rate of the metabolic diseases such as diabetes, obesity, arteriosclerosis, fatty liver raises year by year.According to IDF (InternationalDiabetesFederation, IDF) statistics, by 2014, global diabetes number reached 3.82 hundred million, expects 2035, the puzzlement that the whole world will have 5.92 hundred million people to be subject to diabetes.At present, China diabetic subject number has reached 0.96 hundred million.Body is in hyperglycemia state for a long time can bring out fat metabolic disturbance, causes hyperlipemia, increases the sickness rate of cardiovascular disorder.According to the display of the World Health Organization (WorldHealthOrganization, WHO) publish data, within 2012, the whole world about has 1,750 ten thousand people to die from cardiovascular disorder, accounts for 31% of the dead sum in the whole world.The 2014 Chinese cardiovascular diseases reports of issuing, China's cardiovascular patient number is 2.9 hundred million, and cardiovascular disorder is occupied first of people's various diseases cause of the death.All there is the untoward reactions such as hepar damnification, lactic acidosis and gastrointestinal discomfort in the medicine of the metabolic diseases such as current treatment diabetes and hyperlipidemia, therefore develops natural, safe, to have hypoglycemic biologic activity efficiently food source functional factor in the urgent need to seeking.
The newborn resource that Qu Lashi China is exclusive, it is the yak milk rough cheese that spontaneous fermentation is obtained after degreasing, and its casein content is up to more than 70%.The multiple bioactive sequences that yak milk casein contains, utilizes protease hydrolysis technology to discharge, and produces the polypeptide with biologic activity such as hypotensive, anti-oxidant, antibacterial, hypoglycemics.
The dissolving of yak milk casein needs under alkaline pH system, and under neutrality and slightly acidic pH system, solubleness is very low.Therefore, in order to fully dissolve yak milk casein, realize proteolytic enzyme to its effectively hydrolyzing, patented technology all adds a large amount of alkali lye solubilising casein mostly at present, but this processing mode can cause salt content in enzymolysis solution too high, not only affect the quality of product, and the absorption of high salt also can threaten the health of human body.Also have utilize nanofiltration to preparation yak milk casein antihypertensive peptide carry out desalination, but this method complex operation and consuming time, efficiency is low, is unfavorable for suitability for industrialized production.
In addition, can produce a series of molecular weight at below 6000Da after casein hydrolysis, and containing the polypeptide of hydrophobic amino acid, this kind of polypeptide, with bitter taste in various degree, affects its application in field of food.Therefore, need to carry out debitterize process to enzymolysis product.Can carry out debitterize process to the trypsin hydrolyzing liquid of yak milk casein with active carbon adsorption, but this method can be lost the functional component in protein polypeptide and reduce its biologic activity; Take cycloheptaamylose as the bitterness problem that carrier also can solve hydrophobic peptides in yak milk casein peptide, but, adding of cycloheptaamylose, not only exogenous material is introduced to protein peptide system, but also system can be made to produce certain be charred peculiar smell.
Summary of the invention
The object of the present invention is to provide a kind of based on bent casein hypoglycemic peptide drawn of yak milk and its preparation method and application.
To achieve these goals, the technical scheme taked of the present invention is as follows:
Based on a preparation method for the casein hypoglycemic peptide that yak milk song draws, comprise the following steps:
(1) draw raw material pulverizing to 30-120 order yak milk song, by weight: volume ratio is 1g:(10-30ml) add in the water of 30-50 DEG C, soak 1-5h, obtain yak milk song and draw the aqueous solution; After adopting physics assist in dissolving technology to draw aqueous solution pre-treatment to yak milk song, regulate its pH to 4-7, stir 30-90min; Then centrifugal degreasing, removal impurity, filter after collecting supernatant liquor, collect filtrate, obtain yak milk song and draw casein solution;
(2) regulate yak milk song to draw the pH to 2-4 of casein solution, add stomach en-, under 25-65 DEG C of condition, carry out enzyme digestion reaction, the reaction times is 1-3h;
(3) the temperature-resistant of enzyme digestion reaction system is kept, the pH to 6-8 of adjustment reaction system, add trypsinase, after continuing enzymolysis 1-3h, reaction system is warming up to 75-95 DEG C, keep 10-20min, deactivation stomach en-wherein and trypsinase termination reaction, obtain yak milk song and draw casein hydrolyzate;
(4) drawn by yak milk song casein hydrolyzate to cool rear centrifuging and taking supernatant liquor, and be the ultra-filtration membrane of 3000-10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that yak milk song draws;
(5) the casein hypoglycemic peptide solution that yak milk song draws being concentrated into mass concentration is 30%-60%, adjust the temperature to 20-65 DEG C, add proteolytic enzyme and carry out plastein reaction, after reaction 0.5-3h, heat the enzyme that goes out, obtain the yak milk casein hypoglycemic peptide solution that bitter taste is light;
(6) drying treatment is carried out to the casein hypoglycemic peptide solution that yak milk song draws, obtain the casein hypoglycemic Gly-His-Lys that yak milk song draws.
The mode of the physics assist in dissolving technology described in step (1) is: be 200-600MPa at pressure, and temperature is process 15-30min under the condition of 35-65 DEG C; Or be 1000-1500W at ultrasonic power, temperature is process 20-40min under the condition of 40-60 DEG C; Or be 100-700W at microwave power, temperature is process 10-300s under the condition of 40-100 DEG C.
Pepsic add-on described in step (2) is that every 100g yak milk song draws the stomach en-adding 500-1000KU; Wherein, 1U is defined as the amount that per minute caseinhydrolysate at 40 DEG C produces 1 μ g tyrosine.
Tryptic add-on described in step (3) is that every 100g yak milk song draws the trypsinase adding 500-1000KU; Wherein, 1U is defined as the amount that per minute caseinhydrolysate at 40 DEG C produces 1 μ g tyrosine.
Proteolytic enzyme described in step (5) is stomach en-, trypsinase, flavor protease, neutral protease or papoid.
Described in step (5), the add-on of proteolytic enzyme is: the casein hypoglycemic peptide that every 100g yak milk song draws adds the proteolytic enzyme of 50-1000KU; Wherein, 1U is defined as the amount that per minute caseinhydrolysate at 40 DEG C produces 1 μ g tyrosine.
Described a kind of hypoglycemic peptide obtained based on the preparation method of the bent casein hypoglycemic peptide drawn of yak milk.
Described hypoglycemic peptide is preparing the application in food source property food for reducing blood sugar and blood fat or healthcare products.
Described hypoglycemic Toplink be used alone or with the composite use of natural hypoglycemic activeconstituents.
Beneficial effect of the present invention is: described preparation method by ultra-high voltage/ultrasonic wave/microwave assisted techniques in conjunction with simulating gastrointestinal digestion hydrolysis pattern, effectively improve caseic degree of hydrolysis, achieve the controllable release of active amino acid sequence, improve its stomach and intestine stability; The plastein reaction adopted modifies the bitterness problem that debitterness technology solves hypoglycemic peptide on the basis of harmless polypeptide biologic activity; And the prices of raw and semifnished materials that described preparation method adopts are cheap, reduce production cost, the hypoglycemic peptide obtained is of high nutritive value, has no side effect, good absorbing effect, good assistant hypoglycemic effect for reducing fat can be played to diabetes and hyperlipidaemia, have a good application prospect preparing in food source property food for reducing blood sugar and blood fat or healthcare products.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.
Embodiment 1:
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 3h of 200mL40 DEG C, obtain yak milk song and draw the aqueous solution; Be 300MPa at pressure, temperature is process after yak milk song draws aqueous solution 20min under the condition of 50 DEG C, regulates its pH to 5, stirs 30min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 2.0 of casein solution, add stomach en-50KU, under 40 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 1h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 50KU, and after continuing hydrolysis 2h, reaction system is warming up to 85 DEG C, keep 10min, enzyme termination reaction of going out, obtains yak milk song and draw casein hydrolyzate, and the degree of hydrolysis measuring now enzymolysis solution is 22.8%;
3) after the centrifugal 15min of 4000r/min, supernatant liquor is got after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 150mL yak milk song draws, wherein the content of hypoglycemic peptide is 7.5g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 20mL, adjustment solution temperature to 50 DEG C, add papoid 45KU and react 1h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 2.
In above-mentioned experimental procedure, each experimental data obtains according to following measuring method:
(1) mensuration (trinitro-benzene-sulfonic acid method) of degree of hydrolysis:
Draw the gone out yak milk song of enzyme of 0.5mL and draw casein hydrolyzate, after cooling, utilize 1% sodium lauryl sulphate to be settled to 25mL, leave standstill.The 0.1% trinitro-benzene-sulfonic acid solution getting the above-mentioned enzymolysis solution diluent of 0.125mL and 1mL phosphoric acid buffer (pH8.2,0.2mol/L) and 1mL mixes, lucifuge reaction on 50 DEG C of shaking baths.Add the HCl solution termination reaction of 2mL0.1mol/L after 60min, after room temperature leaves standstill 30min, under 420nm, measure its absorbance.Utilize 0-5.0 × 10
-3the L-Leu drawing standard curve of mol/L concentration.Following formula is utilized to calculate degree of hydrolysis.
In formula, h: to represent in hydrolyzate every gram of cleaved peptide bond mmole number (meq/gprotein); Htot: represent peptide bond mmole number total in every gram of material protein.
(2) mensuration (Method of burning of high temperature) of ash content:
Take the crucible that the bent casein hypoglycemic Gly-His-Lys drawn of 3g yak milk is placed in constant weight, add 1mL24% magnesium acetate solution, make the casein hypoglycemic Gly-His-Lys complete wetting that yak milk song draws.After placing 10min, by after moisture evaporate to dryness in water-bath, the casein hypoglycemic Gly-His-Lys first with little fire heating, yak milk song being drawn on hot plate fully carbonizes to smokelessly, is then placed in retort furnace, at 550 DEG C ± 25 DEG C calcination 4h.Be cooled to about 200 DEG C, take out, put into moisture eliminator and cool 30min, weigh.
In formula: X
1the content of ash content in-sample, g/100g;
M
0the quality of-magnesium oxide (after magnesium acetate calcination resultant), g;
M
1the quality of-crucible and ash content, g;
M
2the quality of-crucible, g;
M
3the quality of-crucible and sample, g;
(3) mensuration (sensory evaluation method) of bitterness value:
By soluble in water with 5% mass concentration for testing sample (the casein hypoglycemic Gly-His-Lys that yak milk song draws), carry out gradient dilution in 1:1 ratio; Support samples is soluble in water by 5% mass concentration, carry out gradient dilution in 1:1 ratio; Utilize 3 laboratory methods to carry out sensory evaluation, by dilution on an equal basis two support samples and a testing sample random number, be supplied to experimenter, detect the bitterness can distinguishing them; Represent bitterness value with the minimum extension rate can not tasting bitter taste difference, the larger then bitterness value of this extension rate is higher, illustrates that the bitterness of product is heavier.
Embodiment 2:
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 3h of 200mL40 DEG C, obtain yak milk song and draw the aqueous solution; Be 350MPa at pressure, temperature is process after yak milk song draws aqueous solution 20min under the condition of 50 DEG C, regulates its pH to 5.5, stirs 30min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 3.0 of casein solution, add stomach en-80KU, under 45 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 1h; The pH to 6.0 of adjustment enzyme digestion reaction system, adds trypsinase 60KU, after continuing hydrolysis 1.5h, reaction system is warming up to 85 DEG C, keeps 10min, enzyme termination reaction of going out, obtain yak milk song and draw casein hydrolyzate, the degree of hydrolysis measuring now enzymolysis solution is 20.5%;
3) supernatant liquor is got after the centrifugal 15min of 4000r/min after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 150mL yak milk song draws, wherein the content of hypoglycemic peptide is 7.1g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 15mL, adjustment temperature to 50 DEG C, add flavor protease 55KU and react 0.5h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 3.The measuring method of each data is with embodiment 1.
Embodiment 3:
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 1.5h of 200mL45 DEG C, obtain yak milk song and draw the aqueous solution; Be 1500W at ultrasonic power, temperature is process after yak milk song draws aqueous solution 20min under the condition of 40 DEG C, regulates its pH to 6.5, stirs 50min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 2.5 of casein solution, add stomach en-100KU, under 40 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 0.5h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 80KU, and after continuing hydrolysis 1h, reaction system is warming up to 85 DEG C, keep 10min, enzyme termination reaction of going out, obtains yak milk song and draw casein hydrolyzate, and the degree of hydrolysis measuring now enzymolysis solution is 21.2%;
3) supernatant liquor is got after the centrifugal 15min of 4000r/min after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 8000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 150mL yak milk song draws, wherein the content of hypoglycemic peptide is 7.2g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 18mL, adjustment temperature to 40 DEG C, add trypsinase 40KU and react 1.5h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 5.The measuring method of each data is with embodiment 1.
Embodiment 4:
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 2h of 150mL45 DEG C, obtain yak milk song and draw the aqueous solution; Be 1000W at ultrasonic power, temperature is process after yak milk song draws aqueous solution 40min under the condition of 50 DEG C, regulates its pH to 6.0, stirs 60min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 3.5 of casein solution, add stomach en-60KU, under 37 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 2h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 100KU, after continuing hydrolysis 2h, reaction system is warming up to 85 DEG C, keeps 10min, enzyme termination reaction of going out, obtain yak milk song and draw casein hydrolyzate, the degree of hydrolysis measuring now enzymolysis solution is 22.5%;
3) supernatant liquor is got after the centrifugal 15min of 4000r/min after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 100mL yak milk song draws, wherein the content of hypoglycemic peptide is 7.2g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 20mL, adjustment temperature to 35 DEG C, add stomach en-60KU and react 1h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 6.The measuring method of each data is with embodiment 1.
Embodiment 5:
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 2h of 300mL45 DEG C, obtain yak milk song and draw the aqueous solution; Be 400W at microwave power, temperature is process after yak milk song draws aqueous solution 100s under the condition of 80 DEG C, regulates its pH to 5.5, stirs 90min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 4.0 of casein solution, add stomach en-100KU, under 37 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 1.5h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 50KU, and after continuing hydrolysis 3h, reaction system is warming up to 85 DEG C, keep 10min, enzyme termination reaction of going out, obtains yak milk song and draw casein hydrolyzate, and the degree of hydrolysis measuring now enzymolysis solution is 23.8%;
3) supernatant liquor is got after the centrifugal 15min of 4000r/min after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 6000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 250mL yak milk song draws, wherein the content of hypoglycemic peptide is 6.8g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 15mL, adjustment temperature to 50 DEG C, add flavor protease 35KU and react 3h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 4.The measuring method of each data is with embodiment 1.
Embodiment 6:
1) take 10g to be crushed to 80 object yak milk songs and to draw, add the water soaking 2h of 250mL40 DEG C, obtain yak milk song and draw the aqueous solution; Be 500W at microwave power, temperature is process after yak milk song draws aqueous solution 70s under the condition of 60 DEG C, regulates its pH to 6.0, stirs 45min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 4.0 of casein solution, add stomach en-50KU, under 40 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 0.5h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 80KU, and after continuing hydrolysis 2h, reaction system is warming up to 85 DEG C, keep 10min, enzyme termination reaction of going out, obtains yak milk song and draw casein hydrolyzate, and the degree of hydrolysis measuring now enzymolysis solution is 21.1%;
3) supernatant liquor is got after the centrifugal 15min of 4000r/min after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 6000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 200mL yak milk song draws, wherein the content of hypoglycemic peptide is 5.0g;
4) drawn by yak milk song casein hypoglycemic peptide solution to be concentrated into 10mL, adjustment temperature to 50 DEG C, add papoid 50KU and react 0.5h, heat the enzyme that goes out.Drying obtains the casein hypoglycemic Gly-His-Lys that yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 3.The measuring method of each data is with embodiment 1.
Embodiment 7 (comparative example 1):
Take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 3h of 200mL40 DEG C, obtain yak milk song and draw the aqueous solution, regulate its pH to 9.0, stir 30min; Then be centrifugal 20min under the condition of 4000r/min at rotating speed, filter after collecting supernatant, collect filtrate, obtain yak milk song and draw casein solution.
Adjustment yak milk song draws the pH to 2.0 of casein solution, and add stomach en-50KU, under 37 DEG C of water bath condition, be hydrolyzed reaction, reaction 2h; Then adjust the pH to 7.0 of reaction system, add trypsinase 50KU, continue hydrolysis 2h, constantly add in hydrolytic process alkali lye maintain reaction system pH constant; Reaction system is warming up to 85 DEG C, keep 10min, enzyme termination reaction of going out, obtains yak milk song and draws casein hydrolyzate, measures now enzymolysis solution and is hydrolyzed to 15.4%.
After being drawn by yak milk song casein hydrolyzate to cool, get supernatant liquor after the centrifugal 15min of 4000r/min, and be the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, adjustment solution temperature to 50 DEG C after concentrated, adds papoid 45KU and reacts 1h, heat the enzyme that goes out.The casein hypoglycemic peptide that yak milk song draws is obtained after drying.Measure the ash oontent of product more than 5%, bitterness value is 3.Measuring method is with embodiment 1.
Embodiment 8 (comparative example 2):
1) take 10g to be crushed to 60 object yak milk songs and to draw, add the water soaking 3h of 200mL40 DEG C, obtain yak milk song and draw the aqueous solution; Be 300MPa at pressure, temperature is process after yak milk song draws aqueous solution 25min under the condition of 60 DEG C, regulates its pH to 5.5, stirs 40min; Then be under the condition of 4000r/min at rotating speed, centrifugal 20min, filters after collecting supernatant, collects filtrate, obtains yak milk song and draw casein solution;
2) regulate yak milk song to draw the pH to 2.0 of casein solution, add stomach en-40KU, under 40 DEG C of water bath condition, carry out enzyme digestion reaction, reaction 1.5h; The pH to 7.0 of adjustment enzyme digestion reaction system, adds trypsinase 60KU, after continuing hydrolysis 1.5h, reaction system is warming up to 75 DEG C, keeps 20min, enzyme termination reaction of going out, obtain yak milk song and draw casein hydrolyzate, the degree of hydrolysis measuring now enzymolysis solution is 25.3%;
3) after the centrifugal 15min of 4000r/min, supernatant liquor is got after being drawn by yak milk song casein hydrolyzate to cool, and be the ultra-filtration membrane of 10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that 150mL yak milk song draws, wherein the content of hypoglycemic peptide is 7.0g;
4) take 1.8g cycloheptaamylose, add 100mL water, make it fully dissolve, obtain carrier soln; Then the casein hypoglycemic peptide solution that yak milk song draws is joined in carrier soln, after stirring at room temperature 2h, the casein hypoglycemic Gly-His-Lys that spray-dried obtained yak milk song draws.The ash oontent measuring product is less than 2%, and bitterness value is 15, and with certain peculiar smell (being charred taste).By above-described embodiment and comparative example known, method of the present invention effectively can improve the degree of hydrolysis of yak milk casein, significantly reduces the ash oontent in polypeptide products and bitterness value.Method of the present invention, without the need to follow-up loaded down with trivial details desalination link, is beneficial to suitability for industrialized production, and obtained product special flavour is good.
Embodiment 9: the effect measuring of the casein hypoglycemic peptide that yak milk song draws
1. experiment material and method
1.1 experiment material
1) the bent casein hypoglycemic peptide drawn of yak milk
The bent casein hypoglycemic peptide drawn of yak milk that embodiment 1 is obtained.
2) laboratory animal and feed
C57BL/6 mouse, male, 4 week age, 40, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.; High lipid food and normal diet, purchased from ResearchDiets company of the U.S..
3) main agents
Glucose testing kit, total cholesterol test kit, Triglyceride Reagent box, high density lipoprotein cholesterol test kit and low density lipoprotein cholesterol test kit, build up Bioengineering Research Institute purchased from Nanjing.
1.2 experimental technique
1) foundation of obese model
After 40 C57BL/6 mouse feed 1 week with basal feed adaptability, carry out random packet according to body weight.32 C57BL/6 mouse are fed high lipid food 12 weeks continuously, set up fat C57BL/6 mouse model.8 C57BL/6 mouse are fed chow diet, as Normal group.
2) experimental design
By 32 C57BL/6 obesity mices, be divided into 4 groups, often organize 8, be set to obese model control group respectively, dosage group and yak milk song draw in casein hypoglycemic peptide low dose group that yak milk song draws, the casein hypoglycemic peptide that draws of yak milk song casein hypoglycemic peptide high dose group.Specific experiment animal grouping and dosage as described in Table 1.
The grouping of table 1 laboratory animal and given low
| Group | Feed for nursing | Gavage material | 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 |
| Dosage group in yak milk casein peptide | 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
Often organize 8 C57BL/6 mouse, be divided into 2 mouse cages to raise, 5 groups of experiments are carried out simultaneously, to feed DIFFERENT FEED, normally feed water according to the design of table 1.
4) administering mode
Measure rat body weight before Per-Hop behavior, according to the different dosing dosage of different group, be dissolved in the distilled water of same volume, with the mode administration of gavage.Timing every day gastric infusion once.Test continuous 6 weeks, experimental session mouse freely drinks water and ingests.
5) index determining
After experiment terminates, fasting 12h, measures body weight.After anesthesia, broken end gets blood, collect serum, measure fasting plasma glucose, triglyceride level (TC), total cholesterol (TG), high density lipoprotein cholesterol (HDL-c) and low density lipoprotein cholesterol (LDL-c) in serum.
2. experimental result and analysis
Different treatment group C57BL/6 Mouse Weight and the every biochemical indicator of blood, as described in Table 2.
Table 2 different treatment group C57BL/6 Mouse Weight and the every biochemical indicator of blood
Note: numerical value represents with the form of mean+SD; Different letter represents significant difference, p<0.05.
Before experiment, hyperlipidemia model control group and each dosage group Mice Body weight average are significantly higher than Normal group (p<0.05), illustrate that high lipid diet is fed mouse continuously and successfully can be set up Mice model of obesity in 12 weeks.After experiment, compared with hyperlipidemia model group, each dosage group Mice Body weight average significantly declines (p<0.05), and, present dose-dependent effect.
Compared with hyperlipidemia model group, the fasting plasma glucose of middle dosage group and high dose group mouse is significantly lower than hyperlipidemia model group (p<0.05), and difference remarkable (p>0.05) between Normal group.
Each dosage group mouse TG, TC and LDL-c content is significantly lower than hyperlipidemia model group (p<0.05), and between each dosage group, difference is not significantly (p>0.05).
Each dosage group mouse HDL-c content is significantly higher than hyperlipidemia model group (p<0.05), middle dosage group, difference remarkable (p>0.05) between high dose group and Normal group.
3. experiment conclusion
The casein hypoglycemic peptide that draws of yak milk song of 100mg/kgb.w. dosage, can the increase of effective obesity controlling C57BL/6 Mouse Weight, reduces TG, TC and LDL-c content in its fasting plasma glucose and blood, raises the HDL-c content in its blood.The casein hypoglycemic peptide that long-term taking yak milk of the present invention song draws has certain auxiliary curative effect to hyperglycemia and hyperlipidaemia.
The foregoing is only preferred embodiment of the present invention; embodiments of the present invention are not restricted to the described embodiments; other are any do not deviate from spirit of the present invention and principle under do change, modification, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1., based on a preparation method for the bent casein hypoglycemic peptide drawn of yak milk, it is characterized in that, comprise the following steps:
(1) draw raw material pulverizing to 30-120 order yak milk song, by weight: volume ratio is 1g:(10-30ml) add in the water of 30-50 DEG C, soak 1-5h, obtain yak milk song and draw the aqueous solution; After adopting physics assist in dissolving technology to draw aqueous solution pre-treatment to yak milk song, regulate its pH to 4-7, stir 30-90min; Then centrifugal degreasing, removal impurity, filter after collecting supernatant liquor, collect filtrate, obtain yak milk song and draw casein solution;
(2) regulate yak milk song to draw the pH to 2-4 of casein solution, add stomach en-, under 25-65 DEG C of condition, carry out enzyme digestion reaction, the reaction times is 1-3h;
(3) the temperature-resistant of enzyme digestion reaction system is kept, the pH to 6-8 of adjustment reaction system, add trypsinase, after continuing enzymolysis 1-3h, reaction system is warming up to 75-95 DEG C, keep 10-20min, deactivation stomach en-wherein and trypsinase termination reaction, obtain yak milk song and draw casein hydrolyzate;
(4) drawn by yak milk song casein hydrolyzate to cool rear centrifuging and taking supernatant liquor, and be the ultra-filtration membrane of 3000-10000Da by the molecular weight that dams by supernatant liquor, collect ultrafiltrated, obtain the casein hypoglycemic peptide solution that yak milk song draws;
(5) the casein hypoglycemic peptide solution that yak milk song draws being concentrated into mass concentration is 30%-60%, adjust the temperature to 20-65 DEG C, add proteolytic enzyme and carry out plastein reaction, after reaction 0.5-3h, heat the enzyme that goes out, obtain the yak milk casein hypoglycemic peptide solution that bitter taste is light;
(6) drying treatment is carried out to the casein hypoglycemic peptide solution that yak milk song draws, obtain the casein hypoglycemic Gly-His-Lys that yak milk song draws.
2. a kind of preparation method based on the bent casein hypoglycemic peptide drawn of yak milk according to claim 1, it is characterized in that, the mode of the physics assist in dissolving technology described in step (1) is: be 200-600MPa at pressure, and temperature is process 15-30min under the condition of 35-65 DEG C; Or be 1000-1500W at ultrasonic power, temperature is process 20-40min under the condition of 40-60 DEG C; Or be 100-700W at microwave power, temperature is process 10-300s under the condition of 40-100 DEG C.
3. a kind of preparation method based on the bent casein hypoglycemic peptide drawn of yak milk according to claim 1, it is characterized in that, described in step (2), pepsic add-on is: every 100g yak milk song draws the stomach en-adding 500-1000KU.
4. a kind of preparation method based on the bent casein hypoglycemic peptide drawn of yak milk according to claim 1, it is characterized in that, described in step (3), tryptic add-on is: every 100g yak milk song draws the trypsinase adding 500-1000KU.
5. a kind of preparation method based on the bent casein hypoglycemic peptide drawn of yak milk according to claim 1, it is characterized in that, described in step (5), proteolytic enzyme is stomach en-, trypsinase, flavor protease, neutral protease or papoid.
6. a kind of preparation method based on the bent casein hypoglycemic peptide drawn of yak milk according to claim 1, it is characterized in that, the proteolytic enzyme add-on described in step (5) is: the casein hypoglycemic peptide that every 100g yak milk song draws adds the proteolytic enzyme of 50-1000KU.
7. a kind of hypoglycemic peptide prepared based on the preparation method of the bent casein hypoglycemic peptide drawn of yak milk described in any one of claim 1-6.
8. hypoglycemic peptide according to claim 7 is preparing the application in food source property food for reducing blood sugar and blood fat or healthcare products.
9. application according to claim 8, is characterized in that, described hypoglycemic Toplink be used alone or with the composite use of natural hypoglycemic activeconstituents.
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| CN113061175A (en) * | 2020-12-17 | 2021-07-02 | 西北民族大学 | Calcium coprecipitation protein prepared by taking Triton as raw material and preparation method thereof |
| CN112569347A (en) * | 2020-12-30 | 2021-03-30 | 青海瑞肽生物科技有限公司 | Application of yak collagen peptide in hypoglycemic drugs or hypoglycemic health foods |
| CN112569347B (en) * | 2020-12-30 | 2023-09-22 | 青海瑞肽生物科技有限公司 | Application of yak collagen peptide in hypoglycemic drugs or hypoglycemic health-care foods |
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