CN102863526B - Separation method of yak milk milk fat globule membrane protein - Google Patents
Separation method of yak milk milk fat globule membrane protein Download PDFInfo
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- CN102863526B CN102863526B CN201210374928.7A CN201210374928A CN102863526B CN 102863526 B CN102863526 B CN 102863526B CN 201210374928 A CN201210374928 A CN 201210374928A CN 102863526 B CN102863526 B CN 102863526B
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Abstract
The invention discloses a separation method of yak milk milk fat globule membrane protein, relates to a separation method of protein and solves problems of low purity and unstable properties in terms of cow's milk milk fat globule membrane protein prepared by existing methods. The separation method includes that cane sugar is added in yak milk, a milk fat initial extract is obtained through centrifugation, then centrifugal washing is performed with a sodion phosphate buffer solution and ultrapure water, and finally the centrifugation is performed with a polyethylene glycol octyl phenyl ether solution to obtain the yak milk milk fat globule membrane protein. The prepared yak milk milk fat globule membrane protein has the advantages that the properties are stable, the purity can reach to 93.5%, and problems of low purity and unstable properties in terms of the cow's milk milk fat globule membrane protein prepared by existing separation methods are solved. The separation method is applicable to mass production of the yak milk milk fat globule membrane protein.
Description
Technical field
The present invention relates to a kind of separation method of albumen.
Background technology
Dairy fats is one of main component of cow's milk, dairy fats is scattered in cow's milk with the form of Oil globule, and the film that its surface is 5～10nm by a layer thickness is covered, and accounts for 2%～6% of Oil globule quality, be called milk fat ball film (Milk Fat Globule Menbrane, MFGM).Milk fat ball film is comprised of protein, phosphatide, glycoprotein, triacylglycerol, cholesterol, enzyme and other trace ingredients; wherein; in milk fat ball film albumen, take glycoprotein as main; milk fat ball film albumen accounts for the 25%-60% of milk fat ball film total mass, accounts for the 1%-2% of total milk protein total mass.Research shows, milk fat ball film albumen has good antitumour activity, and the breast cancer susceptibility gene extracting in milk fat ball film egg has the effect that suppresses mammary cancer.The separation method of existing milk fat ball film albumen mainly contains acid deposition method and membrane filter method.Acid deposition method is by using salt acid deposition milk fat ball film albumen, the advantage of the method be simple to operate, cost is low, but hydrochloric acid can affect milk fat ball film protein-active to a certain extent, cause that the purity of the milk fat ball film albumen that separation obtains is on the low side and character is unstable; The milk fat ball film property of protein that membrane filter method obtains is better, but the cost of the method is high, and complicated operation is not suitable for a large amount of productions of milk fat ball film albumen.
Summary of the invention
The present invention will solve the low and unsettled problem of character of the purity of Cow Milk fat globule membrane albumen prepared by existing method, and a kind of separation method of yak milk milk fat ball film albumen is provided.
The separation method of a kind of yak milk milk fat ball film albumen of the present invention carries out according to the following steps:
One, by mass fraction, take the yak milk of 100 parts and the sucrose of 3～8 parts;
Two,, after yak milk step 1 being taken filters with 3～5 layers of gauze, the sucrose that adds step 1 to take, mixes, and obtains cow's milk sucrose mixed solution;
Three, cow's milk sucrose mixed solution step 2 being obtained carries out centrifugal at ambient temperature, obtains the dairy fats primary extract in centrifugal product upper strata; Wherein, centrifugal rotational speed is 3000～4000r/min, and centrifugation time is 20～35min;
Four, in the dairy fats primary extract obtaining in step 3, add 1～3 times to the sodium ion phosphate buffer soln of this dairy fats primary extract volume, after mixing, carry out at ambient temperature centrifuge washing; Wherein, centrifugal rotational speed is 4200～5200r/min, and centrifugation time is 15～25min; The compound method of described sodium ion phosphate buffer soln is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.2～4.4mmol, KCl:2.5～2.8mmol, KH
2pO
4: the ratio of 1.6～1.8mmol and NaCl:135～140mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.2～7.5, obtain sodium ion phosphate buffer soln;
Five,, by the dairy fats primary extract of processing through step 4, by the method repetitive operation of step 4 once, obtain the dairy fats in centrifugal product upper strata;
Six, in the dairy fats obtaining in step 5, add the ultrapure water identical with dairy fats volume, after mixing, carry out at ambient temperature centrifuge washing, obtain the cream in centrifugal product upper strata; Wherein, centrifugal rotational speed is 4200～5200r/min, and centrifugation time is 15～25min;
Seven, cream step 6 being obtained is placed under 2～6 ℃ of conditions, and under this condition standing 12～16h, obtain cream solid;
Eight, cream solid step 7 being obtained is placed under the water bath condition of 40～45 ℃ and melts, and obtains liquid cream;
Nine, in the liquid cream obtaining in step 8, adding volume is the Triton X-100 solution that accounts for liquid state milk oil volume 0.15～0.3%, carries out at ambient temperature centrifugally, and collecting precipitation, obtains yak milk milk fat ball film albumen; Wherein, centrifugal rotational speed is 40000～50000r/min, and centrifugation time is 40～70min; The compound method of described Triton X-100 solution is: by the volume ratio of Triton X-100 and phosphate buffered saline buffer, be that 1:2.5～2.7 take Triton X-100 and phosphate buffered saline buffer, after the two is mixed, be placed in the water-bath of 37～40 ℃, keep 2～3 hours; The concentration of phosphate buffered saline buffer is that 0.07～0.1mol/L, pH are 7.2～7.4.
First the present invention adds sucrose in yak cow's milk, can effectively reduce the loss of milk fat ball film albumen in subsequent separation process; Utilize sodium ion phosphate buffer soln to carry out wash-out to yak milk dairy fats primary extract, sodium ion phosphate buffer soln used in the present invention can wash away casein and the whey-protein being attached on milk fat ball film, has reduced the content of impurity albumen in the milk fat ball film albumen extracting; Utilize nonionic detergent Triton X-100 solution to carry out wash-out to yak milk cream, the nonionic detergent Triton X-100 solution using can lipin dissolving and the character of albumen itself is not impacted, and makes the stable in properties of albumen.Purity to 93～95% of the yak Cow Milk fat globule membrane albumen of preparing by the inventive method, and the purity of yak Cow Milk fat globule membrane albumen prepared by existing method is only 80% left and right, effectively raise the purity of yak Cow Milk fat globule membrane albumen.
The present invention is applicable to a large amount of productions of yak milk milk fat ball film albumen.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the separation method of the yak milk milk fat ball film albumen of present embodiment carries out according to the following steps:
One, by mass fraction, take the yak milk of 100 parts and the sucrose of 3～8 parts;
Two,, after yak milk step 1 being taken filters with 3～5 layers of gauze, the sucrose that adds step 1 to take, mixes, and obtains cow's milk sucrose mixed solution;
Three, cow's milk sucrose mixed solution step 2 being obtained carries out centrifugal at ambient temperature, obtains the dairy fats primary extract in centrifugal product upper strata; Wherein, centrifugal rotational speed is 3000～4000r/min, and centrifugation time is 20～35min;
Four, in the dairy fats primary extract obtaining in step 3, add 1～3 times to the sodium ion phosphate buffer soln of this dairy fats primary extract volume, after mixing, carry out at ambient temperature centrifuge washing; Wherein, centrifugal rotational speed is 4200～5200r/min, and centrifugation time is 15～25min; The compound method of described sodium ion phosphate buffer soln is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.2～4.4mmol, KCl:2.5～2.8mmol, KH
2pO
4: the ratio of 1.6～1.8mmol and NaCl:135～140mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.2～7.5, obtain sodium ion phosphate buffer soln;
Five,, by the dairy fats primary extract of processing through step 4, by the method repetitive operation of step 4 once, obtain the dairy fats in centrifugal product upper strata;
Six, in the dairy fats obtaining in step 5, add the ultrapure water identical with dairy fats volume, after mixing, carry out at ambient temperature centrifuge washing, obtain the cream in centrifugal product upper strata; Wherein, centrifugal rotational speed is 4200～5200r/min, and centrifugation time is 15～25min;
Seven, cream step 6 being obtained is placed under 2～6 ℃ of conditions, and under this condition standing 12～16h, obtain cream solid;
Eight, cream solid step 7 being obtained is placed under the water bath condition of 40～45 ℃ and melts, and obtains liquid cream;
Nine, in the liquid cream obtaining in step 8, adding volume is the Triton X-100 solution that accounts for liquid state milk oil volume 0.15～0.3%, carries out at ambient temperature centrifugally, and collecting precipitation, obtains yak milk milk fat ball film albumen; Wherein, centrifugal rotational speed is 40000～50000r/min, and centrifugation time is 40～70min; The compound method of described Triton X-100 solution is: by the volume ratio of Triton X-100 and phosphate buffered saline buffer, be that 1:2.5～2.7 take Triton X-100 and phosphate buffered saline buffer, after the two is mixed, be placed in the water-bath of 37～40 ℃, keep 2～3 hours; The concentration of phosphate buffered saline buffer is that 0.07～0.1mol/L, pH are 7.2～7.4.
Embodiment two: present embodiment is different from embodiment one: take the yak milk of 20 parts and the sucrose of 1 part by mass fraction in step 1.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment is different from embodiment one or two: in step 3, centrifugal rotational speed is 3800r/min, centrifugation time is 30min.Other step and parameter are identical with embodiment one or two.
Embodiment four: present embodiment is different from one of embodiment one to three: after step 3 and step 4 are centrifugal, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min.Other step and parameter are identical with embodiment one to three.
Embodiment five: present embodiment is different from one of embodiment one to four: the compound method of the sodium ion phosphate buffer soln described in step 4 is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.3mmol, KCl:2.7mmol, KH
2pO
4: the ratio of 1.8mmol and NaCl:137mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.4, obtain sodium ion phosphate buffer soln.Other step and parameter are identical with embodiment one to four.
Embodiment six: present embodiment is different from one of embodiment one to five: in step 4 and step 6, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min.Other step and parameter are identical with embodiment one to five.
Embodiment seven: present embodiment is different from one of embodiment one to six: adding volume in step 9 is the Triton X-100 solution that accounts for liquid state milk oil volume 0.2%.Other step and parameter are identical with embodiment one to six.
Embodiment eight: present embodiment is different from one of embodiment one to seven: in step 9, centrifugal rotational speed is 44910r/min, and centrifugation time is 60min.Other step and parameter are identical with embodiment one to seven.
By following verification experimental verification beneficial effect of the present invention:
Test one: the separation method of a kind of yak milk milk fat ball film albumen of this test carries out according to the following steps:
One, the fresh yak breast of selecting healthy yak, takes the yak milk of 20 parts and the sucrose of 1 part by mass fraction;
Two,, after yak milk step 1 being taken filters with 4 layers of gauze, the sucrose that adds step 1 to take, mixes with magnetic stirring apparatus, obtains cow's milk sucrose mixed solution;
Three, cow's milk sucrose mixed solution step 2 being obtained carries out centrifugal at ambient temperature, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min, obtain the dairy fats primary extract in centrifugal product upper strata; Wherein, centrifugal rotational speed is 3800r/min, and centrifugation time is 30min;
Four, in the dairy fats primary extract obtaining in step 3, add 2 times to the sodium ion phosphate buffer soln of this dairy fats primary extract volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min; The compound method of described sodium ion phosphate buffer soln is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.3mmol, KCl:2.7mmol, KH
2pO
4: the ratio of 1.8mmol and NaCl:137mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.4, obtain sodium ion phosphate buffer soln;
Five,, by the dairy fats primary extract of processing through step 4, by the method repetitive operation of step 4 once, obtain the dairy fats in centrifugal product upper strata;
Six, in the dairy fats obtaining in step 5, add the ultrapure water identical with dairy fats volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, obtain the cream in centrifugal product upper strata; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min;
Seven, cream step 6 being obtained is placed under 4 ℃ of conditions, and under this condition standing 15h, obtain cream solid;
Eight, cream solid step 7 being obtained is placed under the water bath condition of 40～45 ℃ and melts, and obtains liquid cream;
Nine, in the liquid cream obtaining in step 8, adding volume is the Triton X-100 solution that accounts for liquid state milk oil volume 0.2%, carries out at ambient temperature centrifugally, and collecting precipitation, obtains yak milk milk fat ball film albumen; Wherein, centrifugal rotational speed is 44910r/min, and centrifugation time is 60min; The compound method of described Triton X-100 solution is: by the volume ratio of Triton X-100 and phosphate buffered saline buffer, be that 1:2.6 takes Triton X-100 and phosphate buffered saline buffer, after the two is mixed, be placed in the water-bath of 37 ℃, keep 2 hours; The concentration of phosphate buffered saline buffer is that 0.1mol/L, pH are 7.3.
The controlled trial of test one:
One, the fresh yak breast of selecting healthy yak, takes the yak milk of 20 parts and the sucrose of 1 part by mass fraction;
Two,, after yak milk step 1 being taken filters with 4 layers of gauze, the sucrose that adds step 1 to take, mixes with magnetic stirring apparatus, obtains cow's milk sucrose mixed solution;
Three, cow's milk sucrose mixed solution step 2 being obtained carries out centrifugal at ambient temperature, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min, obtain the dairy fats primary extract in centrifugal product upper strata; Wherein, centrifugal rotational speed is 3800r/min, and centrifugation time is 30min;
Four, in the dairy fats primary extract obtaining in step 3, add 2 times to the sodium ion phosphate buffer soln of this dairy fats primary extract volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min; The compound method of described sodium ion phosphate buffer soln is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.3mmol, KCl:2.7mmol, KH
2pO
4: the ratio of 1.8mmol and NaCl:137mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.4, obtain sodium ion phosphate buffer soln;
Five,, by the dairy fats primary extract of processing through step 4, by the method repetitive operation of step 4 once, obtain the dairy fats in centrifugal product upper strata;
Six, in the dairy fats obtaining in step 5, add the ultrapure water identical with dairy fats volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, obtain the cream in centrifugal product upper strata; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min;
Seven, cream step 6 being obtained is placed under 4 ℃ of conditions, and under this condition standing 15h, obtain cream solid;
Eight, cream solid step 7 being obtained is placed under the water bath condition of 40～45 ℃ and melts, and obtains liquid cream;
Nine, liquid cream step 8 being obtained stirs 10min with agitator, then adds the deionized water identical with liquid state milk oil volume, and separation obtains butter and buttermilk;
Ten, butter step 9 being obtained is heating and melting in the water-bath of 60 ℃, then adds the deionized water identical with butter volume, and centrifugal 20min under the condition that is 4800r/min at rotating speed, discards upper strata butter, obtains subnatant;
11, the buttermilk that subnatant step 10 being obtained obtains with step 10 mixes, with the HCL that concentration is 0.01mol/L, regulate its pH to 4.8, under room temperature condition after standing 30min, centrifugal 15min under the condition that is 12000r/min at rotating speed, collecting precipitation thing and suspended substance, obtain yak milk milk fat ball film albumen.
By high performance liquid chromatography (HPLC), the yak milk milk fat ball film albumen of testing the yak milk milk fat ball film albumen obtaining and controlled trial and obtaining is analyzed.In the yak milk milk fat ball film albumen that in the test one yak milk milk fat ball film albumen obtaining, the peak area of impurity peaks only obtains for controlled trial 60% of impurity peaks peak area, and assorted peak α
s2the low levels Caseinum componemt of-CN, κ-CN disappears substantially, and yak milk milk fat ball film property of protein is stable.By calculating the purity of the test one yak milk milk fat ball film albumen obtaining, be 93.5%, and the purity of the yak milk milk fat ball film albumen that controlled trial obtains is only 82.7%, illustrate that the purity of the test one yak milk milk fat ball film albumen obtaining is significantly improved.
Claims (1)
1. a separation method for yak milk milk fat ball film albumen, is characterized in that the separation method of yak milk milk fat ball film albumen carries out according to the following steps:
One, the fresh yak breast of selecting healthy yak, takes the yak milk of 20 parts and the sucrose of 1 part by mass fraction;
Two,, after yak milk step 1 being taken filters with 4 layers of gauze, the sucrose that adds step 1 to take, mixes with magnetic stirring apparatus, obtains cow's milk sucrose mixed solution;
Three, cow's milk sucrose mixed solution step 2 being obtained carries out centrifugal at ambient temperature, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min, obtain the dairy fats primary extract in centrifugal product upper strata; Wherein, centrifugal rotational speed is 3800r/min, and centrifugation time is 30min;
Four, in the dairy fats primary extract obtaining in step 3, add 2 times to the sodium ion phosphate buffer soln of this dairy fats primary extract volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, centrifugal product is placed under 4 ℃ of conditions, and under this condition standing 10min; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min; The compound method of described sodium ion phosphate buffer soln is: by containing Na in every 1L sodium ion phosphate buffer soln
2hPO
4: 4.3mmol, KCl:2.7mmol, KH
2pO
4: the ratio of 1.8mmol and NaCl:137mmol, by Na
2hPO
4, KCl, KH
2pO
4join in deionized water with NaCl, and regulate its pH to 7.4, obtain sodium ion phosphate buffer soln;
Five,, by the dairy fats primary extract of processing through step 4, by the method repetitive operation of step 4 once, obtain the dairy fats in centrifugal product upper strata;
Six, in the dairy fats obtaining in step 5, add the ultrapure water identical with dairy fats volume, after mixing with magnetic stirring apparatus, carry out at ambient temperature centrifuge washing, obtain the cream in centrifugal product upper strata; Wherein, centrifugal rotational speed is 4800r/min, and centrifugation time is 20min;
Seven, cream step 6 being obtained is placed under 4 ℃ of conditions, and under this condition standing 15h, obtain cream solid;
Eight, cream solid step 7 being obtained is placed under the water bath condition of 40～45 ℃ and melts, and obtains liquid cream;
Nine, in the liquid cream obtaining in step 8, adding volume is the Triton X-100 solution that accounts for liquid state milk oil volume 0.2%, carries out at ambient temperature centrifugally, and collecting precipitation, obtains yak milk milk fat ball film albumen; Wherein, centrifugal rotational speed is 44910r/min, and centrifugation time is 60min; The compound method of described Triton X-100 solution is: by the volume ratio of Triton X-100 and phosphate buffered saline buffer, be that 1:2.6 takes Triton X-100 and phosphate buffered saline buffer, after the two is mixed, be placed in the water-bath of 37 ℃, keep 2 hours; The concentration of phosphate buffered saline buffer is that 0.1mol/L, pH are 7.3.
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| CN105455109B (en) * | 2015-12-18 | 2021-11-12 | 光明乳业股份有限公司 | Fat globule membrane powder and preparation method and application thereof |
| CN108299552A (en) * | 2018-02-27 | 2018-07-20 | 吉林大学 | The extracting method of goat dairy milk fat globule membrane protein for proteomics research |
| CN111227039A (en) * | 2018-11-29 | 2020-06-05 | 内蒙古伊利实业集团股份有限公司 | Extraction method of fat globule membrane, yoghourt rich in fat globule membrane and preparation method |
| CN113892649A (en) * | 2020-06-18 | 2022-01-07 | 湖南农业大学 | Probiotic microcapsule and preparation method thereof |
| CN112753770B (en) * | 2020-12-28 | 2023-06-16 | 西藏农牧学院 | Processing technology for improving stability of yak yoghourt |
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| WO2001035760A1 (en) * | 1999-11-16 | 2001-05-25 | Gorewit Ronald C | Bulk preparation of milk fat globule membranes |
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