BETA-CASEIN ANALYSIS OF MILK AND MILK PRODUCTS

Abstract

A method for testing for the presence and quantification of A1-type beta-casein variants or A2-type beta-caseins, in milk and milk derived dairy products, using chymotrypsin digestion followed by LC-MS analysis to determining the concentrations of beta-casein digestion peptides and using the concentrations to calculate the amounts of A1-type beta-casein variants or A2-type beta-casein variants present.

Claims

1. A method for determining the amounts of A1-type beta-casein variants or A2-type beta-casein variants in a composition with greater than 80% accuracy, where the A1-type beta-casein variants are beta-casein variants having histidine at position 67 of the beta-casein amino acid sequence and the A2-type beta-casein variants are beta-casein variants having proline at position 67 of the beta-casein amino acid sequence, and where the composition is milk or a product prepared from milk, comprising the steps: (i) contacting the composition with chymotrypsin under conditions enabling digestion of beta-caseins in the composition by the chymotrypsin; (ii) obtaining an aqueous solution of beta-casein digestion peptides; (iii) determining the concentrations of: a) two or more beta-casein digestion peptides present in the solution, wherein each peptide comprises 8 to 20 amino acid residues and has histidine at a position corresponding to position 67 of the beta-casein amino acid sequence; and b) two or more beta-casein digestion peptides present in the solution, wherein each peptide comprises 8 to 20 amino acid residues and has proline at a position corresponding to position 67 of the beta-casein amino acid sequence; by analysing the mixture using liquid chromatography-mass spectrometry; and (iv) calculating the concentration of A1-type beta-casein variants in the composition or the concentration of A2-type beta-casein variants in the composition using the concentrations of the beta-casein digestion peptides determined in step (iii).

2. A method as claimed in claim 1, wherein the A1-type beta-casein variants comprise any one or more of A1 beta-casein, B beta-casein, C beta-casein, F beta-casein and G beta casein.

3. A method as claimed in claim 1 or claim 2, wherein the A1-type beta-casein variants comprise solely A1 beta-casein.

4. A method as claimed in any one of claims 1 to 3, wherein the A2-type beta-casein variants comprise any one or more of A2 beta-casein, A3 beta-casein, D beta-casein, E beta-casein, H1 beta-casein, H2 beta-casein and I beta-casein.

5. A method as claimed in any one of claims 1 to 4, wherein the A2-type beta-casein variants comprise solely A2 beta-casein.

6. A method as claimed in any one of claims 1 to 5, wherein the A1-type beta-casein variants comprise solely A1 beta-casein and the A2-type beta-casein variants comprise solely A2 beta-casein.

7. A method as claimed in any one of claims 1 to 6, wherein the concentrations of three or four beta-casein digestion peptides having histidine at a position corresponding to position 67 of the beta-casein amino acid sequence are determined.

8. A method as claimed in claim 7, wherein the milk is bovine milk and the beta-casein digestion peptides are selected from the group comprising: TABLE-US-00009 (i) VYPFPGPIHN; (ii) SLVYPFPGPIHN; (iii) VYPFPGPIHNSLPQ; and (iv) SLVYPFPGPIHNSLPQ.

9. A method as claimed in any one of claims 1 to 6, wherein the concentrations of three or four beta-casein digestion peptides having proline at a position corresponding to position 67 of the beta-casein amino acid sequence are determined.

10. A method as claimed in claim 8, wherein the milk is bovine milk and the beta-casein digestion peptides are selected from the group comprising: TABLE-US-00010 (i) VYPFPGPIPN; (ii) SLVYPFPGPIPN; (iii) VYPFPGPIPNSLPQ; and (iv) SLVYPFPGPIPNSLPQ.

11. A method as claimed in any one of claims 1 to 10, wherein the mixture analysed using liquid chromatography-mass spectrometry contains isotopically labelled peptides corresponding to each of the peptides from a) and b) of step (iii).

12. A method as claimed in claim 11, wherein the isotopically labelled peptides correspond to the digestion peptides as follows: TABLE-US-00011 Digestion Peptide Isotopically Labelled Peptide VYPFPGPIHN (*V)YPFPGPIHN SLVYPFPGPIHN SL(*V)YPFPGPIHN VYPFPGPIHNSLPQ VYPFPGPIHNS(*L)PQ SLVYPFPGPIHNSLPQ SL(*V)YPFPGPIHNSLPQ

13. A method as claimed in claim 11, wherein the isotopically labelled peptides correspond to the digestion peptides as follows: TABLE-US-00012 Digestion Peptide Isotopically Labelled Peptide VYPFPGPIPN (*V)YPFPGPIPN SLVYPFPGPIPN SL(*V)YPFPGPIPN VYPFPGPIPNSLPQ VYPFPGPIPNS(*L)PQ SLVYPFPGPIPNSLPQ SL(*V)YPFPGPIPNSLPQ

14. A method as claimed in claim 1, wherein the milk is bovine milk and the concentrations of the following peptides are determined in step (iii): TABLE-US-00013 (i) VYPFPGPIHN; (ii) SLVYPFPGPIHN; (iii) VYPFPGPIHNSLPQ; (iv) SLVYPFPGPIHNSLPQ; (v) VYPFPGPIPN; (vi) SLVYPFPGPIPN; (vii) VYPFPGPIPNSLPQ; and (viii) SLVYPFPGPIPNSLPQ.

15. A method as claimed in any one of claims 1 to 14, wherein the amounts of A1-type beta-casein variants and A2-type beta-casein variants in the composition are determined with greater than 90% accuracy.

16. A method as claimed in any one of claims 1 to 15, wherein the amounts of A1-type beta-casein variants and A2-type beta-casein variants in the composition are determined with greater than 95% accuracy.

17. A method as claimed in any one of claims 1 to 16, wherein the amounts of A1-type beta-casein variants and A2-type beta-casein variants in the composition are determined with greater than 99% accuracy.

18. A method as claimed in any one of claims 1 to 17, wherein the composition is bovine milk or a milk product prepared from bovine milk.

19. A method as claimed in claim 18, wherein the milk is fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk, or non-pasteurised milk.

20. A method as claimed in claim 18, wherein the milk product is infant formula, cream, yoghurt, quark, cheese, butter, or ice cream.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 shows the proportion of A1-type beta-caseins in a series of relative milk standards analysed using the method of the invention, as well as intact HPLC analysis.

[0035] FIG. 2 shows the amount of beta-casein in a series of milk samples and milk products as measured by the method of the invention, compared to the theoretical beta-casein level determined from the protein content of the samples.

DETAILED DESCRIPTION

Definitions

[0036] The term “beta-casomorphin-7” or “BCM-7” refers to the protein fragment Tyr-Pro-Phe-Pro-Gly-Pro-Ile, a heptapeptide produced on enzymatic digestion of bovine beta-casein variants that have a histidine, rather than a proline, at position 67 of the amino acid sequence.

[0037] The term “A1-type beta-casein variant” means any beta-casein variant having a histidine residue at position 67 of its amino acid sequence, and includes any one or more of A1 beta-casein, B beta-casein, C beta-casein, F beta-casein, and G beta casein.

[0038] The term “A2-type beta-casein variant” means any beta-casein variant having a proline residue at position 67 of its amino acid sequence, and includes A2 beta-casein, A3 beta-casein, D beta-casein, E beta-casein, H1 beta-casein, H2 beta-casein and I beta-casein.

[0039] The term “position 67” of the beta-casein amino acid sequence means the position numbered 67 counting amino acid residues of the sequence from the N terminus of the beta-casein (after removal of the signal peptide).

[0040] The term “AQ”, sometimes referred to as “AQUA”, means “absolute quantitation” and indicates that the method of the invention can be used to measure the absolute concentration of beta-caseins as a fraction of a sample (e.g. mass fraction), in contrast to other methods which do not measure absolute concentrations, but measure the proportion of beta-caseins relative to another component in a sample, or the proportion of one variant of beta-casein relative to another.

[0041] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0042] It is intended that any reference to a range of numbers described in this specification (e.g., 1 to 10) also incorporates reference to all related numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and therefore all sub-ranges of all ranges described in this specification are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this specification in a similar manner.

[0043] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0044] Those skilled in the art will appreciate that the invention described in this specification is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps or features referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0045] The invention is not to be limited in scope by the specific examples described in this specification, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention.

[0046] Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field.

Testing Methodology

[0047] The invention provides a method for determining the amounts of A1-type beta-casein variants and A2-type beta-casein variants in a composition. Importantly, the method enables the amounts of the two types of beta-casein variants to be determined with greater than 80% accuracy, and even up to 100% accuracy. The degree of accuracy has become critical as the demand for milk and milk products free of A1 beta-casein (and related variants) has increased dramatically in the past decade. Consumers as well as manufacturers and suppliers need to know whether or not a product is substantially free of A1-type beta-casein variants.

[0048] The method involves the digestion of beta-casein proteins in a composition with the serine protease enzyme chymotrypsin. The applicant has determined that the digestion process produces specific identifiable peptides. The presence and amounts of each peptide producing from the composition can be determined and the amounts then used to calculate the percentage amounts of A1-type beta-casein variants and A2-type beta-casein variants in the composition. The number of peptides (e.g., 2, 3, 4 or more) analysed determines the degree of accuracy. For example, analysis of only two peptides may produce a final determination of percentage amounts of the two types of beta-casein variants in the composition to an accuracy level of only 80%. Analysis of three peptides can produce an accuracy level of about 90%, and analysing for four peptides can produce an accuracy level of about 95%.

[0049] The composition to be analysed may be milk from any animal or a product prepared from animal milk. The composition may also be a non-milk product containing beta-casein that has been obtained from milk.

[0050] The milk may be in the form of fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk, or non-pasteurised milk, or any other form of milk.

[0051] The method of the invention is best suited for analysing bovine milk and bovine milk products, but can readily be adapted for milk and milk products from a wide variety of animals including humans, sheep, goats, horses, pigs, buffalo, deer, and any other mammals that produces caseins in their milk.

[0052] The composition may also be any milk-derived product such as infant formula, cream, yoghurt, quark, cheese, butter, or ice cream.

[0053] Where the composition to be analysed is milk, minimal sample preparation is needed. It is preferable to denature proteins contained in the milk sample, for example by treatment with a denaturing agent such as guanidine hydrochloride or urea.

[0054] In the case of milk products that are solid or semi-solid, sample preparation typically involves weighing an homogenised amount of the product into a separate vessel and extracting the casein proteins into a known volume of solution.

[0055] Digestion of the beta-caseins in the sample using chymotrypsin may be carried out under conditions suitable for enabling effective digestion. The sample is typically incubated at around 37° C. for several hours before quenching with a reagent, such as formic acid, to deactivate the chymotrypsin.

[0056] Once the sample has undergone chymotrypsin digestion, and any post-digestion sample preparation, certain isotopically labelled peptides are added to the sample prior to LC-MS (liquid chromatography—mass spectrum) analysis. The isotopically labelled peptides are selected based on the applicant's prior determination that chymotrypsin breaks down A1-type beta-casein variants to produce the digestion peptides:

[0057] VYPFPGPIHN, SLVYPFPGPIHN, VYPFPGPIHNSLPQ, and SLVYPFPGPIHNSLPQ

and breaks down A2-type beta-casein variants to produce the digestion peptides:

[0058] VYPFPGPIPN, SLVYPFPGPIPN, VYPFPGPIPNSLPQ, and SLVYPFPGPIPNSLPQ

The isotopically labelled peptides may be from the group:

[0059] (*V)YPFPGPIHN, SL(*V)YPFPGPIHN, VYPFPGPIHNS(*L)PQ, SL(*V)YPFPGPIHNSLPQ, (*V)YPFPGPIPN, SL(*V)YPFPGPIPN, VYPFPGPIPNS(*L)PQ, and SL(*V)YPFPGPIPNSLPQ, where *V represents an isotopically labelled valine residue and *L represents an isotopically labelled leucine residue. It should be appreciated that any, or multiple, amino acid residues of a peptide may be isotopically labelled. However, isotopically labelled valine or leucine are generally preferred.

[0060] In some embodiments of the invention, the isotopically labelled peptides used correspond to the digestion peptides according to Table 1.

TABLE-US-00003 TABLE 1 Digestion peptides and their isotopically labelled equivalents Digestion Peptide Isotopically Labelled Peptide VYPFPGPIHN (*V)YPFPGPIHN SLVYPFPGPIHN SL(*V)YPFPGPIHN VYPFPGPIHNSLPQ VYPFPGPIHNS(*L)PQ SLVYPFPGPIHNSLPQ SL(*V)YPFPGPIHNSLPQ VYPFPGPIPN (*V)YPFPGPIPN SLVYPFPGPIPN SL(*V)YPFPGPIPN VYPFPGPIPNSLPQ VYPFPGPIPNS(*L)PQ SLVYPFPGPIPNSLPQ SL(*V)YPFPGPIPNSLPQ

[0061] The method of the invention requires adding at least two of the above isotopically labelled peptides to the sample. However, for greater accuracy, three, four, or possibly more, isotopically labelled peptides are added to the sample.

[0062] LC-MS/MS enables ready identification of the presence of any of the digestion peptides based on the retention time of the peptides and the ion ratios of the fragments produced when analysed in MS/MS mode. This is alternately referred to as multiple reaction monitoring (MRM) mode, or selected reaction monitoring (SRM) mode. Comparison of the responses of each peptide to an isotopically labelled internal standard peptide present in the mixture and to a concurrently run standard curve allows the concentration of each peptide to be determined, from which the concentrations of the A1-type beta-casein variants in the composition and the A2-type beta casein variants in the composition can be calculated.

Discussion of the Examples

[0063] Examples of the experimental steps are provided below. Example 1 shows how samples may be prepared for analysis. This generally includes the extraction of beta-caseins into an aqueous state, wherein they can be unfolded by the action of a chaotropic denaturing agent such urea, so that the later action of chymotrypsin on the sample is more efficient. In the case of liquid samples such as whole milk or UHT milk, this simply involves the transfer of an aliquot of the sample to a vessel and the addition of the denaturing agent. For solid or semi-solid samples, this involves the preparation of an accurately defined solution of the solid dissolved in an extraction buffer, typically an aqueous buffer. Subsequently, the samples are treated in the same manner as for liquid samples.

[0064] Example 2 describes the chymotrypsin digestion step. The denatured samples are exposed to a solution of chymotrypsin under carefully controlled conditions. These conditions typically include a buffer to control the pH of the solution, as proteolytic enzymes are only active within a narrow pH range. Conditions typically also include mechanical agitation of the digest to reduce or eliminate mass transfer limitations, and heat to accelerate the reaction. This results in a controlled cleavage of the beta-caseins, and other proteins within the matrix, to yield a set of peptides which can be uniquely identified as originating from a specific protein. After a pre-determined period of time the reaction is halted (‘quenched’) by the addition of a concentrated acid solution in organic solvent, which immediately stops the digestion. The samples are then fortified by the addition of isotopically-labelled internal standard peptides which are subsequently used for calculating the concentrations of the beta-caseins.

[0065] Example 3 describes the analysis of samples using liquid chromatography with triple quadrupole mass spectrometric detection. This results in spatial and temporal separation of the peptides from one another, which can be individually quantified by passage of the eluent to a mass spectrometer operated in multiple reaction monitoring mode, or an equivalently specific detection procedure such as high resolution selected ion monitoring. The peak areas of target peptides are determined by integration, as well as the isotopically-labelled internal standards. The peak area ratios are then used to determine the concentration of the target peptides by comparison against a standard curve. To ensure the peptides are correctly identified, quality control criteria such retention time matching and ion ratio matching are employed in the procedure.

[0066] In Example 4, the concentrations of the peptides are used to calculate the concentrations of the parent beta-caseins. In general, conversion factors are derived based on the ratio of the mass of the peptide to the mass of the parent protein, and are used to convert from concentration of peptide to concentration of protein. As the target peptides have overlapping sequences, the concentrations can then be summed to find the total A1-type beta-casein content, and the total A2-type beta-casein content, as well as figures derived from these values, such as the proportion of A1-type beta-casein in the sample relative to the total beta-casein.

[0067] Example 5 shows application of the method to the analysis of a range of milk products. The results are compared and contrasted with results from existing methods for determining the percentage of A1-type beta-caseins in a sample of milk or a milk product. For a range of matrix types, it is shown that the concentration of beta-caseins in the sample is consistent with the expected values based on the labelled protein content, and thus the proportion of A1-type beta-caseins in the samples is correct. Moreover, the proportion of A1-type beta-caseins is consistent with previous methods for simple matrices like milk or milk powder, but provides a more accurate estimate for heavily modified products such as infant formula. The results are shown in Table 6.

[0068] Column 1 of Table 6 indicates the identity of the products analysed. Column 2 shows the total protein content of each sample as determined by Kjeldahl nitrogen analysis. This may be compared to column 3, which shows the protein content as specified on the label of the product. In general there is good agreement between these two measures, indicating that the labelled protein content is accurate. Column 4 shows the theoretical beta-casein content of each sample, determined by multiplication of the labelled protein value by 0.32, a commonly accepted conversion factor representing the mass fraction of beta-casein in milk products relative to the total protein content.

[0069] Column 9 shows the total beta-casein content, expressed in g/100 g, as the sum of A1-type beta-caseins and A2-type beta-caseins, as determined by the method of the invention. These show generally good agreement with the values in column 4, indicating that the method is accurate and fit for purpose.

[0070] Column 10 shows the percentage of A1-type beta-caseins of the total beta-casein content, calculated as the sum of A1-type beta-caseins and A2-type beta-caseins. These results may be compared to the results in column 5, which presents the % A1 as determined by intact protein analysis using high performance liquid chromatography with ultraviolet detection. For milk and milk products which are not significantly modified, there is good agreement between these two figures. However, there are significant discrepancies in the case of infant formula samples, which by nature of their manufacture, are unsuitable for intact analysis and produce erroneous results. Similar trends are seen when Column 10 is compared to column 6, which shows the percentage of A1-type beta-caseins determined using a primitive enzymatic method utilising a tryptic digestion of samples with subsequent calibration against volumetrically-diluted relative milk standards.

[0071] FIG. 1 shows the proportion of A1-type beta-caseins in a series of relative milk standards analysed using the method of the invention, as well as intact HPLC analysis. The samples were created by volumetric dilution of milk from two individual animals, one verified to be of the A1/A1 genotype and thus producing milk containing pure A1-type beta-casein, and the other of the A2/A2 genotype thus producing milk containing pure A2-type beta-casein. Tight clustering of data points around the best fit line indicates the current method is precise, as indicated by the correlation coefficient (R.sup.2=0.9996). The slope of the line (0.9861) is interpreted to indicate the current method is also accurate.

[0072] FIG. 2 shows the amount of beta-casein in a series of milk samples and milk products as measured by the method of the invention, compared to the theoretical beta-casein level determined from the protein content of the samples. The good clustering of data points about the best fit line indicates the method is precise, also indicated by the correlation coefficient (R.sup.2=0.9075). The slope of the line (0.9075) also indicates the method is accurate.

[0073] 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.

EXAMPLES

Example 1: Sample Preparation

[0074] Liquid samples—For non-viscous liquid samples, e.g. milk, the liquid (40 μL) was transferred to a 2 mL centrifuge tube. Urea solution (120 μL, 500 g/L) was added to denature proteins, and the mixture then mixed at 2000 RPM for 5 min.

[0075] Solid samples—For solid samples or viscous liquid samples, the sample (5.00±0.01 g) was weighed into a falcon tube (50 ml). Ammonium bicarbonate solution (46.6 ml, 4 g/L) was added and the mixture shaken to wet all the powder. After incubation at 50° C. for 15 min, the mixture was shaken until homogeneous. In the case of infant formula, a sample (100 μL) was transferred to a 2 mL centrifuge tube, casein precipitant (150 g/L ammonium acetate pH 4.2, 1000 μL) was added and the mixture mixed at 2000 RPM for 5 min and then centrifuged at 5000 RCF for 5 min. After decanting the supernatant, casein precipitant (1000 μL) was added to the solid pellet and the mixture mixed at 2000 RPM for 5 min and then centrifuged at 5000 RCF for 5 min before decanting the supernatant. Water (1000 μL) was added to the solid pellet, the mixing and centrifuging steps repeated and the supernatant decanted. Urea solution (120 μL, 500 g/L) was added to denature proteins, and the mixture then mixed at 2000 for 5 min.

Example 2: Chymotrypsin Digestion

[0076] Chymotrypsin solution (1000 μL) was added to a sample prepared in accordance with Example 1. The mixture was incubated at 37° C. for 8 hours while shaking at 200 RPM. Quench solution (120 μL, 500 g/L formic acid solution) was added and the mixture mixed at 2000 RPM for 5 min and centrifuged at 10,000 RCF for 10 min. Sample (980 μL) was transferred to an LC vial, a solution of isotopically labelled peptide standards (20 μL) was added, and the mixture vortexed for 10 sec. The solution of labelled peptide standards (Biomatik, custom order, >95.0%) was prepared by weighing each of the peptides (2.00±0.10 mg) in Table 1 into a 2 mL polyethylene centrifuge tube. The peptides were dissolved by adding of 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, then 1.90 mL of solution was added. The labelled peptide standards of Table 2 correspond with the target peptides of Table 3.

TABLE-US-00004 TABLE 2 Isotopically labelled peptide standards [M + H].sup.+ Peptide Sequence Formula Ion (m/z) A1C-VN(10)-H (*V)YPFPGPIHN [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.51H.sub.77N.sub.11O.sub.13 1146.32 A1C-SN(12)-H SL(*V)YPFPGPIHN [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.60H.sub.93N.sub.14O.sub.16 1346.56 A1C-VQ(14)-H VYPFPGPIHNS(*L)PQ [.sup.13C.sub.6, .sup.15N.sub.1]-C.sub.69H.sub.108N.sub.17O.sub.19 1572.81 A1C-SQ(16)-H SL(*V)YPFPGPIHNSLPQ [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.79H.sub.124N.sub.19O.sub.22 1772.16 A2C-VN(10)-H (*V)YPFPGPIPN [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.50H.sub.77N.sub.10O.sub.13 1106.29 A2C-SN(12)-H SL(*V)YPFPGPIPN [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.59H.sub.93N.sub.12O.sub.16 1306.53 A2C-VQ(14)-H VYPFPGPIPNS(*L)PQ [.sup.13C.sub.6, .sup.15N.sub.1]-C.sub.68H.sub.108N.sub.15O.sub.19 1532.78 A2C-SQ(16)-H SL(*V)YPFPGPIPNSLPQ [.sup.13C.sub.5, .sup.15N.sub.1]-C.sub.78H.sub.124N.sub.17O.sub.22 1732.02

TABLE-US-00005 TABLE 3 Target peptides [M + H].sup.+ Peptide Sequence Formula Ion (m/z) A1C-VN(10) VYPFPGPIHN C.sub.56H.sub.77N.sub.13O.sub.13 1140.28 A1C-SN(12) SLVYPFPGPIHN C.sub.65H.sub.93N.sub.15O.sub.16 1340.65 A1C-VQ(14) VYPFPGPIHNSLPQ C.sub.75H.sub.108N.sub.18O.sub.19 1565.81 A1C-SQ(16) SLVYPFPGPIHNSLPQ C.sub.84H.sub.124N.sub.20O.sub.22 1766.16 A2C-VN(10) VYPFPGPIPN C.sub.55H.sub.77N.sub.11O.sub.13 1100.39 A2C-SN(12) SLVYPFPGPIPN C.sub.64H.sub.93N.sub.13O.sub.16 1300.62 A2C-VQ(14) VYPFPGPIPNSLPQ C.sub.74H.sub.108N.sub.16O.sub.19 1525.78 A2C-SQ(16) SLVYPFPGPIPNSLPQ C.sub.83H.sub.124N.sub.18O.sub.22 1726.13

Example 3: LC-MS/MS Analysis

[0077] Samples (1 μL) were injected onto a Waters Xselect HSS T3 C18 column (2.5 um×2.1 mm×50 mm) with attached Phenomenex KrudCatcher using a Thermo Vanquish LC system. Separation parameters are summarised in Table 4. Briefly, a gradient was run from 20% acetonitrile up to 40% acetonitrile to separate the target peptides, during which point the eluent was passed to a mass spectrometer for detection. The column was then washed at elevated flow prior to commencing the next injection. During this wash period the unwanted components of the sample were diverted to waste, and the acquisition turned off.

TABLE-US-00006 TABLE 4 Gradient and selected acquisition parameters Time % A % B Flow Divert MS Acquisition 0.0 80 20 0.5 1-6 Off 0.5 80 20 0.5 1-2 On 4.0 60 40 0.5 1-2 Off 4.5 0 100 0.8 1-6 Off 6.0 0 100 0.8 1-6 Off 6.5 80 20 0.8 1-6 Off 7.5 80 20 0.5 1-6 Off 8.0 80 20 0.5 1-6 Off

[0078] Mass spectral acquisition was performed using a Thermo TSQ Altis Mass Spectrometer, operating in position ion MRM mode. Each peptide has both quantifier and qualifier transitions, resulting in a total of 32 MRMs. These were all monitored continuously over the acquisition period, without the use of scheduling. The dwell time was set to 10 ms, the Q1 and Q3 resolutions to 2 Da, and the CID gas to 2 mTorr. A list of the transitions and associated parameters is shown in Table 5.

TABLE-US-00007 TABLE 5 MRM data for the sixteen target peptides monitored method Designation Transition(s) Usage CE (V) Rf (V) A1C-VN(10)  571.2 .fwdarw. 440.1 Quantifier 16 60  571.2 .fwdarw. 634.3 Qualifier 22 60 A1C-SN(12)  671.2 .fwdarw. 878.6 Quantifier 21 60  671.2 .fwdarw. 300.2 Qualifier 22 60 A1C-VQ(14)  783.9 .fwdarw. 244.4 Quantifier 27 70  783.9 .fwdarw. 652.5 Qualifier 22 70 A1C-SQ(16)  883.5 .fwdarw. 244.4 Quantifier 28 70  883.5 .fwdarw. 1523.1 Qualifier 25 70 A1C-VN(10)-H  574.2 .fwdarw. 440.1 Quantifier 16 60  574.2 .fwdarw. 634.3 Qualifier 22 60 A1C-SN(12)-H  674.3 .fwdarw. 878.6 Quantifier 21 60  674.3 .fwdarw. 306.2 Qualifier 22 60 A1C-VQ(14)-H  787.4 .fwdarw. 244.4 Quantifier 27 70  787.4 .fwdarw. 656.0 Qualifier 22 70 A1C-SQ(16)-H  886.8 .fwdarw. 244.4 Quantifier 28 70  886.8 .fwdarw. 1523.1 Qualifier 25 70 A2C-VN(10) 1100.6 .fwdarw. 594.3 Quantifier 39 60 1100.6 .fwdarw. 838.4 Qualifier 37 60 A2C-SN(12) 1300.7 .fwdarw. 594.4 Quantifier 42 60 1300.7 .fwdarw. 838.4 Qualifier 42 60 A2C-VQ(14)  763.4 .fwdarw. 244.2 Quantifier 20 70  763.4 .fwdarw. 655.4 Qualifier 19 70 A2C-SQ(16)  863.5 .fwdarw. 244.2 Quantifier 24 70  863.5 .fwdarw. 655.3 Qualifier 20 70 A2C-VN(10)-H 1107.0 .fwdarw. 594.3 Quantifier 39 60 1107.0 .fwdarw. 838.4 Qualifier 37 60 A2C-SN(12)-H 1307.1 .fwdarw. 594.4 Quantifier 42 60 1307.1 .fwdarw. 838.4 Qualifier 42 60 A2C-VQ(14)-H  767.4 .fwdarw. 244.2 Quantifier 20 70  767.4 .fwdarw. 662.4 Qualifier 19 70 A2C-SQ(16)-H  867.0 .fwdarw. 244.2 Quantifier 24 70  867.0 .fwdarw. 655.3 Qualifier 20 70

Example 4: Concentration Determinations for A1-Type Beta-Casein Variants and A2-Type Beta-Casein Variants

[0079] Calculations were performed automatically using a pre-designed digital spreadsheet, though they can be done manually. The concentration of A1-beta-casein variants was calculated by summing the products of the concentration of each peptide by the quotient of the parent protein mass and the peptide mass, then multiplying by the preparation factor:

[00001]$C_{A1-\beta CN}=\left(\left(C_{A1C-VN\left(10\right)}\times \frac{M{w}_{A1-\beta CN}}{M{w}_{A1C-VN\left(10\right)}}\right)+\left(C_{A1C-SN\left(12\right)}\times \frac{M{w}_{A1-\beta CN}}{M{w}_{A1C-SN\left(12\right)}}\right)+\left(C_{A1C-VQ\left(14\right)}\times \frac{M{w}_{A1-\beta CN}}{M{w}_{A1C-VQ\left(14\right)}}\right)+\left(C_{A1C-SQ\left(16\right)}\times \frac{M{w}_{A1-\beta CN}}{M{w}_{A1C-SQ\left(16\right)}}\right)\right)\times P$

[0080] The same calculation was used for calculating the concentration of A2-beta-casein variants, but with masses adjusted accordingly:

[00002]$C_{A2-\beta CN}=\left(\left(C_{A2C-VN\left(10\right)}\times \frac{M{w}_{A2-\beta CN}}{M{w}_{A2C-VN\left(10\right)}}\right)+\left(C_{A2C-SN\left(12\right)}\times \frac{M{w}_{A2-\beta CN}}{M{w}_{A2C-SN\left(12\right)}}\right)+\left(C_{A2C-VQ\left(14\right)}\times \frac{M{w}_{A2-\beta CN}}{M{w}_{A2C-VQ\left(14\right)}}\right)+\left(C_{A2C-SQ\left(16\right)}\times \frac{M{w}_{A2-\beta CN}}{M{w}_{A2C-SQ\left(16\right)}}\right)\right)\times P$

[0081] The preparation factor, P, was calculated as follows:

[00003]$P=\frac{v_1}{m_1}\times \frac{v_2}{v_3}\times \frac{v_4}{v_5}$

[0082] where v.sub.1 is the final volume of the product after extraction, m.sub.1 is the mass of product weighed, v.sub.2 is the volume in the digestion tube after quenching, V.sub.3 is the volume of extract added to the digestion tube, V.sub.4 is the final volume in the LC vial, and V.sub.5 is the volume of digest transferred to the LC vial.

[0083] The total beta-casein concentration was calculated by summing the concentrations of the two polymorphs:

C.sub.Total βCN=C.sub.A1βCN+C.sub.A2βCN

[0084] The percentage of A1-beta-casein variants in the product was calculated by dividing the A1-beta-casein variants concentration by the sum of the A1- and A2-beta-casein variants concentrations:

[00004]$\%A1=\frac{C_{A1\beta CN}}{C_{A1\beta CN}+c_{A2\beta CN}}\times 100\%$

Example 5: Milk and Milk Product Analysis

[0085] Various fresh milk, milk powder, infant formula and UHT milk samples were tested according to the method outlined in Examples 1-4. A series of milk standards were also prepared and tested. The results are shown in Table 4, wherein results determined according to the method of the invention are compared to other methods for the determination of beta-casein content.

TABLE-US-00008 TABLE 6 Concentration data for beta-casein variants in milk and milk products Kjeldahl Labelled Calculated Intact AQ Total Protein Protein bCN UV Tryptic AQ A1 AQ A2 bCN AQ Sample (g/100 g) (g/100 g) (g/100 g) % A1 % A1 (g/100 g) (g/100 g) (g/100 g) % A1 Milk (non-homogenised, A1/A2) 3.62 3.80 1.22 39.79 18.26 0.34 0.84 1.18 28.77 Milk (full fat, A1/A2) 3.17 3.30 1.06 40.93 18.10 0.03 0.07 1.09 28.59 Milk (reduced fat, A1/A2) 3.09 3.30 1.06 42.04 19.28 0.35 0.80 1.15 30.26 Milk (fat-free, A1/A2) 3.57 4.00 1.28 38.53 18.54 0.34 0.85 1.19 28.52 Milk Powder (full fat, A2) 21.3 23.80 7.62 0.00 0.00 0.01 8.11 8.12 0.13 Milk Powder(reduced fat, A2) 29.9 33.60 10.75 0.00 0.00 0.02 10.89 10.90 0.15 Milk Powder (full fat, A1/A2) 21.8 24.00 7.68 35.83 16.03 2.29 6.09 8.39 27.35 Milk Powder (reduced fat, A1/A2) 29.3 32.80 10.50 34.77 16.78 3.10 7.70 10.81 28.71 Infant Formula (Stage 1, A2) 9.41 10.15 1.51 28.24 0.00 0.18 2.15 2.33 7.64 Infant Formula (Stage 2, A2) 13.3 15.25 2.82 32.44 0.00 0.14 4.52 4.66 2.94 Infant Formula (Stage 3, A2) 12.8 13.91 2.60 38.32 0.67 0.18 3.89 4.07 4.52 Infant Formula (Stage 1, A1/A2) 9.6 10.55 1.56 39.56 14.02 0.72 1.85 2.57 28.19 Infant Formula (Stage 2, A1/A2) 13.3 15.03 2.80 32.79 14.67 1.08 2.67 3.74 28.71 Infant Formula (Stage 3, A1/A2) 15.7 16.10 2.97 43.00 23.37 2.19 4.25 6.44 34.02 UHT Milk (reduced fat, A1/A2) 4.82 3.90 1.25 42.96 18.62 0.44 1.12 1.57 28.38 UHT Milk (full fat, A1/A2) 3.73 4.00 1.28 39.20 18.14 0.39 0.91 1.30 30.12 Relative milk standard (0.0% A1) — — — 0.14 0.00 0.00 1.51 1.52 0.16 Relative milk standard (0.5% A1) — — — 0.44 0.43 0.01 1.54 1.55 0.48 Relative milk standard (1.0% A1) — — — 0.96 — 0.01 1.45 1.47 0.92 Relative milk standard (2.0% A1) — — — 1.73 2.55 0.03 1.45 1.48 1.91 Relative milk standard (5.0% A1) — — — 4.60 4.41 0.07 1.45 1.52 4.87 Relative milk standard (10.0% A1) — — — 9.68 9.38 0.15 1.41 1.56 9.80 Relative milk standard (20.0% A1) — — — 20.93 20.93 0.30 1.22 1.52 20.00 Relative milk standard (40.0% A1) — — — 40.01 37.86 0.57 0.94 1.51 37.99 Relative milk standard (60.0% A1) — — — 59.88 64.97 0.81 0.59 1.39 58.02 Relative milk standard (80.0% A1) — — — — — 1.08 0.29 1.37 78.76 Relative milk standard (100% A1) — — — 98.58 — 1.41 0.00 1.41 99.93

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