HU231308B1

HU231308B1 - Method for separating major protein fractions in milk by hplc

Info

Publication number: HU231308B1
Application number: HUP2100384A
Authority: HU
Inventors: Henrietta Buzás; Szafner Gábor Dr.
Original assignee: Magyar Tejgazdasági Kísérleti Intézet Kft.
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2022-11-28
Also published as: HUP2100384A1

Description

Method for separating the main protein fractions in milk by HPLC

FIELD OF THE INVENTION

The subject of the invention is an improved method for the fractionation of milk proteins, which enables the simultaneous qualitative and quantitative separation of the main protein fractions in milk, in particular the separation of the A1 and A2 genetic variants of the beta-casein fraction.

POSITION OF TECHNIQUE

The dairy industry is a significant source of income for many people around the world, and dairy products are a significant source of food in the form of milk, cheese, yogurt and other products. Milk, as an animal product that can be produced efficiently and in large quantities, plays a significant role in the protein supply of mankind. Milk contains proteins, essential amino acids, and fats that are essential for humans, to such an extent that 0.3 liters of milk can cover 20-60% of a person's daily need for these nutrients. Cows, goats, sheep and mares that produce milk produce almost indispensable food for humans from forages that are unfit for human consumption, and with the exception of ruminants and horses, it would be difficult for other farm animals to utilize them. At least part of the success of the dairy industry is due to the increased efficiency of milk production by various breeds of cattle, as well as the improved efficiency of milk processing capabilities.

Foods of animal origin are absolutely necessary to satisfy human nutritional needs, because plant foods usually contain little or not the right proportion of essential amino acids, fatty acids, macro- and micro-elements for humans. The constant composition of milk, which is balanced in terms of the nutritional supply of the human body, meets the requirements to supplement plant-based foods in an appropriate way.

Breast milk occupies a special place among foods, because it is almost the only food for a newborn in the first period after birth. Breast milk contains all the important nutrients, especially protein and minerals, that a newborn baby needs for growth and development. If it is not possible to feed the baby with breast milk, it is possible to feed it with different - typically cow's milk-based - formulas. Although breast milk (or cow's milk-based formula) is no longer the child's exclusive food after infancy, cow's milk or dairy products entering the diet will continue to play an important role in supplying the young body with nutrients and meeting its nutritional needs. Milk and milk products also play an important role in the nutrition of adults, not only because they contain all important nutrients, but also because they are rich in the components that the adult body may need. Without milk and milk products, it is almost impossible to ensure adequate nutrition for the body. It is worth mentioning that, when combined in the right proportion, different foods can complement each other and provide a balanced, ideal nutritional composition. From this point of view, milk is also important, because it can properly complement the vegetables.

The composition of milk and the ratio of its components depend on many factors, and of course it can be fundamentally different depending on the animal species and breed.

The proportion of proteins in cow's milk is approx. 3.1-3.4%. In general, the protein content is always 0.3-0.5% less than the fat content. Two large groups of proteins can be distinguished in milk, which can be further divided into components: (i) casein proteins (aS1-, aS2-, β-, κ-casein): phosphorus-containing proteins, the total protein content is approx. 80%, they are characterized by the fact that they denature under the influence of acid (at pH 4.6) and quencher, thereby causing the coagulation of milk, which is the basis of the technology for the production of sour preparations and partly curds and cheeses, as well as (ii) the whey proteins, most of which do not denature under the influence of acid and quencher, but are sensitive to heat, which heat denaturation approx. It starts at 60 °C and becomes more significant above 90 °C, its main components are: - blood serum albumin, - lactalbumin (precipitates easily due to heat), α-, β-lactoglobulin, - immunoglobulins, and (iii) other proteins, membrane and coat proteins, which account for only 1% of all proteins, are globular, do not precipitate under quenching, but precipitate at 100 °C in the presence of Ca ions and under the action of acid (below pH 4.6) and are also important as secondary emulsifiers.

Milk protein consists of different fractions, of which casein makes up 80% of milk protein, while whey proteins make up 20% of milk protein. Casein can be divided into four fractions: aS1-, aS2-, β- and κ-casein. The individual casein fractions differ significantly in their phosphorus content. α and β-casein contain 1.0 and 0.6% phosphorus, while κ- and γ-casein contain 0.2 and 0.1% phosphorus. The phosphorus content plays a significant role in the stability of the micelle. The main whey protein fractions of milk are serum albumin, β-lactoglobulin, α-lactalbumin and globulins. The proteose-peptone fraction, which contains 11% carbohydrates and, unlike the other whey protein fractions, does not precipitate when heated to 100 °C after being acidified to pH=4.7, is also considered part of the whey protein. In addition to the described protein fractions, there are many variants and genotypes that differ from each other in their amino acid composition and certain properties, so the number of milk protein components can be put at more than 50. Some protein variants are considered genetic variants because they occur in different amounts in different populations, and there are genetic variants that do not occur at all in some populations. Some of these variants are assumed to be genetically related to another milk component or milk property, such as e.g. heat stability, grafting ability or resistance to mastitis.

Table 1. Protein fractions of milk

Protein fraction Variants % in milk protein % in casein or in whey protein Average (%) Outliers (%) Average (%) Outliers (%) Casein fractions αs-casein A B C D 43.5 35-63 54.2 48-60 κ-casein A, B 10.7 8-15 13.3 7-21 β-casein A1, A2, A3 B, Bz, (B12) 24.2 19-35 30.1 26-40 C, D, E Whey protein fractions Serum albumin 0.9 0.5-1.3 4.6 2-8 β-lactoglobulin A, B, C, D, Dr 9.6 7-14 48.7 44-59 α-lactalbumin A, B, C 3.7 2-5 18.8 17-22 Globulins IgG, IgM, IgA 2.2 1-4 11.2 8-17 Proteose peptone 4 factions 3.3 2-6 16.8 10-19

Table 1 shows the protein fractions of milk, their average concentration and extreme values. The data are averages of a large number of literature data. Variants of the main protein fractions can also be found in the table.

Several methods are known in the state of the art for determining individual protein fractions of raw milk. For example, US Patent No. US 6096870A (Sepragen Corporation, 2000-08-01) describes a process for successive chromatographic separation of whey proteins. The proteins are bound on a cation exchange column, washing out the components that do not contain lactose, minerals, lactic acid and nitrogen. After that, immunoglobulin and beta-lactoglobulin are eluted, and alpha-lactalbumin, serum albumin, lactoferrin, and lactoperoxidase are separated from this in separate fractions. The patent description describes an additional complicated two-column process for the separation of the above proteins. The patent description only describes the separation of whey proteins, so there is no mention of additional proteins found in milk and their separation, so there is no mention of the high-resolution separation of casein either. The separation procedure consists of several successive separation steps, so it cannot be said to be fast either.

The Spanish patent description No. ES 2144356 B1 (Universidad de Alcalá, 2000-06-01) describes a process for the rapid HPLC separation of cow's milk whey proteins. The separation procedure uses a three-step linear and binary gradient with an eluent containing a mixture of acetonitrile-water-trifluoroacetic acid. The flow rate is 3 ml/min. During the applied gradient, B% increases from 5 to 25% in 1.7 minutes, B% increases from 25 to 34% in 0.3 minutes, B% increases from 34 to 41% in 3 minutes increases to , and finally in 1 minute the B% decreases to 5% and another 1 minute is required to equalize. In total, separation takes 7 minutes. The temperature of the column is 60°C, the detection takes place at a wavelength of 254 nm. The procedure enables the rapid detection of cow's milk whey proteins from dairy products. However, the patent description only describes the separation of whey proteins, so there is no mention of additional proteins found in milk and their separation, so the high-resolution separation of casein is not described either.

Chinese patent document CN 105044222 A (UNIV SHANGHAI JIAOTONG, 2015-11-11) describes a method for identifying bioactive polypeptides in fermented milk. The identification of bioactive polypeptides is based on the mapping of genetic information and the procedural steps include complex tests other than the HPLC separation procedure.

The Bobe et al. Separation and Quantification of Bovine Milk Proteins by ReversePhase High-Performance Liquid Chromatography, J. Agric. The publication Food Chem. 1998, 46, 458-463 describes an analytical procedure for the separation and quantification of milk proteins for the six most important cow's milk proteins and their genetic variants, where the HPLC separation procedure can be performed in 2 hours by omitting the filtration step. During sample preparation, the milk sample to be tested is diluted 1:1 by volume with sample buffer A, the sample is incubated at room temperature for 1 hour, and then centrifuged at 16,000 g for 5 minutes. After removing the fat layer, the sample is diluted 1:3 with the sample buffer and then injected onto the column without filtration. Composition of sample buffer A: 0.1 M BisTRis buffer, 6 M Guanidine hydrochloride, 5.37 mM sodium citrate, 19.5 mM dithiothreitol. Composition of sample buffer AB: 4.5 M guanidine hydrochloride A dissolved in eluent A. The composition of A eulens: 100:900:1 volume ratio acetonitrile:water:trifluoroacetic acid. AB eluent composition: 900:100:1 volume ratio acetonitrile:water:trifluoroacetic acid. For HPLC separation, a C-18 column operates at room temperature, the sample flow rate is 1.2 ml/min, and the detection wavelength is 220 nm. The separation procedure is carried out by gradient elution, which starts at 27 B% and then rises to 32 B% in 2 minutes. After that, there is a slow linear increase in the proportion of eluent B, rising to 48.4 B% in 34.9 min, then to 50.2 B% in 4 min, after which it returns to the initial value in 2.1 min and for 9 min takes to re-equilibrate the column, which ultimately results in a run time of 52 minutes. Based on the information presented in the publication, some genetic variants of milk proteins cannot be completely separated, so for example the A1 and A2 variants of beta-casein elute from the column under one peak at the same time, so the separation of these two important genetic variants is not possible with this method. The time for the separation of proteins is not short enough to meet the demand that a bulk milk sample can be tested for milk protein within a day.

Ma et al. The publication rapid analytical method of major milk proteins by reversed-phase highperformance liquid chromatography, Animal Science Journal (2017) describes a rapid and automated test procedure for the HPLC separation of the most important cow's milk proteins. The duration of the procedure is 30 minutes. The sample preparation solutions and eluents are the same as in the previous document, while the eluent gradient is different: B% increases from 10 to 50% in 10 minutes, then B% increases from 50% to 80% in 20 minutes. The detection wavelength is 214 nm and the temperature of the thermostat is 40°C. Although the procedure results in the rapid and automated separation of the most important milk proteins, its resolution falls short of the solution according to the previous document, and it is also clear from the chromatogram that it does not allow the separation of the individual genetic variants, especially the A1 and A2 variants of beta-casein.

The Yüksel et al. Detection of the milk proteins by RP-HPLC The publication GIDA / The Journal of FOOD 2010, Vol. 35, Issue 1, p5-11 describes the separation of the most important milk proteins using RP-HPLC. The sample preparation solutions and eluents are the same as Bobe et al. according to the publication, the total run time is 30 minutes, the column temperature is 25°C, the flow rate is 1.0 ml/min, and the detection wavelength is 220 nm. The eluent gradient starts at 20% B%, which rises to 46% B% in 27.8 minutes and then returns to the starting value in 2.4 minutes. In addition to the fact that some of the separation parameters differ, the final value of B% rises to 46%. However, it is not clearly stated in the description whether this value is kept at this value from the first moment, or whether this value is reached by a continuous increase until the end of the elution. The disadvantage of the described method is that the genetic variants of the milk proteins, especially the A1 and A2 variants of beta-casein, are not separated using this method.

The Vincent et al. Quantitation and identification of intact major milk proteins for High-Throughput LC-ESI-Q-TOF MS Analyzes PLOS ONE, 17.10.2016. The publication DOI:10.1371/journal.pone.0163471 describes the development of an HPLC and MS separation method for the separation of the most important milk proteins and their genetic variants. During the examination of the method development, a number of parameters described in the state of the art are examined in connection with the separation, where in the HPLC separation step, Bobe et al. the solutions and parameters described in the document are used as a basis and the effect of different gradient elution ratios on the resolution is also investigated. As a result of the method development, it can be said that the RP-HPLC separation alone was not sufficient for the perfect separation and quantification of the genetic variants of milk proteins, especially the A1 and A2 variants of beta casein. This could only be achieved by MS analysis of the separated samples, which significantly increases the costs of the test and the test time.

The Rotkaja et al. Reversed-Phase High-Performance Liquid Chromatography Analysis of beta-Lactoglobulin and alpha Lactalbumin in Different Types of milk Material Science and Applied Chemistry, 08.10.2016, doi: 10.1515/msac-2016-0007, 2016/33, 36-39 describes an HPLC procedure for the separation of alpha-lactalbumin and beta-lactoglobulin whey proteins. The test does not cover the rapid separation of all milk protein fractions and their variants.

Nowadays, many researches are devoted to the investigation of the effect of individual milk protein fractions and their variants, primarily the A1 and A2 variants of beta-casein, on the physiological properties.

Type A1 beta-casein is associated - both in human and mouse experiments - with intestinal inflammation, irritable bowel syndrome (IBS), the development of type 1 diabetes (Robert Elliott 1993 New Zealand and Bryndis Eva Birgisdottir 2006 Iceland), increased digestion time , with elevated inflammation markers in serum. In China, in 2014, a research/clinical trial was conducted on 104 applicants, of which 23 had lactose intolerance and 45 admittedly had cow's milk intolerance. The two groups took part in a double blind experiment, where the applicants in one group received only milk containing A2 beta casein, while in the second group the applicants received milk containing a mixture of A1 and A2 beta casein (60-40 %). The researchers' hypothesis was that the consumption of milk containing A1 beta-casein causes digestive system complaints and results in a higher concentration of inflammatory markers in the blood serum. The assumption was confirmed, because the research proved that in the case of milk containing both A1 beta-casein and A2 beta-casein (60-40%), digestive tract complaints worsened, the absorption time increased, the concentrations of inflammatory markers in the blood increased, decreased the total short-chain fatty acid content of the feces, while no negative effects could be detected in the case of milk containing only A2 beta-casein, which proved that in the case of milk containing both types of casein, only A1 beta-casein could cause the increasing negative effects.

In the case of lactose-intolerant patients, it was shown that milk containing both beta-caseins aggravated the patients' digestive system complaints and gastrointestinal transit time to a greater extent than in the case of lactose-tolerant patients, whereas milk containing only A2 beta-casein it did not increase complaints in the lactose intolerant group. From this, it can be concluded that, in the case of lactose intolerant patients, the intensification of complaints was probably caused by A1 beta-casein.

International PCT Publication No. WO 2015/187795 A1 (Abbott Laboratories, 2015-12-10) describes a method and compositions for improving the metabolic health of consumers. It is hypothesized that the beta-casein A1 genetic variant may be associated with type 1 diabetes. The object of the invention is to provide preparations which contain the genetic variant of cow's milk beta-casein A2 together with lipophilic substances. The patent description does not describe the rapid and high-resolution fractionation of milk proteins, on the basis of which the proteins and their genetic variants could be separated.

US 2003/0017250 A1 (Elliott et al., 2003-01-23) describes a process for the selection and processing of non-diabetogenic milk products and the breeding of non-diabetogenic milk-producing cows. Diabetogenic protein variants are separated by SDS PAGE gel electrophoresis, which is long and complicated.

In addition to the publications listed above, however, several publications are known in the literature in which the researchers made opposite findings. Truswell's 2005 review article found no association between the A1 and A2 variants of beta-casein and diabetes, cardiovascular or other digestive diseases.

Daniloski et al. (2021) also did not find the correlation between the A1 and A2 variants of beta-casein and different diabetogenic propensities and other digestive disorders to be clearly established.

It is also clear from the presented literature that the variants of the individual protein fractions in this case are beta-casein A1 and A2 - currently a topic of great interest to researchers, however, the current position of science is not uniform, yet there is a need for a separation method where the two variants separation takes place in a well-defined manner.

The state-of-the-art documents and their content are considered part of the description, especially regarding the description of the definitions used.

Based on the above, a number of patent documents describe a process used to separate the protein fractions of milk. Some of these are HPLC liquid chromatography processes, where one of the goals of the further development of the processes is fast analytical separation, while the other goal, in addition to fast run time, is analytical testing with the highest possible resolution and the most accurate separation of the protein fraction.

From the exploration of the state of the art, it is obvious to a specialist that there is still a great demand for separation methods that enable the most appropriate separation of the protein fraction of milk, as well as their more accurate quantitative analysis.

The purpose of the invention is to eliminate the errors of the previous solutions and to provide a process that is capable of separating the protein fractions of milk faster than before, but with a higher resolution. With the solution according to the invention, it was possible to develop a separation process capable of qualitatively and quantitatively separating the main protein fractions of milk in a short time in such a way that it also provides qualitative and quantitative data for the protein fractions, especially β-casein A1 and A2 variants.

GENERAL DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

There is therefore still a need for a process that provides a fast and very accurate separation method for the qualitative and quantitative separation of the main protein fractions of milk. State-of-the-art solutions do not provide methods suitable for high-resolution rapid separation in which the main protein fractions of milk, especially the A1 and A2 variants of β-casein, can be qualitatively and quantitatively separated.

SOLVING THE PROBLEM

In the course of our research, we found that the protein fractions of milk can be qualitatively and quantitatively separated with good resolution within a short time, under the system conditions determined by further development of the previously known methods, by using a specific elution gradient. As a result, the composition of the protein fractions of any milk sample can be determined quickly, for example, within a period of 20 minutes to 1 hour. In addition to the separation of the main protein fractions of milk, the genetic variants A1 and A2 of β-casein can also be well separated with the method according to the invention.

Our inventive realization is, therefore, that we have developed a high-efficiency HPLC method using gradient elution, which is able to eliminate the problems described above.

BRIEF DESCRIPTION OF THE INVENTION

The subject of the invention is a method for the fractionation of milk proteins, which enables the simultaneous qualitative and quantitative determination of the main protein fractions in milk, as well as the separation of the A1 and A2 genetic variants of the beta-casein fraction, which method includes the following steps:

a) prepare the sample,

b) inject the prepared sample onto the column without filtering,

c) gradient elution is performed using eluents A and B,

d) the main protein fractions are separated based on the peaks appearing in the chromatogram, characterized by the fact that in step c) the temperature of the column is thermostated at 35-45°C, preferably 40°C, the flow rate is 1.0-1.2 ml /min, preferably 1.1 ml/min, and the gradient elution is as follows:

- 70.9% eluent A and 29.1% eluent B for 0.5 minutes, then

- in 20 minutes, the amount of eluent A decreases evenly to 54.0%, while the amount of eluent B increases to 46.0%, and

- In 0.1 minutes, the amount of eluent A increases to 70.9% and the amount of eluent B decreases to 29.1%.

The subject of the invention is also the use of the method for the identification of genetic variants A1 and A2 of beta-casein.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The protein fractions in milk and their variants are summarized in Table 1. The main protein fractions of milk are proteins whose average amount is greater than 3% of the milk proteins. These include, but are not limited to, αs1- and αs2-casein, βcasein, preferably its A1 and A2 genetic variants, κ-casein, β-lactoglobulin A and B and αlactalbumin.

The terms HPLC or high-performance liquid chromatography is a chromatographic procedure often used in biochemistry and analytical chemistry for the separation, identification, and quantification of compounds. The HPLC system consists of the following components: the column containing the chromatographic charge (stationary phase), the pump pushing the mobile phase (mobile phase, eluent) through the column, and the detector indicating the retention time of the molecules. The retention time depends on the interactions between the stationary phase, the tested molecule and the mobile phase. The sample to be tested is dissolved and a small volume of the solution is introduced (injected) into the flowing mobile phase. As the sample travels through the column, it lags behind the liquid stream (retarded) through specific chemical or physical interactions with the stationary phase. The degree of retention depends on the properties of the material to be tested and the stationary phase, as well as the composition of the mobile phase. The period during which a given substance elutes (appears at the end of the column) is called the retention time. In a given system, the retention time is a relatively unique characteristic of each tested compound. By applying a higher pressure, the linear flow rate can be increased, so that the components have less time to diffuse within the column, which improves the separation between the individual materials. The mobile phase is usually water and various organic liquids, most often methanol or acetonitrile is used. Better separation of the components of the sample can be helped by buffers or salts added to the water, or other substances, such as trifluoroacetic acid used as an ion pair generator.

Another possibility for the HPLC technique is to change the composition of the mobile phase during measurement, which is called gradient elution.

In the case of reversed phase HPLC (RP-HPLC or RPC), the surface of the stationary phase is apolar, while the mobile phase is polar, typically a mixture of water and an organic solvent. A frequently used stationary phase is silica treated with RMe2SiCl, where R is some straight-chain alkyl group, e.g. C18H37 or C8H17. Using such stationary phases, the retention time of more apolar molecules is longer, while polar molecules elute faster. Adding an apolar solvent to the mobile phase can reduce it, while adding a more hydrophilic solvent can increase the retention time. RP-HPLC works on the principle of hydrophobic interactions, which are the result of repulsive forces between the polar mobile phase, the relatively apolar substance to be tested and the apolar stationary phase. The binding of the substance to be tested to the stationary phase is proportional to the area of the contact surface formed in the interaction with the ligand in the aqueous mobile phase around the apolar segment of the tested molecule. Retention can be reduced if the surface tension of water is reduced by adding a less polar solvent (methanol, acetonitrile) to the mobile phase.

The separation can be influenced by the type and temperature of the column used for the separation in the HPLC system used, the composition of the mobile phase, gradient, flow rate and the detection wavelength.

During sample preparation, the milk sample to be tested is diluted with sample buffer A at a ratio of 1:0.5-1:2 (v/v), preferably at a ratio of 1:1, incubated at room temperature for approximately 1 hour, then at 12,000-16,000g, preferably at 14,000g centrifuge for a short time, preferably for 2-10 minutes, more preferably for 5 minutes, and remove the fat layer. As part of sample preparation, the prepared sample is diluted with sample buffer B in a ratio of 1:3-4, preferably 1:3 (v/v). The

Sample buffer contains 0.1 M BisTris buffer, 6 M Guanidine hydrochloride, 5.37 mM sodium citrate and 19.5 mM dithiothreitol in high purity water, while sample buffer B contains 4.5 M Guanidine hydrochloride dissolved in eluent A.

The structure of the stationary phase of the column used is preferably C18, but it can also have a C8 structure. The column used is packed with large-pore, 3.6 μm average diameter, shell-structured particles, such as Phenomenex Aeris WIDEPORE XB-C18 and C4 liquid chromatography cartridges, which are also commercially available.

The temperature of the column used during the separation is 24-50°C, preferably around 40°C.

The following mobile phases were used during the gradient elution: eluent A contains acetonitrile:water:trifluoroacetic acid in a volume ratio of 100:900:1, eluent B contains acetonitrile:water:trifluoroacetic acid in a volume ratio of 900:100:1.

The flow rate of the mobile phase can be between 1.0 and 1.2, preferably 1.1 ml/min.

The detection wavelength is 214 nm.

The gradient of the mobile phase is preferably the following:

- 70.9% eluent A and 29.1% eluent B for 0.5 minutes, then

The values given in the patent description may differ by ±5%, preferably ±1%, which values are also considered to be within the scope of patent protection.

BRIEF DESCRIPTION OF THE FIGURES

Hereinafter, exemplary preferred embodiments of the invention are described with drawings, where

Figure 1 shows the RP-HPLC chromatogram of the main protein fraction standards of cow's milk.

Figure 2 shows the RP-HPLC chromatogram of Sample 14_ENAR 8104 cow's milk containing the A1 beta-casein variant.

Figure 3 shows the RP-HPLC chromatogram of Sample 2_ENAR 8111 cow's milk containing A1 and A2 beta-casein variants.

Figure 4 shows the RP-HPLC chromatogram of Sample 6_ENAR 7869 cow's milk containing A2 beta-casein variant.

EXECUTION EXAMPLES

EXAMPLE 1

Materials

Acetonitrile (Molar Chemicals Kft.) was of HPLC grade, and the water used was deionized and distilled. All other chemicals were of analytical grade. For separation, BisTris buffer (VWR Life Science, lot 0715), DL-dithiothreitol >99% (DTT, G-5545, Sigma-Aldrich), Guanidine hydrochloride >99% (GdnHCl, G4505, Sigma-Aldrich), sodium citrate >99% (S4641) trifluoroacetic acid >99.0% (302031, Sigma-Aldrich) was used.

Purified protein fraction standards were as follows (purity in parentheses): κcasein (>70%,1), as1-casein and as2-casein (>70%, 3 and 2), β-casein (>98%, 4.5 ), α-lactalbumin (>85%, 6), β-lactoglobulin A (>90%, 7) β-lactoglobulin B (>90%, 8) (all Sigma-Aldrich).

Sample preparation

During sample preparation, the room temperature milk sample to be tested was diluted 1:1 (v/v) with sample buffer A, incubated at room temperature for 1 hour, then centrifuged at 14,000g for 5 minutes and the fat layer was removed with a spatula. The thus prepared and degreased sample was diluted 1:3-4 (v/v) with sample buffer B, then injected without filtration onto a C-18 RP-HPLC column Aeris Widepore XB-C18, 250 mm x 4.6 mm column. Sample buffer A contained 0.1 M BisTris buffer, 6 M Guanidine hydrochloride, 5.37 mM sodium citrate, and 19.5 mM dithiothreitol dissolved in water, while sample buffer B contained 4.5 M Guanidine hydrochloride dissolved in eluent A. Eluent A contained acetonitrile:water:trifluoroacetic acid in a volume ratio of 100:900:1, and eluent B contained acetonitrile:water:trifluoroacetic acid in a volume ratio of 900:100:1. A gradient elution was performed using eluents A and B, where the temperature of the column was thermostated at 40°C, the flow rate was 1.1 ml/min, and the detection wavelength was 214 nm.

Gradient elution was performed as follows:

- 70.9% eluent A and 29.1% eluent B for 0.5 minutes, then

- in 20 minutes, the amount of eluent A was uniformly reduced to 54.0%, while the amount of eluent B was increased to 46.0%, and

- In 0.1 minutes, the amount of eluent A was increased to 70.9% and the amount of eluent B was reduced to 29.1%. The individual protein fractions and genetic variants were separated based on the peaks appearing on the chromatogram. The chromatogram of the sample containing the standards is shown in Figure 1.

Components of RS HPLC system

The HPLC system consisted of two HPLC pumps (Flexar FX-15 UHPLC Pump), an automatic sample feeder (Flexar FX Peltier UHPLC Autosampler), an absorbance detector (Flexar FX-PDA UHPLC Detector) and a processing and presentation unit. The equipment was operated through a software (Chromera Software) which controlled the solvent gradient, data collection and data processing. A C-18 RP-HPLC column Aeris Widepore XB-C18, 250 mm x 4.6 mm column was used to separate the proteins. The solutions were filtered through a filter.

Eluent gradient

The following gradient was used during the separation procedure:

- 70.9% eluent A and 29.1% eluent B for 0.5 minutes, then

Chromatogram of cow's milk protein fractions checked with standards (Figure 1)

In the case of a raw cow's milk sample, during the validation of the method, we proved that the performance characteristics of the method satisfy the requirements of the analytical method. During the validation of the method, the method according to the invention was carried out in accordance with the validation protocols. In our case, we therefore examined the linearity, repeatability and reproducibility of the method according to the invention.

The milk protein fractions were identified based on commercially available casein and whey protein standards (as-casein, β-casein, κ-casein, β-lactoglobulin A and β-lactoglobulin B). The retention time of the chromatographic peaks obtained during the examination of the sample solutions containing individual casein and whey protein fractions was compared with the retention time of the peaks obtained by the analysis of the standard protein solutions. The β-casein A1 and A2 fractions, in the absence of a standard, were identified by the retention times obtained by examining milk samples with genetically confirmed beta-casein status.

The quantitative information is obtained from the area under the peak given by the time integral of the peaks (elution curves). The calibration curves we recorded for the different protein fractions contain the concentration dependence of the area under the peak of 6 standard solutions with known concentrations. Based on the fitted curves, the concentration of the protein fraction in the sample can be determined. The correlation coefficient of the calibration curve of each protein fraction is always r>0.99.

Linearity

Different volumes (5, 10, 20, 40 and 80 μl) of the same cow's milk sample were injected onto the column, with three repetitions. Based on the measurements, the measurement curves taken for each fraction were considered to be straight in the examined range. The correlation coefficient of the measuring curve of the fractions is r>0.99.

Repeatability

We tested the same cow's milk sample with the same injection volume (20 μl) with ten parallel measurements, and then calculated the retention times and the RSD% below the peak for the individual fractions.

Reproducibility

With the same injection volume (20 μl), 10 individual samples of cow's milk were examined with three parallel measurements over four days.

The RSD% values of the retention times and the areas under the peak were determined for each fraction. Based on the obtained results, we found that the method according to the invention is a reliable method for the separation and quantitative determination of the main fractions and genetic variants of milk protein, especially β-casein A1 and A2 variants.

Examination of beta casein variants in individual milk samples.

We examined milk samples for the presence of beta-casein A1 and A2 genetic variants. For each milk sample, the customer specified that the given milk sample is

Which of the A1 and A2 variants contains beta-casein protein. Using the method according to the invention, we checked which of the A1 and A2 variants of beta casein protein the milk samples contained (Figures 2-4). The results are summarized in Table 2.

As a result of the measurements, we verified whether the examined milk samples contain the A1 and/or A2 beta-casein variants. We proved that 1-4. samples only the A1 variant, the 5-10. samples contained both the A1 and A2 variants, while samples 11-16 contained only the A2 variant. The area under the peaks found at the retention time of each variant is proportional to the amount of the given variant.

With the above examples, we proved that the method we developed is suitable for the separation of the main milk protein fractions and the identification of the A1 and A2 variants of beta casein. We have also proven that the method according to the invention can be repeated and reproduced, so it can be validated as a test method for milk samples.

Table 2. Results of the examination of cows' milk with certified beta-casein status

Serial number ENAR number Sample number Presumed Beta-casein status Confirmed Beta-casein status HPLC Beta-casein A1 tRA1 (min) Beta-casein A2 tRA2 (min) 1. 8028 10 A1A1 A1A1 13.656 — 2. 8041 17 A1A1 A1A1 13.619 — 3. 8068 13 A1A1 A1A1 13,650 - 4. 8104 14 A1A1 A1A1 13.629 - 5. 8111 2 A1A2 A1A2 13.726 14.123 6. 8010 19 A1A2 A1A2 13.748 14.140 7. 8107 15 A1A2 A1A2 13.675 14.071 A1A2 A1A2 13,746 14,142 10. 8122 8 A1A2 A1A2 13.770 14.276 11. 7869 6 A2A2 A2A2 - 13.972 12. 7902 5 A2A2 A2A2 - 14.005 13. 8008 12 A2A2 A2A2 - 14.014 14. 8092 — 14,013 16. 8212 9 A2A2 A2A2 — 14,060

Claims

PATENT CLAIMS

1. A method for the fractionation of the main protein fractions in milk and their genetic variants, where the method includes the following steps:

a) prepare the sample;

b) the prepared sample is injected without filtration onto a C-18 RP-HPLC column;

c) A gradient elution is performed using eluents A and B, where eluent A contained acetonitrile:water:trifluoroacetic acid in a volume ratio of 100:900:1, and eluent B contained acetonitrile:water:trifluoroacetic acid in a volume ratio of 900:100:1; and

d) the main protein fractions are separated based on the peaks appearing in the chromatogram, characterized by

- the fractionation of the main protein fractions in milk and their genetic variants takes place simultaneously and includes the separation of the A1 and A2 genetic variants of the beta-casein fraction,

- in step c), the temperature of the column is thermostated at 35-45°C, preferably 40°C, the flow rate is 1.0-1.2 ml/min, preferably 1.1 ml/min, and

- the gradient elution is as follows:

- 70.9% eluent A and 29.1% eluent B for 0.5 minutes, then