Analysis of β-casein in milk and milk products

Abstract

A method for testing milk and milk-derived milk products for the presence and quantification of β-casein variant A1 and β-casein variant A2 using chymotrypsin digestion followed by LC-MS analysis to determine the concentration of β-casein digest peptides and use this concentration to calculate the amount of β-casein variant A1 and β-casein variant A2 present.

Description

Technical Field

[0001] The present invention relates to the analysis of beta-casein in animal milk and dairy products obtained from animal milk. In particular, the present invention relates to testing milk and milk-derived dairy products for the presence and quantification of beta-casein types A1 and A2 and related beta-casein variants. Background of the Invention

[0003] Milk, primarily bovine milk, consumed by the world's population is the main source of protein in the human diet. Bovine milk typically contains about 30 to 40 grams of protein per liter. Casein constitutes the largest component of this protein (80%), and beta-casein accounts for about 37% of the casein. Over the past two decades, a growing body of evidence has shown that casein (especially beta-casein) is involved in many physiological or biological effects (some of which are related to health diseases).

[0004] β-casein can be classified as type A1 β-casein and type A2 β-casein. A1 β-casein and A2 β-casein are the main β-caseins in bovine milk consumed by most people. Many β-casein variants can be classified as type A1 or type A2 based on the amino acid residue at position 67 of the 209 amino acid sequence of β-casein. A1 β-casein differs from A2 β-casein by only one amino acid. The histidine amino acid is located at position 67, while proline is located at the same position in A2 β-casein. However, this single amino acid difference is crucial for the enzymatic digestion of β-casein in the intestine. The presence of histidine at position 67 allows the production of a protein fragment containing seven amino acids, called β-casomorphin-7 (BCM-7), by enzymatic digestion. Therefore, BCM-7 is a digestion product of A1 β-casein. In the case of A2 β-casein, position 67 is occupied by proline, which hinders the cleavage of the amino acid bond at this position. Therefore, BCM-7 is not a digestion product of A2 β-casein.

[0005] Other β-casein variants, such as the B, C, F, G and H1 variants, also have histidine at position 67, while the A3, D, E, H2 and I variants have proline at position 67 like the A2 variant. However, these variants are only found at very low levels or not at all in milk from cows of European origin. Therefore, in the context of the present invention, the term A1 β-casein may refer to any β-casein with histidine at position 67, while the term A2 β-casein may refer to any β-casein with proline at position 67.

[0006] BCM-7 is an opioid peptide that binds and activates opioid receptors throughout the body and has been shown to limit glutathione / antioxidant production, leading to alterations in cellular function and gene expression. 1BCM-7 has the ability to cross the gastrointestinal wall and enter the circulation, enabling it to affect systemic and cellular activities through opioid receptors. Applicants and others have previously identified a link between the intake of A1 β-casein found in milk or milk products and type I diabetes, 2 Coronary heart disease, 3 Neurological disorders, 4 enteritis, 5 Lactose intolerance symptoms, 6 Regulation of blood sugar levels, 7 cognitive function, 8 and intestinal flora 9 There is an adverse association between them.

[0007] These findings all relate to the effects of BCM-7 or β-casein variants that produce BCM-7 when digested on behavioral characteristics of individuals with clinically diagnosed diseases or conditions. The findings do not relate to healthy individuals. Furthermore, it is well known that findings based on cell culture experiments cannot be reliably extrapolated to in vivo or clinical effects because there are many variables regarding BCM-7 production, transport, and metabolism that affect tissue exposure and therefore biological responses. For example, it is known that the concentration of opioids and their affinity for opioid receptors are important for cellular responses.

[0008] The growing body of scientific evidence linking β-casein variants to adverse effects of consuming milk and milk products has led to the importance of being able to reliably and accurately analyse milk and milk products for the presence of β-casein variants, in particular A1 β-casein and its related variants having histidine at position 67 of the amino acid sequence.

[0009] The determination of total β-casein levels in bovine milk is relatively trivial. The most common method involves separation of the intact protein by liquid chromatography (LC) followed by detection using ultraviolet light (UV) or by mass spectrometry (MS). This approach was recently described by Vincent et al. 10 For studies of milk in Australia, and by Givens et al. 11 used to study milk in the United Kingdom. In both cases, the major β-casein variants were detected separately, but because purified β-casein variants are not commercially available as analytical standards, they cannot be quantified individually. In addition, both methods require the use of high-resolution mass spectrometers and are not sensitive enough to distinguish trace amounts of the A1 β-casein variant in the presence of excess A2 β-casein variant.

[0010] An alternative approach involves digesting milk proteins and quantifying the resulting peptides. This approach was adopted by Lutter et al. 12, they used trypsin to digest various foods to determine the concentrations of major milk proteins. The samples were analyzed by LC-MS / MS using internal calibration. Although this method is more sensitive and specific than the intact protein method, it lacks the critical ability to distinguish between β-casein variants.

[0011] Although the above disclosed method may be applicable to the analysis of fresh milk, there is no method to meet all necessary analytical requirements of modern single variant milk products. This is especially true for products such as UHT milk or infant formula milk powder, which can produce significant changes in protein during processing. In the analytical process, the behavior of casein in these matrices may be different from the "wild type" casein from fresh milk, and therefore the analytical procedure must be adaptively changed according to the matrix. For example, the complete beta-casein from raw milk samples is analyzed using LC-UV to produce a separate peak for each beta-casein variant, which is all applicable to qualitative and quantitative analysis, because they can easily identify and integrate. On the contrary, the same procedure applied to infant formula milk powder produces a broad peak, which can not be distinguished from each other and is not suitable for qualitative or quantitative analysis.

[0012] The use of analytical methods that digest with proteolytic enzymes may also encounter problems that prevent their use in the analysis of β-casein. The most widely used protease is trypsin, which, when it contacts β-casein, produces a long peptide containing 48 residues. The considerable length of this peptide means that it is prone to multiple modifications, which leads to changes in mass, which in turn substantially complicates the detection using mass spectrometry. In addition, its length makes it difficult to synthesize this peptide for use as an analytical standard, and therefore, means that the digestion method based on trypsin is not suitable for the quantitative analysis of β-casein in some dairy products. Therefore, for the analysis of β-casein, the use of alternative enzymes is necessary.

[0013] The applicant has now developed a method for the absolute determination of commercially relevant levels of the A1 β-casein and A2 β-casein variants in milk and milk products.

[0014] It was therefore an object of the present invention to provide a method for analysing beta-casein variants in animal milk and products derived from animal milk, or at least to provide a useful alternative to existing methods. SUMMARY OF THE INVENTION

[0016] In a first aspect of the present invention, there is provided a method for determining the content of type A1 β-casein variant or type A2 β-casein variant in a composition with an accuracy of more than 80%, wherein type A1 β-casein variant is a β-casein variant having histidine at position 67 of the β-casein amino acid sequence, and type A2 β-casein variant is a β-casein variant having proline at position 67 of the β-casein amino acid sequence, and wherein the composition is milk or a product obtained from milk, comprising the steps of:

[0017] (i) contacting the composition with chymotrypsin under conditions capable of digesting β-casein in the composition by chymotrypsin;

[0018] (ii) obtaining an aqueous solution of β-casein digest peptides;

[0019] (iii) analyzing the mixture using liquid chromatography-mass spectrometry to determine the concentrations of:

[0020] a) two or more β-casein digest peptides present in the solution, wherein each peptide comprises 8 to 20 amino acid residues and has a histidine at a position corresponding to position 67 of the β-casein amino acid sequence; and

[0021] b) two or more β-casein digest peptides present in the solution, wherein each peptide comprises 8 to 20 amino acid residues and has a proline at a position corresponding to position 67 of the β-casein amino acid sequence; and

[0022] (iv) calculating the concentration of the A1 type β-casein variant in the composition or the concentration of the A2 type β-casein variant in the composition using the concentration of the β-casein digestive peptide determined in step (iii).

[0023] In certain embodiments of the present invention, the A1 β-casein variant includes any one or more of A1 β-casein, B β-casein, C β-casein, F β-casein, G β-casein and H1 β-casein. For example, the A1 β-casein variant may include only A1 β-casein.

[0024] In certain embodiments of the present invention, the A2 type β-casein variant includes any one or more of A2 β-casein, A3 β-casein, D β-casein, E β-casein, H2 β-casein and I β-casein. For example, the A2 type β-casein variant may include only A2 β-casein.

[0025] In some embodiments, the A1 β-casein variant includes only A1 β-casein, and the A2 β-casein variant includes only A2 β-casein.

[0026] In some embodiments of the invention, the concentration of three or four β-casein digestive peptides having histidine at a position corresponding to position 67 of the β-casein amino acid sequence is determined. In some embodiments, where the milk is bovine milk, the β-casein digestive peptide is selected from the group consisting of VYPFPGPIHN, SLVYPFPGPIHN, VYPFPGPIHNSLPQ and SLVYPFPGPIHNSLPQ

[0027] In some embodiments of the invention, the concentrations of three or four β-casein digestive peptides having proline at a position corresponding to position 67 of the β-casein amino acid sequence are determined. In some embodiments, where the milk is bovine milk, the β-casein digestive peptide is selected from VYPFPGPIPN, SLVYPFPGPIPN, VYPFPGPIPNSLPQ and SLVYPFPGPIPNSLPQ.

[0028] In certain embodiments of the invention, the mixture analyzed using liquid chromatography-mass spectrometry contains isotope-labeled peptides corresponding to each of the peptides of a) and b). The isotope-labeled peptides may be added to the composition before treatment with chymotrypsin in step (i) or after treatment with chymotrypsin in step (i).

[0029] In some embodiments of the invention, where the milk is bovine milk, the isotope-labeled peptides may correspond to the following digestive peptides:

[0030]

[0031]

[0032] In some embodiments of the invention, where the milk is bovine milk, the isotope-labeled peptides may correspond to the following digestive peptides:

[0033] Digestive Peptides Isotope-labeled peptides VYPFPGPIPN (*V)YPFPGPIPN SLVYPFPGPIPN SL(*V)YPFPGPIPN VYPFPGPIPNSLPQ VYPFPGPIPNS(*L)PQ SLVYPFPGPIPNSLPQ SL(*V)YPFPGPIPNSLPQ

[0034] In some embodiments of the invention, where the milk is bovine milk, the concentrations of the following peptides are determined in step (iii): VYPFPGPIHN, SLVYPFPGPIHN, VYPFPGPIHNSLPQ, SLVYPFPGPIHNSLPQ, VYPFPGPIPN, SLVYPFPGPIPN, VYPFPGPIPNSLPQ and SLVYPFPGPIPNSLPQ.

[0035] In some embodiments of the invention, the content of the A1 β-casein variant and the A2 β-casein variant in the composition is determined with an accuracy of greater than 90%, greater than 95%, or greater than 99%.

[0036] In some embodiments of the invention, the composition is cow's milk or a milk product made from cow's milk.

[0037] In some embodiments of the present invention, milk is fresh milk, raw milk, milk powder, liquid milk reconstituted from milk powder, skim milk, homogenized milk, concentrated milk, evaporated milk, flavored milk, UHT milk, pasteurized milk or unpasteurized milk. In some embodiments of the present invention, the dairy product is infant formula, cream, yogurt, quark, cheese, butter or ice cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Shown is the proportion of type A1 β-casein in a series of relevant milk standards analyzed using the method of the invention and complete HPLC analysis.

[0040] Figure 2 Shown is the content of beta-casein in a series of milk samples and milk products measured by the method of the invention compared to the theoretical beta-casein level determined from the protein content of the samples.

[0041] Details

[0042] definition

[0043] The term "β-casomorphin-7" or "BCM-7" refers to the protein fragment Tyr-Pro-Phe-Pro-Gly-Pro-Ile, a heptapeptide produced upon enzymatic digestion of a bovine β-casein variant having histidine instead of proline at position 67 of the amino acid sequence.

[0044] The term "A1 type β-casein variant" means any β-casein variant having a histidine residue at position 67 of its amino acid sequence, and includes any one or more of A1 β-casein, B β-casein, C β-casein, F β-casein and G β-casein.

[0045] The term "A2 type β-casein variant" refers to any β-casein variant having a proline residue at position 67 of its amino acid sequence, and includes A2 β-casein, A3 β-casein, D β-casein, E β-casein, H1 β-casein, H2 β-casein and I β-casein.

[0046] The term "position 67" of the β-casein amino acid sequence refers to the position numbered 67 when counting the amino acid residue of the sequence from the N-terminus of β-casein (after removing the signal peptide).

[0047] The term "AQ", sometimes referred to as "AQUA", stands for "absolute quantification" and indicates that the methods of the invention can be used to measure the absolute concentration of β-casein as a fraction (e.g., mass fraction) of a sample, as opposed to other methods that do not measure absolute concentration but rather measure the ratio of β-casein relative to another component in the sample, or the ratio of one β-casein variant relative to another.

[0048] Throughout the specification, unless specifically stated otherwise or the context requires otherwise, reference to an individual step, composition of matter, group of steps or group of compositions of matter shall be considered to include one or several (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0049] Any reference to a numerical range described in this specification (e.g., 1 to 10) is also intended to include reference to all related numbers within the range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any reasonable numerical range within the range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and therefore includes all subranges of all ranges described in this specification that are explicitly disclosed. These are examples for specific purposes only, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be explicitly stated in this specification in a similar manner.

[0050] Throughout the specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to mean the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0051] Those skilled in the art will recognize that the invention described in this specification may be subject to changes and modifications in addition to those specifically described. It should be understood that the present invention includes all such changes and modifications. The present invention also includes, individually or collectively, all steps or features mentioned or indicated in this specification, as well as any and all combinations or any two or more of the steps or features.

[0052] The scope of the present invention is not limited by the specific embodiments described in this specification, which are provided for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention.

[0053] Any reference to prior art documents in this specification should not be considered as an admission that the prior art is known in the art or forms part of the common general knowledge in the art.

[0054] Test Method

[0055] The present invention provides a method for determining the content of A1 type β-casein variant and A2 type β-casein variant in a composition. Importantly, the method is capable of determining the content of two types of β-casein variants with an accuracy higher than 80%, and even up to 100%. Because the demand for milk and milk products that do not contain A1 β-casein (and related variants) has increased significantly in the past decade, the degree of accuracy has become important. Consumers as well as manufacturers and suppliers need to know whether a product is substantially free of A1 type β-casein variants.

[0056] The method relates to the beta-casein protein in the composition digested with the serine protease chymotrypsin. The applicant has determined that the digestion process produces specific identifiable peptides. The presence and content of each peptide produced from the composition can be determined and the content is subsequently used to calculate the percentage content of A1 type beta-casein variants and A2 type beta-casein variants in the composition. The number of peptides analyzed (e.g., 2,3,4 or more) determines the degree of accuracy. For example, the final determination of the percentage content of the beta-casein variants of two types in the composition produced by the analysis of only two peptides is only 80% accuracy level. The analysis of three peptides can produce an accuracy level of about 90%, and analyzing four peptides can produce an accuracy level of about 95%.

[0057] The composition to be analyzed may be milk from any animal or a product made from animal milk. The composition may also be a non-dairy product containing beta-casein that has been obtained from milk.

[0058] The milk may be fresh milk, raw milk, powdered milk, liquid milk reconstituted from powdered milk, skim milk, homogenized milk, concentrated milk, evaporated milk, flavored milk, UHT milk, pasteurized milk or unpasteurized milk, or any other form of milk.

[0059] The methods of the present invention are most suitable for the analysis of bovine milk and bovine milk products, but can also be readily adapted to milk and milk products from a variety of animals including humans, sheep, goats, horses, pigs, buffalo, deer and any other mammal that produces casein in its milk.

[0060] The composition may also be any milk-derived product such as infant formula, cream, yoghurt, quark, cheese, butter or ice cream.

[0061] In case the composition to be analysed is milk, minimal sample preparation is required.The proteins contained in the milk sample are preferably denatured, for example by treatment with a denaturing agent such as guanidine hydrochloride or urea.

[0062] Where the dairy product is a solid or semi-solid, sample preparation typically involves weighing a uniform amount of product into a separate container and extracting the casein protein into a known volume of solution.

[0063] Digestion of β-casein in a sample using chymotrypsin can be performed under conditions suitable for efficient digestion. The sample is typically incubated at about 37°C for several hours, followed by quenching with a reagent (such as formic acid) to inactivate chymotrypsin.

[0064] Once the sample has been subjected to chymotrypsin digestion and any post-digestion sample preparation, certain isotope-labeled peptides are added to the sample and then subjected to LC-MS (liquid chromatography-mass spectrometry) analysis. The isotope-labeled peptides are selected based on the applicant's previous determination that chymotrypsin breaks down type A1 β-casein variants to produce digestion peptides VYPFPGPIHN, SLVYPFPGPIHN, VYPFPGPIHNSLPQ and SLVYPFPGPIHNSLPQ, and breaks down type A2 β-casein variants to produce digestion peptides VYPFPGPIPN, SLVYPFPGPIPN, VYPFPGPIPNSLPQ and SLVYPFPGPIPNSLPQ.

[0065] Isotope-labeled peptides may be from the following group: (*V)YPFPGPIHN, SL(*V)YPFPGPIHN, VYPFPGPIHNS(*L)PQ, SL(*V)YPFPGPIHNSLPQ, (*V)YPFPGPIPN, SL(*V)YPFPGPIPN, VYPFPGPIPNS(*L)PQ, and SL(*V)YPFPGPIPNSLPQ, wherein *V represents an isotope-labeled valine residue and *L represents an isotope-labeled leucine residue. It should be appreciated that any one or more of the amino acid residues of the peptide may be isotope-labeled. However, isotope-labeled valine or leucine are generally preferred.

[0066] In some embodiments of the invention, the isotopically labeled peptides used correspond to the digested peptides according to Table 1.

[0067] Table 1: Digested peptides and their isotope-labeled equivalents

[0068]

[0069]

[0070] The method of the present invention requires at least two of the above isotope-labeled peptides to be added to the sample. However, for higher accuracy, three, four or possibly more isotope-labeled peptides are added to the sample.

[0071] LC-MS / MS can easily identify the presence of any one digested peptide based on the retention time of the peptide and the ion ratio of the fragments produced when analyzed in MS / MS mode. This is alternatively referred to as multiple reaction monitoring (MCM) mode, or selected reaction monitoring (SRM) mode. Comparison of the response of each peptide to an isotopically labeled internal standard peptide present in the mixture and a standard curve run simultaneously allows the determination of the concentration of each peptide, from which the concentration of the A1 type β-casein variant in the composition and the A2 type β-casein variant in the composition can be calculated.

[0072] Discussion of Examples

[0073] The example of experimental procedure is provided below. Example 1 shows how to prepare samples for analysis. This generally includes extracting beta-casein into an aqueous state, where they can be unfolded by the action of a chaotropic denaturant (such as urea), so that the action of chymosin on the sample is more effective afterwards. In the case of liquid samples (such as whole milk or UHT milk), this simply involves transferring aliquots of the sample into a container and adding a denaturant. For solid or semi-solid samples, this involves the preparation of an accurately defined solution of the solid dissolved in the extraction buffer (usually an aqueous buffer). Subsequently, the sample is treated in the same manner as for the liquid sample.

[0074] Example 2 describes the chymotrypsin digestion step. The denatured sample is exposed to a chymotrypsin solution under carefully controlled conditions. These conditions typically include a buffer to control the pH of the solution, because the proteolytic enzyme is active only within a narrow pH range. The conditions typically also include mechanical agitation of the digestion to reduce or eliminate material transfer restrictions, and heating to accelerate the reaction. This results in a controlled decomposition of β-casein and other proteins in the matrix to produce a group of peptides that can be uniquely identified as being derived from a specific protein. After a predetermined period of time, the reaction is stopped ("quenching") by adding a concentrated acid solution in an organic solvent, which immediately stops the digestion. The sample is then strengthened by adding an isotope-labeled internal standard peptide, which is subsequently used to calculate the concentration of β-casein.

[0075] Example 3 describes the analysis of samples using liquid chromatography using triple quadrupole mass spectrometry detection. This results in the spatial and temporal separation of the peptides, which can be quantified individually by passing the eluent through a mass spectrometer operated in multiple reaction monitoring mode or an equivalent specific detection procedure (such as high resolution selected ion monitoring). The peak area of ​​the target peptide is determined by integration and isotope-labeled internal standards. The peak area ratio is then used to determine the concentration of the target peptide by comparison with a standard curve. To ensure that the peptides are correctly identified, quality control criteria such as retention time matching and ion ratio matching are used in the procedure.

[0076] In Example 4, the concentration of the peptide is used to calculate the concentration of the parent β-casein. Typically, a conversion factor is derived based on the ratio of the peptide mass to the parent protein mass and used to convert from the peptide concentration to the protein concentration. Because the target peptides have overlapping sequences, the concentrations can then be summed to find the total A1 β-casein content, and the total A2 β-casein content, as well as graphs derived from these values, such as the ratio of A1 β-casein to total β-casein in the sample.

[0077] Example 5 shows the application of this method in the analysis of a range of dairy products. The results are compared and contrasted with the results of existing methods for determining the percentage of type A1 beta-casein in milk or dairy product samples. For a range of matrix types, it is shown that the concentration of beta-casein in the sample is consistent with the expected value based on the protein content of the marker, and therefore the proportion of type A1 beta-casein in the sample is correct. In addition, the proportion of type A1 beta-casein is consistent with previous methods for simple matrices (such as milk or milk powder), but can provide more accurate estimates for severely modified products such as infant formula milk. The results are shown in Table 6.

[0078] Column 1 of Table 6 indicates the identity of the product analyzed. Column 2 shows the total protein content of each sample as determined by Kjeldahl analysis. This can be compared with column 3, which shows the protein content specified on the product label. In general, there was good agreement between the two measurements, indicating that the labeled protein content is accurate. Column 4 shows the theoretical β-casein content of each sample, which was determined by multiplying the labeled protein value by 0.32, a recognized conversion factor representing the mass fraction of β-casein in a dairy product relative to the total protein content.

[0079] Column 9 shows the total β-casein content, expressed in g / 100 g, as the sum of type A1 β-casein and type A2 β-casein, as determined by the method of the invention. These generally show good agreement with the values ​​in column 4, indicating that the method is accurate and suitable for the purpose.

[0080] Column 10 shows the percentage of beta-casein type A1 of the total beta-casein content, calculated as the sum of beta-casein type A1 and beta-casein type A2. These results can be compared with those in column 5, which present the A1%, which is determined by intact protein analysis using high performance liquid chromatography and ultraviolet detection. For milk and milk products that have not been significantly altered, there is good agreement between the two figures. However, in the case of infant formula samples, there are significant differences, due to the nature of their manufacture, they are not suitable for intact analysis and produce erroneous results. A similar trend is seen when comparing column 10 with column 6, which shows the percentage of beta-casein type A1 determined using the original enzymatic method using trypsin digestion of the sample and subsequent calibration with relative milk standards diluted relative to the volume.

[0081] Figure 1 The proportion of A1 β-casein in a series of relative milk standards analyzed using the method of the present invention is shown, as well as the complete HPLC analysis. The samples were prepared by volume dilution of milk from two separate animals, one of which was confirmed to be of the A1 / A1 genotype and therefore produced milk containing pure A1 β-casein, and the other of the A2 / A2 genotype and therefore produced milk containing pure A2 β-casein. As shown by the correlation coefficient (R 2 =0.9996), the tight clustering of data points around the best fit line indicates that the current method is accurate. The slope of the interpretation line (0.9861) indicates that the current method is also accurate.

[0082] Figure 2 The β-casein content in a series of milk samples and milk products measured by the method of the present invention is shown compared to the theoretical β-casein level determined from the protein content of the samples. The good clustering of the data points about the best fit line shows that the method is accurate, as is the correlation coefficient (R2=0.9075). The slope of the line (0.9075) also shows that the method is accurate.

[0083] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by the examples. Example

[0084] Example 1: Sample preparation

[0085] Liquid samples - For non-viscous liquid samples, such as milk, transfer the liquid (40 μL) to a 2 mL centrifuge tube. Add urea solution (120 μL, 500 g / L) to denature the protein, and then mix the mixture at 2000 RPM for 5 min.

[0086] Solid sample-For solid sample or viscous liquid sample, sample (5.00 ± 0.01g) is weighed in falcon tube (50ml).Ammonium bicarbonate solution (46.6ml, 4g / L) is added, and the mixture is vibrated to wet all powders.After incubation at 50 ℃ for 15min, the mixture is vibrated to uniformity.In the case of infant formula milk powder, sample (100 μL) is transferred in 2mL centrifuge tube, casein precipitation agent (150g / L ammonium acetate pH4.2, 1000 μL) is added, and the mixture is mixed at 2000RPM for 5min, then centrifuged at 5000RCF for 5min.After decanting the supernatant, casein precipitation agent (1000 μL) is added in solid precipitate, and the mixture is mixed at 2000RPM for 5min, then centrifuged at 5000RCF for 5min before decanting the supernatant. Water (1000 μL) was added to the solid precipitate, the mixing and centrifugation steps were repeated, and the supernatant was decanted. Urea solution (120 μL, 500 g / L) was added to denature the protein, and the mixture was mixed at 2000°C for 5 min.

[0087] Example 2: Chymotrypsin digestion

[0088] Chymotrypsin solution (1000 μL) was added to the sample prepared according to Example 1. The mixture was incubated at 37°C for 8 hours while shaking at 200RPM. A quenching solution (120 μL, 500 g / L formic acid solution) was added, and the mixture was mixed at 2000RPM for 5 min and centrifuged at 10,000RCF for 10 min. The sample (980 μL) was transferred to an LC vial, an isotope-labeled peptide standard solution (20 μL) was added, and the mixture was vortexed for 10 sec. A labeled peptide standard solution (Biomatik, custom made, ≥95.0%) was prepared by weighing each peptide in Table 1 (2.00 ± 0.10 mg) into a 2 mL polyethylene centrifuge tube. The peptides were dissolved by adding 20% ​​acetonitrile containing 0.1% formic acid. The volume was adjusted to obtain a concentration of 1,000.0 mg / L. For example, if only 1.90 mg was weighed, 1.90 mL of solution was added. The labeled peptides in Table 2 correspond to the target peptides in Table 3.

[0089] Table 2: Isotope-labeled peptide standards

[0090]

[0091] Table 3: Target peptides

[0092]

[0093]

[0094] Example 3: LC-MS / MS analysis

[0095] The sample (1 μL) was injected onto a Waters Xselect HSS T3 C18 column (2.5 um × 2.1 mm × 50 mm) connected to a Phenomenex KrudCatcher using a Thermo Vanquish LC system. The separation parameters are summarized in Table 4. In brief, a gradient was run from 20% acetonitrile to 40% acetonitrile to separate the target peptides, during which the eluent was passed to the mass spectrometer for detection. The column was then washed at an increased flow rate before the next injection was started. During this wash, unwanted components in the sample were diverted to waste and acquisition was turned off.

[0096] Table 4: Gradients and selected acquisition parameters

[0097] time ％A ％B Flow rate Transfer MS acquisition 0.0 80 20 0.5 1-6 close 0.5 80 20 0.5 1-2 open 4.0 60 40 0.5 1-2 close 4.5 0 100 0.8 1-6 close 6.0 0 100 0.8 1-6 close 6.5 80 20 0.8 1-6 close 7.5 80 20 0.5 1-6 close 8.0 80 20 0.5 1-6 close

[0098] Mass spectra were acquired using a Thermo TSQ Altis mass spectrometer, operating in positional ion MRM mode. Each peptide had quantitative and qualitative transitions, with a total of 32 MRMs acquired. All of these were monitored continuously during acquisition without the use of scheduling. The dwell time was set to 10 m, Q1 and Q3 resolution was set to 2 Da, and the CID gas was set to 2 mTorr. A list of transitions and related parameters is shown in Table 5.

[0099] Table 5: MRM data of the sixteen target peptide monitoring methods

[0100]

[0101]

[0102] Example 4: Concentration determination of type A1 β-casein variant and type A2 β-casein variant

[0103] Although the calculations can be done manually, they can be automated using a pre-designed digital spreadsheet. The concentration of the A1-β-casein variant is calculated by summing the concentration of each peptide times the product of the quotient of the parent protein mass and the peptide mass, and then multiplying by the preparation factor:

[0104]

[0105] The same calculation was used to calculate the concentration of the A2-β-casein variant, but using the correspondingly adjusted mass:

[0106]

[0107] The preparation factor, P, is calculated as follows:

[0108]

[0109] where v1 is the final volume of the product after extraction, m1 is the weighed mass of the product, v2 is the volume in the digestion tube after quenching, v3 is the volume of the extract added to the digestion tube, v4 is the final volume in the LC vial, and v5 is the volume of the digestion solution transferred to the LC vial.

[0110] The total β-casein concentration was calculated by adding the concentrations of the two polymorphs:

[0111] C 总βCN =C A1βCN +C A2βCN

[0112] The percentage of the A1-β-casein variant in the product is calculated by dividing the A1-β-casein variant concentration by the sum of the A1- and A2-β-casein variant concentrations:

[0113]

[0114] Example 5: Analysis of milk and milk products

[0115] Various fresh milk, milk powder, infant formula and UHT milk samples were tested according to the methods outlined in Examples 1 to 4. A series of milk standards were also prepared and tested. The results are shown in Table 4, where the results determined according to the method of the present invention are compared with other methods for determining the content of β-casein.

[0116]

[0117]

[0118] References

[0119] 1. Trivedi, MS, Shah, JS, A1-Mughairy, S., Hodgson, N.W., Simms, B., Trooskens, G., Van Criekinge, W., and Deth, RC, J. Nutr. Biochem., 2014, 25, 1011-1018.

[0120] 2. WO 1996 / 014577

[0121] 3. WO 1996 / 036239

[0122] 4. WO 2002 / 019832

[0123] 5. WO 2014 / 193248

[0124] 6. WO 2015 / 005804

[0125] 7. WO 2015 / 026245

[0126] 8. WO 2017 / 171563

[0127] 9. WO 2018 / 063008

[0128] 10. Vincent, D., Elkins, A., Condina, M.R., Ezernieks, V. and Rochfort, S., 2016, PLOS ONE 11, e0163471.

[0129] 11. Givens, I., Aikman, P., Gibson, T. and Brown, R., Food Chemistry, 2013, 139, 549 - 552.

[0130] 12. Lutter, P., Parisod, V. and Weymuth, H., J.AOAC Int., 2011, 94, 1043 - 1059.

Claims

1. A method for determining the content of type A1 β-casein variant or type A2 β-casein variant in a composition with an accuracy of more than 80%, wherein type A1 β-casein variant is a β-casein variant having histidine at position 67 of the β-casein amino acid sequence, and type A2 β-casein variant is a β-casein variant having proline at position 67 of the β-casein amino acid sequence, and wherein the composition is milk or a product obtained from milk, comprising the steps of: (i) contacting the composition with chymotrypsin under conditions capable of digesting β-casein in the composition by chymotrypsin; (ii) obtaining an aqueous solution of β-casein digest peptides; (iii) adding isotope-labeled peptides corresponding to three or four of the following peptides: VYPFPGPIHN; SLVYPFPGPIHN; VYPFPGPIHNSLPQ; and SLVYPFPGPIHNSLPQ; (iv) adding isotope-labeled peptides corresponding to three or four of the following peptides: VYPFPGPIPN; SLVYPFPGPIPN; VYPFPGPIPNSLPQ; and SLVYPFPGPIPNSLPQ; (v) determining the concentration of the three or four peptides from (iii) present in the solution and the concentration of the three or four peptides from (iv) present in the solution by analyzing the mixture using liquid chromatography-mass spectrometry; and (vi) calculating the concentration of the A1 β-casein variant in the composition or the concentration of the A2 β-casein variant in the composition using the concentration of the peptide determined in step (v).

2. The method as claimed in claim 1, wherein the A1 type β-casein variant comprises any one or more of A1 β-casein, B β-casein, C β-casein, F β-casein and G β-casein.

3. A method as claimed in claim 1 or claim 2, wherein the A1 β-casein variant comprises only A1 β-casein.

4. The method as claimed in claim 1, wherein the A2 type β-casein variant comprises any one or more of A2 β-casein, A3 β-casein, D β-casein, E β-casein, H1 β-casein, H2 β-casein and I β-casein.

5. A method as claimed in claim 1, wherein the A2 type β-casein variant comprises only A2 β-casein.

6. A method as claimed in claim 1, wherein the A1 β-casein variant comprises only A1 β-casein and the A2 β-casein variant comprises only A2 β-casein.

7. A method as claimed in claim 1, wherein the isotope-labeled peptide corresponds to a digested peptide of: 。 8. A method as claimed in claim 1, wherein the isotope-labeled peptide corresponds to a digested peptide of: 。 9. A method as claimed in claim 1, wherein in step (v) the concentration of the following peptides is determined: (i) VYPFPGPIHN; (ii) SLVYPFPGPIHN; (iii) VYPFPGPIHNSLPQ; (iv)SLVYPFPGPIHNSLPQ; (v)VYPFPGPIPN; (vi)SLVYPFPGPIPN; (vii) VYPFPGPIPNSLPQ; and (viii)SLVYPFPGPIPNSLPQ.

10. A method as claimed in claim 1, wherein the content of the A1 beta-casein variant and the A2 beta-casein variant in the composition is determined with an accuracy of more than 90%.

11. A method as claimed in claim 1, wherein the composition is cow's milk or a milk product obtained from cow's milk.

12. A method as claimed in claim 1, wherein the milk is fresh milk, raw milk, milk powder, liquid milk reconstituted from milk powder, skimmed milk, homogenized milk, concentrated milk, evaporated milk, flavored milk, UHT milk, pasteurized milk or unpasteurized milk.

13. A method as claimed in claim 11, wherein the dairy product is infant formula, cream, yoghurt, quark, cheese, butter or ice cream.

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