AU2021100819A4

AU2021100819A4 - Method for detecting different beta-casein variants in cow milk

Info

Publication number: AU2021100819A4
Application number: AU2021100819A
Authority: AU
Inventors: Yaping Gao; Yundong GAO; Jinming Huang; Qiang Jiang; Zhihua JU; Lingling Wang; Xiuge WANG; Xiaochao WEI; Yaran Zhang; Jifeng ZHONG
Original assignee: Dairy Cattle Research Center Shandong Academy of Agricultural Science; Shandong Ox Livestock Breeding Co Ltd
Current assignee: Dairy Cattle Research Center Shandong Academy of Agricultural Science; Shandong Ox Livestock Breeding Co Ltd
Priority date: 2021-02-10
Filing date: 2021-02-10
Publication date: 2021-04-22
Anticipated expiration: 2029-02-10

Abstract

The present invention provides a method for detecting different p-casein variants in cow milk. The method comprises: Si. extracting a protein from a sample to be tested; S2. enzymatically hydrolyzing the extracted protein by using trypsin and a V8 protease; S3. detecting the peptide fragments obtained from the enzymatically hydrolyzing of the protein by mass spectrometry to identify the p-casein variants. The detection method of the present invention has a reasonable design, can effectively detect different p-casein variants (Al, A2, A3, B, C, E, F, HI, I) in a dairy product, and has high detection accuracy and simple operations. The present invention can determine an A2 type p-casein variant of the terminal dairy product of dairy cows, and can regulate the A2 milk dairy product market. The present invention is a good application of protein detection technology in production practice, and can produce huge economic and social benefits.

Description

METHOD FOR DETECTING DIFFERENT p-CASEIN VARIANTS IN COW MILK

Field of the Invention The present invention belongs to the technical field of animal breeding and food detection, and specifically relates to a method for simultaneously detecting p-casein A1, A2, A3, B, C, E, F, H and I variants in dairy products.

Background of the Invention The content of this section only provides background information related to the present invention, and does not necessarily constitute prior art. Casein and whey protein are the two main proteins in milk. Among them, casein accounts for about 80% of the total protein in milk, including four types: a s, a s2, P, and K. p-casein (CSN2) is an important source of amino acids, accounting for about % of the total protein. p-casein presents different protein variants, because p-casein gene contains multiple single nucleotide polymorphism (SNP) sites. Reportedly, it has at least 12 compositional forms of different amino acid sequence, such as Al, A2, A3, B, C, D, E, F, H, H2,I, G and other genetic protein variants. Studies have found that different p-casein variants are an important cause of symptoms such as abdominal pain and diarrhea after drinking milk. This is mainly due to the base changes of p-casein, leading to changes in corresponding amino acids, which ultimately affects the digestion process of the human digestive enzymes to milk. A2 homozygous p-casein milk is abbreviated as A2 milk, which helps to eliminate the symptoms of intestinal reactions in some people who drink milk and effectively avoid adverse reactions of infants and young children after drinking milk. In terms of molecular detection, there are related technical methods that can distinguish different SNP sites of the p-casein gene at the same time, thereby indirectly identifying different p-casein variants at the gene level (Application No.: 201710423750.3; Application No.: 201710438948.9). At the level of protein detection, there are no methods for simultaneously detecting multiple variants of p-casein in dairy products. Although the invention patents (Application No.: 2016107841806; Application No. 2016109833819) disclose methods for detecting Al type p-casein and A2 type p-casein in dairy products, these two protein detection methods distinguish Al and A2 milk only based on detection of one p-casein amino acid mutation site. Since A2 and A3, E, HI, and I variants have common mutation sites, detecting only at one site will result in false positive results. Currently, A2 dairy products are commercial available, producers, consumers and food regulators urgently need an accurate and rapid identification method to identify different protein variants of j-casein in dairy products, and then identify the authenticity of A2 milk. This identification technology is an urgent need and primary prerequisite to guarantee the production of high-end pure A2 dairy products, an important basis for product quality controlling and preventing fake products, and is of great significance to the healthy development of dairy industry and food safety in China.

Summary of the Invention In view of the above-mentioned shortcomings of the prior art, the present invention provides a method which can be used to detect different p-casein variants (Al, A2, A3, B, C, E, F, HI,I) in dairy products by carefully screening and a large number of tests, in order to more accurately and efficiently identify different p-casein variants in dairy products. The detection method can simultaneously detect multiple amino acid mutation sites, has strong specificity and high sensitivity, and can accurately detect different p-casein variants in dairy products (fresh milk, milk powder). One of the purposes of the present invention is to provide a method for detecting different p-casein variants in cow milk. The second purpose of the present invention is to provide the application of the above method. In order to achieve the above purposes, the present invention relates to the following technical solutions: The first aspect of the present invention provides a method for detecting different p-casein variants in cow milk, the method comprising: Si. extracting a protein from a sample to be tested; S2. enzymatically hydrolyzing the extracted protein by using trypsin and a V8 protease;

S3. detecting the peptide fragments obtained from the enzymatically hydrolyzing of

the protein by mass spectrometry to identify the p-casein variants.

Wherein, in step SI, the sample to be tested comprises but are not limited to a raw

milk sample or a milk powder sample;

Specifically, the method for extracting protein in the sample to be tested comprises:

S1.1 adding a lysis solution into the sample to be tested for lysing on ice, and

centrifuging;

S1.2 adding dithiothreitol and subjecting to water bath, then adding iodoacetamide

and allowing to stand;

S1.3 performing quantitative analysis of the protein and performing gel

electrophoresis.

Further,

in step S1.1, the lysis solution is a mixture of urea and 4-hydroxyethylpiperazine

ethanesulfonic acid, with a molar ratio of 8:0.02 to 0.04 (preferably 8:0.03);

in step S1.2,

the temperature of the water bath is controlled at 55-60°C (preferably 56°C); and then

is stood in a dark room;

in step S1.3,

a quantitative analysis method of proteins is preferably coomassie brilliant blue

staining method (bradford method), and a gel electrophoresis is specifically sodium

dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE);

In step S2, the method specifically comprises:

S2.1 after centrifuging the protein sample and discarding the waste liquid, adding

ammonium bicarbonate to adjust and stabilize pH;

S2.2 adding trypsin and the V8 protease, subjecting to water bath at a water

temperature of 35-40°C (preferably 37C), and centrifuging to obtain digested peptide

fragments;

S2.3 drying the digested peptide fragments in vacuum, and then reconstituting the

peptide fragments with formic acid.

Wherein, in step S2.1, the concentration of ammonium bicarbonate added is 25mM; in step S2.2, the concentration of trypsin and V8 protease added is 1 g/l; in step S2.3, the concentration of formic acid added is 0.1%. The second aspect of the present invention provides the application of the above method in the detection of A2 dairy products. The beneficial effects of the present invention: the detection method of the present invention has a reasonable design, can effectively detect different p-casein variants (Al, A2, A3, B, C, E, F, HI, I) in dairy products, and has high detection accuracy and simple operation; the present invention can determine A2 type p-casein variant of the terminal dairy products of dairy cows, and can regulate A2 milk dairy product market. The present invention is well applicable for protein detection technology in production practice, and can result in huge economic and social benefits.

Brief Description of the Drawings Fig. 1: Schematic diagram of the process of detecting different p-casein variants in dairy products; Fig. 2: Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein; Fig. 3: The amino acid sequence corresponding to A2 type p-casein and the amino acid mutation sites corresponding to different variants; the gray font indicates the mutation sites in the different variant p-casein, and the gray box indicates the amino acid mutation type; Fig. 4: Direct DNA sequencing to identify the p-casein genotypes of dairy cows. In the figure, two raw milk samples correspond to the results of the p-casein (CSN2) gene sequencing of dairy cows. The positions marked by the arrows are mutation sites in the CSN2 gene of different variants. The A indicates the mutation site in sample 2 (363-position nucleotide is mutated from C to A, resulting in the mutation of amino acid 121 from His to Gln).

Detailed Description of Embodiments

It should be pointed out that the following detailed descriptions are all illustrative and

are intended to provide further explanations for the application. Unless otherwise

indicated, all technical and scientific terms used herein have the same meaning as

commonly understood by those skilled in the art to which this application belongs.

It should be noted that the terms used here are only for describing specific

implementations, and are not intended to limit the exemplary implementations

according to the present application. As used herein, unless the context clearly

indicates otherwise, the singular form is also intended to comprise the plural form. In

addition, it should also be understood that when the terms "comprising" and/or

"including" are used in this specification, they indicate that there are features, steps,

operations, devices, components, and/or combinations thereof.

The present invention discloses a method for detecting different p-casein variants in

dairy products. The method can simultaneously detect multiple amino acid mutation

sites of p-casein, accurately identify different p-casein variants in dairy products, and

is easy to operate.

The present invention provides a method for identifying different p-casein variants

(Al, A2, A3, B, C, E, F, H, I) in dairy products using mass spectrometry detection.

Different j-casein variants are determined by multiple amino acid mutation sites

within it, but the existing detection methods only detect one p-casein mutation amino

acid site (p.82 P>H) to distinguish p-casein Al and A2-casein variants in dairy

products, but because A2 and A3, E, HI, and I variants have the same amino acids at

this site, detecting only at this site will result in false positive results. In the actual

application process of A2 dairy food market detection, it has caused interferences to

accurately identify A2 in dairy products. The inventors analyzed the feasibility of

detecting different p-casein variants at the same time based on the existing detection

methods, and proposed the present invention.

The design idea of the present invention is: before the dairy product (raw milk or milk

powder) is tested, it is necessary to ensure that the dairy sample is stored at -80°C,

thereby effectively ensuring the protein activity. First, the dairy samples are lysed, total protein is extracted, and protein is quantified and quality-controlled. Then, the protein extracted from the dairy product is enzymatically hydrolyzed; finally, the peptides produced by enzymatically hydrolyzing protein are tested by mass spectrometry, and the detection results are output. According to the peptides detected by mass spectrometry, the variants corresponding to different amino acid mutation combinations are combined to count p-casein variants (Fig. 1). Different j-casein variants (Al, A2, A3, B, C, E, F, HI, I) are determined based on 8 amino acid mutation sites co-occurring in different p-casein variants (p.51 E > K, p.52 E > K, p.82 P>H, p.103 I > L, p.108 M > L, p.121 H > Q, p.137 S > R, p.167 P > L) (GenBank: AAA30431.1), to achieve the purpose of detecting different p-casein variants. In a specific embodiment of the present invention, a method that can detect different p-casein variants in dairy products is provided. This method is accurate and simple after long-term exploration and optimization. The method comprises the following steps: 1. performing protein extraction and quality control on a raw milk sample or a milk powder sample; 2. enzymatically hydrolyzing the extracted protein above; 3. detecting the peptide fragments of the above-mentioned enzymatically hydrolyzing of protein by mass spectrometry; 4. according to the peptides detected by mass spectrometry, combining the corresponding variants of different amino acid mutation combinations to identify the p-casein variants. In another specific embodiment of the present invention, the step 1 specifically comprises: (1) taking 50 1 of raw milk sample or 30 mg of milk powder sample and adding 500 pl of lysis solution (8 M urea, 30 mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes)), lysing on ice for 10 min and centrifuging at 4C, 20000 g, for 30 min, taking the supernatant without sucking grease;

(2) adding dithiothreitol (DTT) to a final concentration of 10 mM, and subjecting to

water bath at 56°C for 1 h, after taking it out, quickly adding iodoacetamide (IAM) to

a final concentration of 55 mM, and allowing to stand in a dark room for 1 h;

(3) using coomassie brilliant blue staining method (bradford method) to quantify

protein;

(4) performing sodium dodecyl sulfonate-polyacrylamide gel electrophoresis

(SDS-PAGE) on the quantified protein.

In another specific embodiment of the present invention, the step 2 specifically

comprises:

(1) taking a volume of 20 g protein from each sample, adding to a10K ultrafiltration

tube, centrifuging at 14000g, 4°C for 40 min, and discarding the waste solution.

(2) adding 200 1 of 25 mM ammonium bicarbonate (NH4HCO3), centrifuging at

14000g, 4C, for 40min, and discarding the waste liquid.

(3) repeating the above steps twice.

(4) adding 1 g/ l of trypsin (Promega) and 1 1 of V8 protease (Glu-C, Sigma) in

water bath at 37°C for 24 hours, and collecting the digested peptides by centrifuging,

and using appropriate concentrations and proportions of Trypsin and V8 protease for

double enzymatic hydrolysis, which not only improves the enzymatic hydrolysis rate,

but also improves the coverage rate of peptides which is significant higher than that of

single enzymatic hydrolysis by mass spectrometry, thus effectively ensuring the

detection efficiency and accuracy of subsequent mass spectrometry detection.

However, when the amount of V8 protease is increased, the initial reaction speed of

the enzymatic hydrolysis system increases, so that part of the macromolecular protein

is degraded into polypeptides in a short time, and then it forms a competitive

relationship with the protein to form amino acids, which inhibits the enzymatic

hydrolysis process, and reduces the efficiency of enzymatic hydrolysis, and

significantly reduces the coverage of peptides obtained, even inferior to the single

addition of trypsin.

(5) drying the digested peptide in vacuum.

(6) reconstituting the peptide with 0.1% formic acid (FA).

In another specific embodiment of the present invention, the step 3 specifically

comprises:

(1) testing the peptides with the machine, using a Q-Exactive mass spectrometer to

detect the peptide signal;

(2) after completing the mass spectrum scan, obtaining the original mass spectrum

file;

(3) after inputting the original mass spectrum file into PD (Proteome Discoverer 1.3,

thermo) software, screening the mass spectrum by software;

(4) searching the extracted spectrum of PD with the mascot software, after searching,

the PD software outputs the identification results according to the search results of the

mascot software;

(5) according to the peptides detected by mass spectrometry, combining the

corresponding variants of different amino acid mutation combinations to count the

variants of p-casein.

The following examples are used to further explain the present invention, but do not

constitute a limitation to the present invention. It should be understood that these

examples are only used to illustrate the present invention and not to limit the scope of

the present invention. The test methods without specific conditions noted in the

following examples are usually performed under conventional conditions. Among

them, trypsin is purchased from Promega, and V8 protease is purchased from Sigma.

Example 1: Construction of methods for detecting different p-casein variants

1. Performing protein extraction and quality control on cow milk or milk powder

sample.

8 M urea was used to fully dissolve the protein in the sample, and the protein and oil

in the sample were separated by centrifuging, and the obtained protein sample was

reduced by dithiothreitol (DTT) and alkylated by iodoacetamide (IAM). Then

coomassie brilliant blue staining method (bradford method) was used to quantify the

protein, and sodium dodecyl sulfonate-polyacrylamide gel electrophoresis

(SDS-PAGE) was used to identify whether the protein band was clear and without

degradation.

2. Enzymatic hydrolysis of the extracted protein above;

An appropriate amount of the above sample was took and ultracentrifugation was

used to replace the buffer in the sample with 25mM ammonium bicarbonate

(NH4HCO3) to facilitate subsequent enzymatic hydrolysis, and then trypsin and V8

protease were added to digest the protein into peptides. After vacuuming the solution,

the peptide was reconstituted with formic acid (FA) and then loaded for mass

spectrometry test.

3. Detecting enzymatically hydrolyzed peptides of the above-mentioned protein by

mass spectrometry.

A Q-Exactive mass spectrometer was used to detect peptides, the original files

obtained by mass spectrometry were output into PD (Proteome Discoverer 1.3,

thermo) software for screening, and the screened spectra was searched with mascot

software, and finally the identification results were output.

4. Counting the p-casein variants

According to the peptides detected by mass spectrometry, the corresponding variants

of different amino acid mutation combinations were combined to count the p-casein

variants.

Example 2: Testing in the terms of accuracy and repeatability of the method.

The dairy cows whose p-casein genotype has been determined through direct DNA

sequencing were the test objects, and the method of Example 1 was used to identify

different p-casein variants in milk, with an accuracy rate of 100%, showing that this

detection method is accurate and effective.

Milk samples with known p-casein variants were the test objects, and this test method

was used for multiple repeated tests, and the results of the repeated tests were

consistent, showing that the method has good stability.

Example 3: Detection of actual samples.

The method of Example 1 of the present invention was used to detect two raw milk

samples.

1. Protein extraction and quality control;

(1) Taking 50 [ of raw milk sample and adding 500 1 of lysis buffer (8 M urea, 30

mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes)), lysing on ice for 10 min,

centrifuging at 4C, 20000 g, for 30 min, and taking the supernatant without sucking

grease;

(2) adding dithiothreitol (DTT) to a final concentration of 10 mM, subjecting to water

bath at 56°C for 1 h, after taking it out, quickly adding iodoacetamide (IAM) to a final

concentration of 55 mM, and allowing to stand in a dark room for 1 h;

(3) using the coomassie blue staining method (bradford method) to quantify the

protein;

(4) performing sodium dodecyl sulfonate-polyacrylamide gel electrophoresis

(SDS-PAGE) on the quantified protein (Fig. 2).

2. Performing protein enzymatic hydrolysis on the above-mentioned extracted protein;

tube, centrifuging at 14000g, 4°C for 40 min, and discarding the waste solution; (2)

adding 200 1 of 25 mM ammonium bicarbonate (NH4HCO3), centrifuging at 14000g,

4°C for 40min, and discarding the waste liquid;(3) repeating the above steps twice; (4)

adding 1 g/ l of Trypsin and V8 protease (Glu-C) 1 , and subjecting to water bath

at 37°C for 24 h, centrifuging to collect the digested peptides; (5) drying the digested

peptide in vacuum; (6) reconstituting the peptide with 0.1% formic acid (FA).

3. Detecting peptides of the above-mentioned digested protein by mass spectrometry.

detect the peptide signal; (2) after completing the mass spectrum scan, obtaining the

original mass spectrum file; (3) after inputting the original mass spectrum file into PD

(Proteome Discoverer 1.3, thermo) software, screening the mass spectrum by the

software; (4) searching the extracted spectrum of PD with the mascot software, after

searching, the PD software outputs the identification results according to the search

results of the mascot software.

4. According to the peptides with mutation sites detected in the samples (Table 1,

Table 2), the corresponding variants of different amino acid mutation combinations

are combined (Table 3, Fig. 3), and the different p-casein variants are counted. The p-casein of sample 1 is A2 variant, and the p-casein of the sample 2 is A3 variant. It is consistent with the genotype of dairy cow p-casein identified by direct DNA sequencing (Fig. 4).

Sequences of peptides modification/mutation

FQsEEQQQTEDELQDK(51,52) S3(Phospho)

HKEMPFPK(121)

IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPE

VMGVSK(82,103,108)

Table 1. A identification list of sample 1 protein peptide, lowercase letters represent

modification/mutation sites, underlined letters represent amino acid mutation sites in

differentp-casein variants, numbers in brackets represent amino acid positions

Sequences of peptides modification/mutation

FQsEEQQQTEDELQDK(51,52) S3(Phospho)

hKEMPFPKY(121) H2(His->Gln)

IHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPE

VMGVSK(82,103,108)

Table 2. A identification list of sample 2 protein peptide, lowercase letters represent

differentp-casein variants, numbers in brackets represent amino acid positions

mino acid

ites 51 52 82 103 108 121 137 167

p-caseintype

A2 Glu Glu(E) Pro(P) Leu(L) Met(M) His(H) Ser(S) Pro(P)

(E)

Al His(H)

A3 Gln(Q)

B His(H) Arg(R)

C Lys(K) His(H)

E Lys

(K)

F His(H) Leu(L)

HI Ile(I)

I Leu(L)

Table 3. Chart of amino sites corresponding to different p-casein variants

It should be noted that the above examples are only used to illustrate the technical

solutions of the present invention, but do not constitute a limitation to the present

invention. Although the present invention has been described in detail with reference

to the given examples, those skilled in the art can modify or equivalently replace the

technical solutions of the present invention as needed, without departing from the

spirit and scope of the technical solutions of the present invention.

Claims

1. A method for detecting different p-casein variants in cow milk, comprising:

Si. extracting a protein from a sample to be tested;

S2. enzymatically hydrolyzing the extracted protein by using trypsin and a V8

protease;

the protein by mass spectrometry to identify the p-casein variants;

wherein, in step S1, the sample to be tested comprises, but not limited to a raw milk

sample or a milk powder sample.

2. The detection method according to claim 1, wherein the method for extracting the

protein from the sample to be tested in step SI comprises:

centrifuging;

and allowing to stand;

S1.3 performing quantitative analysis of the protein and performing gel

electrophoresis.

3. The detection method according to claim 2, wherein:

ethanesulfonic acid with a molar ratio of 8:0.02 to 0.04 (preferably 8:0.03).

4. The detection method according to claim 2, wherein:

in step S1.2,

the temperature of the water bath is controlled at 55-60°C (preferably 56C); and the

allowing to stand is allowing to stand in a dark room.

5. The detection method according to claim 2, wherein:

in step S1.3,

the performing quantitative analysis of the protein is preferably a coomassie brilliant

blue staining method, and the gel electrophoresis is specifically sodium dodecyl

sulfonate-polyacrylamide gel electrophoresis.

6. The detection method according to claim 1, wherein in step S2, the method

specifically comprises:

Claims

ammonium bicarbonate to adjust and stabilize pH;

S2.2 adding trypsin and the V8 protease, subjecting to water bath at a water

temperature of 35-40°C (preferably 37°C), and centrifuging to obtain digested peptide

fragments;

peptide fragments with formic acid.

7. The detection method according to claim 6, wherein:

in step S2.1, the concentration of ammonium bicarbonate as added is 25 mM.

8. The detection method according to claim 6, wherein:

in step S2.2, the concentration of each of trypsin and the V8 protease as added is 1

g41.

9. The detection method according to claim 6, wherein:

in step S2.3, the concentration of formic acid as added is 0.1.

10. Use of the method according to any one of claims 1-9 in the detection of an A2

dairy product.