CN111077213A

CN111077213A - Method for identifying and breeding milk-producing livestock of type A2 and method for producing milk of type A2

Info

Publication number: CN111077213A
Application number: CN201911418898.3A
Authority: CN
Inventors: 陈莲莲; 吕倩; 何昆; 付书辉; 马庆伟; 向华
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Academy of Military Medical Sciences AMMS of PLA; Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-28

Abstract

The invention discloses a method for identifying and breeding A2 type milk-producing livestock, which identifies A2 type milk-producing livestock through standard characteristic polypeptide groups of A1 β -casein and A2 β casein so as to finish breeding, wherein the milk-producing livestock comprises milk-producing cows and sheep, and the standard characteristic polypeptide sequences of the A1 β -casein and the A2 β casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

Description

Method for identifying and breeding milk-producing livestock of type A2 and method for producing milk of type A2

Technical Field

The invention belongs to the field of biological genetic breeding, and particularly relates to a method for identifying and breeding A2 type milk-producing livestock by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) and a method for producing A2 type milk by using the method.

Technical Field

According to the difference of amino acid composition and electrophoresis behavior, the casein can be divided into α s-casein, β -casein and kappa-casein, wherein β -casein accounts for about 30-35% of the total amount of cow milk casein, β -casein also has a plurality of variants, most commonly A1 β -casein and A2 β -casein, the difference of the two is that the 67 th position of the amino acid chain has a variation, the former is histidine, the latter is proline, the milk containing both A1 β -casein and A2 β -casein is A1/A2 type milk, the milk containing only A2 β -casein is A2 type milk, and the milk containing only A1 β -casein is A1 type milk.

It has now been found that BCM-7 is able to cross the gastrointestinal wall into the blood circulation and to influence systemic and cellular activities via opioid receptors, furthermore BCM-7 and other β -casein derivatives are potent exogenous agonists of opioid receptors-exorphins, where the affinity to μ receptors is greatest, thus BCM-7 may influence the activities of multiple organs/systems, in particular the digestive system and immune cells, furthermore BCM-7 may also be associated with various diseases in infants, such as type I diabetes and respiratory dysfunction, digestive disorders, causing anal fistulas, immune disorders, and affecting central nervous system activities, thus dairy products containing A2 β -casein are more healthy and safer and are also more popular in the domestic and foreign markets.

The dairy industry is an important mark of the national development degree and the modernization level of food consumption, the development of the dairy industry has important significance for optimizing the agricultural structure, promoting the upgrading of the animal husbandry industry, increasing the income of farmers and improving the physical quality of the nation, currently, the dairy industry in China is in the key period of the transformation from the traditional dairy industry to the modern dairy industry, the development of the dairy industry in the future is upgraded from the quantity-focusing type to the quality type, because not all dairy cows can produce the milk only containing the pure A2 type β -casein but not containing the A1 type β -casein, the milk source of the A2 type β -casein is very rare, nowadays, only about 30% of the western dairy cows are pure A2 dairy cows, the milk produced by the dairy cows only contains 100% of the A2 type β -casein, if the identification technology of the milk products of the A2 β -casein in China can master the large-volume dairy industry of the A2-dairy cows according to the resources of the dairy cows in China, and therefore, the development of the A2-dairy industry can not improve the development level of the dairy industry in China.

The Chinese patent application No. 201610784180.6 entitled "method for detecting A1 β -casein and A2 β -casein in cow milk" comprises (1) heating, centrifuging, freezing and thawing a sample to be detected in sequence to obtain a stock solution to be detected, (2) pretreating the stock solution to be detected to obtain a liquid to be detected, wherein the pretreatment comprises mixing the stock solution to be detected with a pretreatment solution, and (3) detecting the liquid to be detected by using a capillary electrophoresis method to determine whether the cow milk contains the A1 β -casein and the A2 β -casein.

The invention discloses a characteristic peptide and a method for detecting the content of A2 β -casein in a milk product, and discloses a method for detecting the content of A2 β -casein in the milk product, which is disclosed by the invention patent application No. 201810487863.7 in China and the invention name, wherein the method comprises the steps of (1) diluting a sample to be detected with water, sequentially carrying out denaturation treatment, trypsin enzymolysis treatment and dimethylation treatment to obtain a dimethylated internal standard peptide solution, (2) carrying out the same treatment on the characteristic internal standard peptide to obtain the dimethylated internal standard peptide solution, (3) mixing the internal standard peptide solution with the sample solution, and detecting the sample to be detected by adopting a high performance liquid chromatography-mass spectrometry combined technology, (4) calculating the peak area ratio of the A2 β -casein characteristic peptide in the sample to be detected and the corresponding internal standard peptide to obtain the content of A2 β -casein in the sample, however, a liquid mass spectrometry adopted by the method needs to design a treatment process aiming at a specific amino acid fragment of A2 β casein, and simultaneously needs to introduce the internal standard peptide with a long detection time, a high detection sample quantity, high detection cost and cannot be adapted to modern industries.

The Chinese patent application No. 201810316627.6 entitled "A1/A2 β -casein mass spectrum detection method" discloses a A1/A2 β -casein mass spectrum detection method, which comprises the following steps of a) obtaining an amino acid sequence of A2 β -casein, b) modifying amino acid proline at position 67 of A2 β -casein into histidine to obtain an amino acid sequence of A1 β -casein, c) utilizing a specific enzymolysis method to cut the protein to be detected into a polypeptide fragment mixture with small molecular weight, utilizing an electrostatic field orbital trap high-resolution mass spectrum full-scan mode to detect the molecular weight and fragment information of each polypeptide in the mixture, comparing the molecular weight and fragment information with the previous amino acid sequence to judge whether a characteristic peptide segment of A1 or A2 β -casein exists, d) selecting the characteristic peptide segment qualitatively as A1 or A2 β -casein, synthesizing an isotope peptide segment thereof, utilizing a series four-level mass spectrum to establish a multi-reaction monitoring method for quantitative analysis, pre-learning processing a specific amino acid sequence, optimizing ESI, and optimizing modern complicated development conditions for milk production.

In conclusion, the detection methods for A1 β -casein and A2 β casein generally have the defects of complicated operation and low flux, so a new method with low detection cost, high detection flux and high detection speed is needed to be established.

Disclosure of Invention

The first invention principle of the invention lies in that through big data analysis, a standard characteristic polypeptide mass spectrum peak capable of characterizing A1 β -casein and A2 β -casein in a dairy product is screened out, and the mass spectrum peak of a sample to be detected is compared with the standard characteristic polypeptide mass spectrum peak, so that whether the dairy product contains A1 β -casein and A2 β -casein is determined.

The second inventive principle of the invention lies in that the relative proportion of A1 β -casein and A2 β -casein of the dairy product can be detected through mass spectrum results for the first time.

The third invention principle of the invention is based on the principle, and the standard characteristic polypeptide of the dairy product is detected, so that the method for identifying and breeding the livestock producing the milk A2 and producing the milk A2 is completed.

Therefore, the first objective of the present invention is to provide a standard characteristic polypeptide set for simultaneously detecting a1 β -casein and a2 β -casein in dairy products, wherein the dairy products comprise cow milk and sheep milk, and the standard characteristic polypeptide sequences of the a1 β -casein and the a2 β -casein are shown in SEQ ID nos. 1 and 2:

SEQ ID No.1：

IHPFAQTQSLVYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK；

SEQ ID No.2：

IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSK；

in one embodiment, the characteristic polypeptide mass spectrum peak of the A1 β -casein is 5360m/z (5 per thousand, representing mass-to-charge ratio deviation tolerance +/-5 per thousand, the same below) or 2680m/z (5 per thousand) with double charges, and when the mass spectrum polypeptide peak of the single charge or the double charge appears, the sample to be detected is A1 type milk or A1/A2 hybrid type milk.

In another embodiment, the peak value of the characteristic polypeptide peak of the A2 β -casein is 5320m/z (5 per thousand) with single charge or 2660m/z (5 per thousand) with double charge, and when the peak value of the polypeptide peak of the single charge or the double charge appears, the sample to be detected is A2 type milk or A1/A2 hybrid type milk.

In the above embodiments, since the signature polypeptide has two charged forms of single charge and double charge, the position of the double charge polypeptide in the mass spectrum differs by half from that of the single charge polypeptide of the same mass unit, i.e. the mass-to-charge ratio of the two is different by half.

The second invention aims to provide a mass spectrum model for simultaneously detecting A1 β -casein and A2 β casein in dairy products, which comprises standard characteristic polypeptide groups of A1 β -casein and A2 β casein, wherein the dairy products comprise cow milk and goat milk, and the standard characteristic polypeptide sequences of the A1 β -casein and the A2 β casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

In one embodiment, the characteristic polypeptide mass spectrum peak value of the A1 β -casein is single-charge 5360m/z (5 per thousand) or double-charge 2680m/z (5 per thousand), and when the single-charge or double-charge mass spectrum polypeptide peak value appears, the sample to be detected is A1 type milk or A1/A2 hybrid type milk.

The third purpose of the invention is to provide a construction method for preparing the mass spectrum model, which comprises the following steps:

1) collecting multiple cases of common pure milk (containing A1 type or A1/A2 heterozygote type) and A2 type milk as two groups of samples for use;

2) performing pretreatment before mass spectrometry on milk protein;

3) performing mass spectrum detection reading on the two groups of pretreated milk proteins to obtain fingerprint spectrums of the two groups of milk polypeptides;

4) standardizing fingerprint spectra of all common pure milk (containing A1 type or A1/A2 heterozygote type) and A2 type milk, and collecting data;

5) and performing quality control treatment on the obtained data, and screening out two characteristic polypeptides with the following mass-to-charge ratio peaks: single charge 5360m/z (5 per thousand) or double charge 2680m/z (5 per thousand), single charge 5320m/z (5 per thousand) or double charge 2660m/z (5 per thousand), and the characteristic polypeptide is subjected to sequence determination, and a qualitative and semi-quantitative mass spectrum model for detecting the dairy product is established according to the two mass-to-charge ratio peaks.

In one embodiment, the method of step 2) pre-treatment comprises enzymatic hydrolysis with trypsin to obtain specific polypeptide fragments in the milk.

In one embodiment, in step 3), the kit is used to perform reduction, alkylation and enzymolysis on the two groups of dairy samples, and polypeptide fragments obtained after the enzymolysis of the dairy are read to obtain fingerprint spectra of the two groups of dairy polypeptides.

In one embodiment, the quality control processing in step 5) retains mass spectrum data with a signal-to-noise ratio greater than 3, and uses an intra-group coefficient of variation to ensure experimental consistency, so as to perform screening according to the allowable range that the coefficient of variation satisfies consistency, wherein the coefficient of variation is 15%.

The fourth object of the present invention is to provide a detection product for detecting a1 β -casein and a2 β -casein in dairy products, which comprises the above characteristic polypeptide group, or comprises the above mass spectrum model, wherein the dairy products comprise cow milk and goat milk.

In one embodiment, the test product comprises a test kit, chip, test reagent, and the like. In a preferred embodiment, the kit consists of a reducing agent, an alkylating reagent, trypsin, a reaction termination reagent, a reaction buffer and a mass spectrometry matrix, wherein each component can use a commercially available kit or related reagents.

In another embodiment, the kit further comprises a standard mass spectrum sample tube containing the characteristic polypeptide, wherein the sample tube can be a sample tube containing a single characteristic polypeptide or a sample tube containing 2 characteristic polypeptides, and a sample in the standard sample tube is used for performing parallel mass spectrum test when performing mass spectrum with a sample to be tested so as to judge whether the sample to be tested contains the characteristic polypeptide.

In another embodiment, the kit may contain software or a chip of the standard database of the characteristic polypeptide (i.e. the characteristic polypeptide database of a1/a2 β casein) described above, and may be used to provide a comparison of standard data or curves when a sample to be tested is subjected to mass spectrometry so as to determine whether the sample to be tested contains the characteristic polypeptide of a1/a2 β casein.

The kit comprises a dairy pretreatment reagent, a mass spectrometry matrix and time-of-flight mass spectrometry A2 β -casein detection software.

The dairy pretreatment reagent in the kit comprises the following components: the sodium bicarbonate buffer solution mainly comprises sodium carbonate and sodium bicarbonate; a reducing agent, the main component of which is dithiothreitol; an alkylating reagent, the main component of which is iodoacetamide; an enzyme, the main component of which is trypsin; the main component of the terminating reagent is formic acid.

The mass spectrometry matrix in the kit comprises the following components: a matrix powder comprising sinapic acid as a major component; the matrix solution mainly comprises acetonitrile, trifluoroacetic acid and water.

The kit can be applied to qualitative and relative quantitative detection of A1 β -casein and A2 β casein.

The fifth invention aim at providing a method for detecting A1 β -casein and A2 β casein by MALDI-TOF-MS, which comprises the following steps:

(1) sequentially carrying out reduction reaction, alkylation reaction and trypsin enzymolysis treatment on a sample to be detected in a buffer solution, and then terminating the reaction to obtain a dairy pretreatment solution;

(2) coating the dairy treatment fluid and the mass spectrum matrix on a mass spectrum special target plate;

(3) detecting the sample by using MALDI-TOF-MS;

(4) according to the detection result, the relative content of A1 β -casein and A2 β -casein is calculated by using a time-of-flight mass spectrum A2 β -casein detection software.

In one embodiment, the reduction reaction in step (1) is to add a reducing agent into a sodium bicarbonate buffer system to make the final concentration not lower than 0.1mol/L, and perform isothermal reaction at 50-70 ℃ for 10-60 min, wherein the reducing agent is Dithiothreitol (DTT) and can hydrolyze disulfide bonds to destroy the spatial structure of the protein.

In another embodiment, the alkylation reaction in step (1) is to add an alkylating reagent into the product to make the final concentration not lower than 0.1mol/L, and react for 10-60 min at 20-30 ℃ in a dark constant temperature, which can completely denature the protein, wherein the alkylating reagent is Iodoacetamide (IAA).

In another embodiment, the trypsin enzymolysis treatment in the step (1) is to add a trypsin solution into the product to enable the final concentration to be not lower than 0.1g/L, and carry out a constant temperature reaction at 37 ℃ for at least 30min, the step uses the characteristic that the trypsin only acts on the specificity of arginine (R) and lysine (K), β -casein is cut into small molecular peptide segments with the molecular weight of less than 10000Da, a termination reagent formic acid solution is added after complete enzymolysis to denature the trypsin, and the reaction is terminated.

In one embodiment, in step (2), SA matrix is used, which has a composition comprising 25mg sinapic acid per ml matrix solution, 500. mu.l acetonitrile, 1. mu.l trifluoroacetic acid, 500. mu.l pure water.

In one embodiment, in step (3), the detection conditions for MALDI-TOF-MS are: the modulation method comprises the following steps: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

In one embodiment, in step (4), the functions of the software include background removal, noise reduction, correction of peak position, peak intensity planning and calculation of the relative amounts of A1 β -casein and A2 β -casein.

In any of the above embodiments, the sample is fresh cow's milk and other animal or dairy products (e.g., goat's milk). In a preferred embodiment, the dairy product is an animal dairy product such as infant formula powder, skim milk powder, whole milk powder, high temperature sterilized milk, pasteurized milk, fermented yogurt, and the like.

It is a sixth object of the present invention to provide a method for identifying and breeding milk-producing livestock of type a2, comprising the steps of:

(1) determining β -casein A2/A1 genotyping of the milk producing livestock according to the aforementioned mass spectrometric signature polypeptide set, mass spectrometric model or test product or identification method;

(2) selecting a milk producing livestock having only the gene encoding β -casein A2;

(3) breeding the selected milk producing livestock until producing or/and forming the required production population.

In one embodiment, step (1) is performed on a sample of cow's milk by mass spectrometry.

In all of the above embodiments, the dairy livestock is a chinese holstein cow, yak, sheep, goat.

The seventh object of the invention is to provide a method for producing β -casein A2 dairy products, which comprises the following steps:

(3) breeding the selected milk producing livestock until producing or/and forming a required production population;

(4) the screened milk-producing animals are milked.

In one embodiment, step (1) is mass spectrometric detection of the milk sample.

In another embodiment, the milk is processed to produce a dairy product comprising step (4).

Technical effects

Compared with the prior art, the invention has the following beneficial effects:

1. the invention adopts MALDI-TOF-MS method, and firstly proposes that the relative quantification of A1 β -casein and A2 β -casein in dairy products is realized by identifying β -casein characteristic mass spectrum peak.

2. By the method, whether the commercial dairy product is A2 milk or not can be accurately identified, two samples to be detected, namely A1 and A2, can be simultaneously detected, and the technical defect that the A1 milk or the A2 milk can only be detected once in the prior detection technology is overcome.

3. The relative quantitative detection method can also quickly know the residual degree of the A1 protein in the dairy product, thereby providing an accurate and scientific quantitative monitoring means for the product quality control of the A2 milk.

4. The invention can detect A2 type milk and goat milk, thereby completing the identification and breeding of A2 type cows, goats and sheep.

5. The method does not need to add an internal standard additionally, and has the advantages of simple and rapid operation, low cost, high throughput and the like.

Drawings

FIG. 1 is a comparison spectrum of the same sample in example 1, wherein the spectrum peak near m/z5360 is the characteristic peak of A1 β -casein, and the spectrum peak near m/z 5320 is the characteristic peak of A2 β -casein.

FIG. 2 is a comparative spectrum of the mixed samples of different proportions in example 2, wherein the spectrum peak near m/z5360 is the characteristic peak of A1 β -casein, the spectrum peak near m/z 5320 is the characteristic peak of A2 β -casein, the relative peak intensity (or peak area) of the A2 peak/the A1 peak is gradually increased with the increase of the A2 protein content, and the proportion is correlated.

FIG. 3 is a MALDI-TOF-MS spectrum of plain pure milk (A1/A2 type) in example 3.

FIG. 4 is a partial enlarged view of a single charge portion of MALDI-TOF-MS spectrum of plain milk (A1/A2 type) in example 3, wherein the spectrum peak near m/z5360 is the characteristic peak of A1 β -casein, and the spectrum peak near m/z 5320 is the characteristic peak of A2 β -casein.

FIG. 5 is a partial enlarged view of the double charge part of the MALDI-TOF-MS spectrum of the normal pure milk (A1/A2 type) in example 3, wherein the spectrum peak near m/z2680 is the characteristic peak of A1 β -casein, and the spectrum peak near m/z 2660 is the characteristic peak of A2 β -casein.

FIG. 6 is the MALDI-TOF-MS spectrum of A2 milk powder in example 4.

FIG. 7 is a partial enlarged view of a single charge part of MALDI-TOF-MS spectrum of A2 milk powder in example 4, wherein the spectrum peak near m/z5360 is the characteristic peak of A1 β -casein, and the spectrum peak near m/z 5320 is the characteristic peak of A2 β -casein.

FIG. 8 is a partial enlarged view of the double charge portion of MALDI-TOF-MS spectrum of milk powder A2 in example 4, wherein the spectrum peak near m/z2680 is the characteristic peak of A1 β -casein, and the spectrum peak near m/z 2660 is the characteristic peak of A2 β -casein.

FIG. 9 is a partial enlarged view of MALDI-TOF-MS spectra of A1 β -casein and A2 β -casein in yak milk and goat milk samples in example 5, wherein a spectral peak near m/z5360 is a characteristic peak of A1 β -casein, and a spectral peak near m/z 5320 is a characteristic peak of A2 β -casein.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

The mass spectrometer adopted by the invention is as follows: matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Clin-TOF-II, Beijing Yixinbo Biotech limited).

The mass spectrometry conditions used in the following examples are as follows:

modulation mode: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

TABLE 1 time of flight Mass Spectrometry Main feature Peak List

Name (R)	Single/double charge	m/z	Allowable offset range
				A1 β -Casein	Single charge		5360	Within +/-5 ‰
A2 β -Casein	Single charge		5320						Within +/-5 ‰
				A1 β -Casein	Double electric charge	2680	Within +/-5 ‰
A2 β -Casein	Double electric charge	2660	Within +/-5 ‰

Example 1 test result reproducibility verification

1. Sample pretreatment

a) The two kinds of milk are uniformly mixed according to the volume ratio of 1:1 by taking commercially available common milk (trade name: three-component pure milk; manufacturer: Beijing three-component food stock Co., Ltd.; producing area: China) and commercially available A2 milk (trade name: extreme A2- β casein fresh milk; manufacturer: Beijing three-component food stock Co., Ltd.; producing area: China).

b) Accurately aspirate 18 identical mixed milk samples, 10. mu.l each, add 920. mu.l sodium bicarbonate buffer to each milk sample, and vortex at 1200rpm on a vortex mixer for 30 s.

c) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

d) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

e) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

f) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

g) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

h) Sucking 1 mul sample matrix mixture liquid and evenly coating the mixture liquid on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

a) The mass spectrometer parameters were set to: modulation mode: linearity; data collection range: m/z is 1000-20000; and (3) spectrum acquisition quantity: collecting 50 spectrograms for each sample point, and bombarding each spectrogram 10 times by using laser; laser frequency: 30 Hz; optimal pulse extraction: 5340 Da.

b) The mass spectrum mass axis is corrected by using the known external standard protein, and the corrected average molecular deviation is less than 500 ppm.

c) The same mass spectral parameters were used for the detection of the sample spot. The results are shown in FIG. 1.

3. Software analysis

a) And (3) importing an ascii file automatically generated by a mass spectrometer into a time-of-flight mass spectrum A2 β -casein detection software, and calculating the peak intensity ratio of characteristic peaks of A1 β -casein and A2 β -casein.

b) TABLE 2 reproducibility of the test results

Type of sample	Peak to intensity ratio mean value	Peak intensity ratio SD	Peak intensity ratio CV
				Milk	0.430	0.015	3.48％

c) The peak intensity ratio CV value was less than 5%, indicating good reproducibility.

Example 2A2- β Casein semi-quantitative assay

1. Sample pretreatment

a) A ternary pure milk sample (trade name: three-component pure milk; the manufacturer: beijing three-component food products, Inc.; the producing area: china) is a1/a2 heterozygote milk in this example;

b) a2 sample of whole milk powder (trade name: a2 full cream adult milk powder; the manufacturer: a2 Co., Ltd; the producing area: new zealand; import business: chinese agro-reclamation shanghai corporation) was the a2 homozygous milk sample in this example;

c) accurately weighing 1.00g of A2 whole milk powder sample, adding purified water, and vortexing at 3000rpm on a vortex mixer for 2min to completely dissolve the milk powder;

d) respectively and uniformly mixing A1/A2 heterozygote type milk and A2 homozygote type milk according to the volume of the following table;

e) TABLE 3 Dairy mix volume table

f) Mu.l of each mixed milk sample was accurately aspirated into 920. mu.l of sodium bicarbonate buffer (pH9.5,0.1M) and vortexed at 1200rpm on a vortex mixer for 30 s.

g) Adding 10 μ l dithiothreitol solution (1M), and reacting at 50 deg.C for 30 min.

h) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

i) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

j) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

k) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

l) sucking 1 mul of sample matrix mixed solution and uniformly coating the sample matrix mixed solution on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

c) The same mass spectral parameters were used for the detection of the sample spot.

3. Software analysis

a) And importing the ascii file automatically generated by the mass spectrometer into time-of-flight mass spectrum A2 β -casein detection software to obtain qualitative and relative quantitative results.

4. And (4) analyzing results:

as shown in FIG. 2, the spectral peak near m/z5360 is A1 β -casein characteristic peak, the spectral peak near m/z 5320 is A2 β -casein characteristic peak, along with the change of the relative content of the sample to be detected, the peak intensity (or peak area) ratio of mass spectral peaks appearing at m/z5360 and m/z 5320 changes.

Example 3 commercially available pure milk assay

1. Sample pretreatment

a) Accurately aspirate 10 μ l of pure milk sample (trade name: three-component pure milk; the manufacturer: beijing three-component food products, Inc.; the producing area: china), add 920 μ l of sodium bicarbonate buffer and vortex at 1200rpm on a vortex mixer for 30 s.

b) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

c) After cooling to room temperature, 30. mu.l of iodoacetamide solution is added, and the mixture is kept standing for 30min in the dark at room temperature.

d) Adding 10 μ l of trypsin solution, and performing enzymolysis at 37 deg.C for more than 2 hr.

e) The reaction was stopped by adding 20. mu.l of pure formic acid. And obtaining a sample treatment solution.

f) Aspirate 20. mu.l of sample treatment fluid and mix with 20. mu.l of SA matrix and vortex on a vortex mixer at 1200rpm for 30 s.

g) Sucking 1 mul sample matrix mixture liquid and evenly coating the mixture liquid on a mass spectrum special target plate.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

4. And (4) analyzing results:

as shown in FIG. 3 and FIG. 4, the spectrum peak near m/z5360 is A1 β -casein characteristic peak, the spectrum peak near m/z 5320 is A2 β -casein characteristic peak, because the sample to be detected has mass spectrum peaks at m/z5360 and m/z 5320 at the same time, the milk to be detected is A1/A2 heterozygote milk, and the ratio of the characteristic peak intensity (or peak area) of A1 β -casein to A2 β -casein has correlation with the contents of the two proteins.

As shown in FIG. 5, the spectral peak near m/z2680 is A1 β -casein double charge peak, the spectral peak near m/z 2660 is A2 β -casein double charge peak, the double charge peak of two proteins simultaneously appears also represents that the two proteins exist simultaneously, namely the tested sample is A1/A2 heterozygote milk.

Example 4 detection of commercially available A2 milk powder

1. Sample pretreatment

a) 1.00g of milk powder (trade name: a2 full cream adult milk powder; the manufacturer: a2 Co., Ltd; the producing area: new zealand; import business: chinese agricultural reclamation shanghai corporation), purified water was added and the milk powder was completely dissolved by vortexing on a vortex mixer at 3000rpm for 2 min.

b) A10. mu.l sample of bovine milk powder lysate was aspirated accurately, 920. mu.l of sodium bicarbonate buffer was added, and vortexed at 1200rpm on a vortex mixer for 30 s.

c) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

And importing the ascii file automatically generated by the mass spectrometer into time-of-flight mass spectrum A2 β -casein detection software to obtain qualitative and relative quantitative results.

4. Analysis of results

The results are shown in FIG. 6 and FIG. 7, wherein the spectrum peak near m/z5360 is A1 β -casein characteristic peak, the spectrum peak near m/z 5320 is A2 β -casein characteristic peak, the mass spectrum 7 only shows mass spectrum peak at m/z 5320, the detected milk powder is A2 type milk powder, which is consistent with the commercial situation.

As shown in FIG. 8, the spectral peak near m/z2680 is A1 β -casein double charge peak, the spectral peak near m/z 2660 is A2 β -casein double charge peak, the mass spectrum 8 only has mass spectrum peak at m/z 2660, which represents that the tested sample does not contain A1 β -casein, namely the tested sample is A2 pure milk.

Example 5 Yak and goat milk sample testing

1. Sample pretreatment

a) Samples of 1ml each of yak and goat milk were randomly selected and 10. mu.l accurately aspirated, 920. mu.l sodium bicarbonate buffer was added and vortexed at 1200rpm on a vortex mixer for 30 s.

b) Adding 10 μ l dithiothreitol solution, and reacting at 50 deg.C for 30 min.

MALDI-TOF-MS Mass Spectrometry detection

3. Software analysis

4. Analysis of results

As a result, as shown in FIG. 9, mass spectra showed mass peaks at m/z5360 and m/z 5320 simultaneously. The two tested milks (yak milk and goat milk) are A1/A2 type milk.

From the relative ratio of the peak intensities (or peak areas) of the two characteristic peaks, the ratio of the A1 peak/A2 peak of yak milk is lower, which indicates that the yak milk contains more A2 β -casein.

Sequence listing

<110> Beijing resolute Xinbo Chuang Biotech Co., Ltd

<120> method for identifying and breeding milk-producing livestock of type A2 and method for producing milk of type A2 thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>49

<212>PRT

<213> cow (2 Ambystoma latex x Ambystoma jeffersonia)

<400>1

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

<210>2

<211>49

<212>PRT

<213> cow (2 Ambystoma latex x Ambystoma jeffersonia)

<400>2

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

Claims

1. A method for identifying and breeding milk-producing livestock of type a2, comprising the steps of:

(1) determining β -casein A2/A1 gene typing of milk producing livestock according to standard characteristic polypeptide groups of A1 β -casein and A2 β casein, or mass spectrum model mass spectrum models or detection products of A1 β -casein and A2 β casein in detection dairy products;

wherein the standard characteristic polypeptide sequences of the A1 β -casein and the A2 β casein are respectively shown as SEQ ID No.1 and SEQ ID No. 2;

the mass spectrum model comprises the standard characteristic polypeptide groups of the A1 β -casein and the A2 β casein.

2. The method of claim 1 wherein said a1 β -casein has a characteristic polypeptide mass spectrum peak at single charge 5360m/z (5% o) or double charge 2680m/z (5% o), and the presence of said single or double charge mass spectrum polypeptide peak is indicative of a dairy livestock type a1 or a dairy livestock type a1/a 2.

3. The method of claim 1 wherein said a2 β -casein has a characteristic polypeptide peak mass spectrum peak at single charge 5320m/z (5% o) or double charge 2660m/z (5% o), and the presence of said single or double charge mass spectrum polypeptide peak indicates that the livestock to be tested for milk production is a milk producing livestock of type a2 or a milk producing livestock of type a1/a2 hybrid.

4. The method of claim 1 or 2 wherein the dairy livestock is chinese holstein cows, yaks, sheep, goats.

5. A method of producing β -casein a2 dairy product, the steps comprising:

(4) the screened milk-producing animals are milked.

6. The method of claim 5, wherein step (1) is mass spectrometric detection of the milk sample.

7. The method of claim 5, wherein step (4) comprises processing the milk to produce a dairy product.

8. The method of any one of claims 5-7 wherein the dairy livestock is a Chinese Holstein cow, yak, sheep, goat.