DETERMINATION AND QUANTIFICATION OF PROTEOSE PEPTONE CONTENT AND / OR BETA-CASEIN CONTENT AND NUTRITIONAL COMPOSITION WITH REDUCED PROTEOSE PEPTONE CONTENT DERIVED FROM BETA-CASEIN.

Abstract

The present invention relates to a method for determining and quantifying proteose peptones derived from β-casein. This method comprises the steps of (i) providing a dairy-based product for analysis; (ii) subjecting the product to liquid chromatography-mass spectrometry analysis; and (iii) determining and / or quantifying the proteose peptones derived from β-casein in the product by detecting compounds with defined m / z values ​​or by deconvolving the mass spectra to calculate monoisotopic masses. The present invention also relates to nutritional compositions having a reduced content of proteose peptones derived from β-casein and their uses, for example, treating, preventing, and / or alleviating abdominal pain in infants.

Description

The present invention relates to a method for determining and quantifying proteose peptones derived from β-casein, such as PP-S, PP8s, and PP8f, and / or β-cssein. The present invention also relates to a nutritional composition and infant formula comprising a reduced content of proteose peptones derived from β-casein, such as PP-5, PP8s, and PP8f. BACKGROUND OF THE INVENTION Dairy cow's milk has long been considered nature's perfect food, providing an important source of nutrients including high-quality protein, carbohydrates, and selected micronutrients. Milk proteins are primarily caseins, with the remaining proteins grouped into the 20 whey protein cluster, the main ones being beta-lactalbumin and alpha-lactalbumin. Among the caseins, beta-casein is the second most abundant protein in cow's milk and has an excellent nutritional balance of amino acids. However, different mutations in the bovine beta-casein gene have resulted in several genetic variants, with the so-called A1 and A2 variants being the most common. The A1 and A2 variants of β-casein differ at amino acid position 67 of the secreted β-casein protein, with histidine (His) in the A1 variant of β-casein and prolin (Pro) in the A2 variant of β-casein, as a result of a single nucleotide difference. This is illustrated in Figure 1. The A2 variant of β-easein is more comparable to human β-casein in terms of digestive degradation. It has been suggested that the A1 variant of β-easein may be associated with cow's milk intolerance, and a statistically significant positive association has been observed between abdominal pain and stool consistency when participants consumed a diet containing A1, but not a diet containing A3 (Ho et al., 2014). The digestive difference observed between variants A1 and A2 may be due to the particular polymorphism described above. This polymorphism leads to a key conformational change in the secondary structure of the β-casein protein. Gastrointestinal proteolytic digestion of variant A1 (but not A2) of β-casein leads to the generation of β-casomorphin 7 (BCM-7), which is an exogenous opioid peptide (exorphin) that can activate opioid receptors throughout the body (EFSA). Raport, 2009), This is illustrated in Figure 2. Originally, all cows produced milk containing only the A2 variant of β-casein, but genetic variation has led to mixed herds. Therefore, the herds of cows typically used to produce milk usually produce a mixture of the A1 and A2 variants of β-casein, but with a different amount of A1 compared to A2. It has been shown that proteolysis of β-casein by plasmin leads to the generation of γ-caseins and proteose peptones (fast PP8, slow PP8 and PP~5). The proteose peptones do not precipitate with the caseins during acidification or enzymatic treatment with rennet and remain in the whey fraction (reviewed in Karamoko et al. 2013). PP8 and PP-5 correspond to sequences 29-105 / 7 and 1-105 / 7, respectively, and both include position 67. Therefore, whey products derived from milk containing β-casein A1 will likely contain proteose peptones of the A1 type. Proteose peptones are derived from the same δ gene and are produced through endogenous events, so they are also considered proteoforms of β-casein. Intact protein analyses by reversed-phase high-performance liquid chromatography (RP-HPLC) or capillary electrophoresis (CE) coupled with UV detection can effectively separate most β-casein variants in raw milk samples (see, for example, de Jong et al. 1993, Vísser et al., 1995, Bonfattí et al.).(Poulsen et al., 2008, 2016). However, in manufactured products, processing conditions induce reactions between proteins and reducing sugars (Maillard reaction) that produce multiple proteoforms containing one or more sugar adducts (Fenaille et al., 2006). As a consequence, the protein peaks divide and amplify, creating overlaps that prevent the analysis of individual proteoforms using UV detection (Vallejo-Cordoba, 1997; Feng et al., 2017). Human breast milk (HBM) is known to contain the A2 form of human beta-casein. By definition, it represents a gold standard in terms of infant nutrition. Therefore, there is a need to provide (synthetic) nutrifocal compositions that closely mimic the composition of HBM.Due to the digestive differences observed in humans regarding the M and A2 variants of bovine β-casein, it would be advantageous to be able to detect and quantify the amount of β-casein and its proteoforms in a simple and efficient manner. Currently, this is not possible. There is a simple and effective method for characterizing and quantifying individual milk proteins and their proteoforms such as proteose peptones in finished products. Furthermore, there is a need to provide a nutritional composition that comprises a relatively low amount or proportion of β-casein or proteose peptones derived from β-casein or that may be devoid of any form of these. There is also a need to provide a nutritional composition comprising whey fractions that are devoid of or reduced by 1G proteose peptones derived from β-casein. BBB®J0^SB!£S!é!iee^^ An objective of the present invention relates to an improved method 15 for the efficient and simple detection of β-casein and its proteoforms, in particular its proteose peptones derived from β-casein, as well as a method for reducing the content of proteose peptones derived from β-asein. An additional objective of the present invention is to provide a nutritional composition such as an infant formula, which can prevent abdominal pain and / or improve stool inconsistency in an infant. Therefore, one aspect of the invention relates to a method for determining and quantifying proteose peptones derived from β-casein and / or β-casein, said method comprising the steps of (i) providing a dairy-based product for analysis; (ii) subject said product using liquid chromatography - mass spectrometry analysis; (iii) determine and / or quantify said proteose peptones derived from β-casein and / or β-casein in said product ai detect compounds of defined m / z values ​​or ai decorate one or more mass spectrometry spectra to calculate monoisotopic masses. A second aspect of the invention relates to a method for producing a whey protein fraction having a reduced content of proteose peptones derived from p-oasema, said method comprising the steps of (i) providing a whey protein fraction; (ü) determine and quantify said proteose peptones derived from β-casein in said whey protein fraction as described herein; and (iii) select said whey protein fraction having at most 10% by weight of proteose peptones derived from β-casein based on the total protein in said whey protein fraction forming a selected whey protein fraction. A third aspect of the invention relates to a method for producing a whey protein fraction having a reduced content of proteose 20 peptones derived from β-casein, said method comprising the steps of (i) providing a whey protein fraction; (ii) reducing the content of proteose peptones derived from β-casein in said whey protein fraction to a concentration of no more than 10% by weight based on the total protein in the whey protein fraction, forming a protein fraction of e reduced tea whey. A fourth aspect of the invention relates to a method for producing a nutritional composition having a reduced content of proteose peptones derived from β-casein, said method comprising the steps of (I) providing a selected whey protein fraction or a reduced whey protein fraction as described herein; (ii) prepare said nutritional composition with a reduced content of proteose peptones derived from β-casein from said selected whey protein fractions, said reduced whey protein fractions or a mixture thereof. A fifth aspect of the invention relates to a whey protein fraction having a reduced content of proteose peptones derived from β-casein, obtainable by the method as described herein. A sixth aspect of the invention relates to a nutritional composition comprising said whey protein fraction having a reduced content of protecse peptones derived from β-casein as described herein or obtainable by the method as described herein. A seventh aspect of the invention relates to a nutritional composition as described herein for use as a medicament. An eighth aspect of the invention relates to an infant formula as described herein for use in the treatment, prevention and / or improvement of abdominal pain in an infant and / or the improvement of bowel comfort in said infant, A ninth aspect of the invention relates to an infant formula as described herein for use in the treatment, prevention and / or improvement of lactose intolerance in an infant. A tenth aspect of the invention relates to the use of infant formula as described herein to improve stool consistency. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a partial sequence alignment between the beta-casein A2 and beta-casein A1 sequences relative to amino acid sequences 59-71. The amino acid at position 67 is highlighted, demonstrating that the difference of one amino acid between beta-casein A1 and beta-casein A2 is His and Pro, respectively: Figure 2 shows a partial sequence alignment between the beta-casein A2 and beta-casein Al sequences in relation to amino acid sequences 59-71. The difference of one amino acid between the two beta-caseins influences the cleavage ability of the two chains, since beta-casein A2 does not cleave due to the presence of Pro at position 67, while beta-casein Al cleaves at the N-terminal side of His forming a possibility for the formation of a long sequence of seven amino acids, i.e., 8eta-casomorone-7 (BCM-7); Figures 3A, 38 and 3C show a description of the steps of the method LC-HRMS: (Fig. 3A) sample preparation, (Fig. 38) deconvoluted mesoisotopic mass spectra for intact β-casein and (Fig. 3C) bubble plot representation of the intact β-casein region (bubble sizes represent signal intensities). Figures 4A and 4B show the global fingerprint and the intact β-casein region. The main proteins of the teche were separated in a global fingerprint (Fig. 4A) of a raw milk sample. Different proteoforms were detected for (Fig. 4B) Intact β-casein. A1, A2 and B are genetic variants, the superscript notations correspond to the number of lactose aducts (L); Figure 5 shows examples of global footprints in samples of raw tea, human milk, infant formula (IF), skimmed milk powder (SMP), and whey; Figure 6 shows bubble charts (intact β-casein region) of seven samples of A2 infant formula either untreated or enriched to 5% with β-casein Al (5 g intact β-casein Al / 100 g total intact β-casein); Figures 7A, 7B, and 7C show how glycation affects MS readings. (Fig. 7A) Bubble plots (intact β-casein region) showed a 1.5 Increase in otosylation during the solid-state gliation experiment. (Reg. 7B) The percentage of non-glycated signal (black bar) decreased over time (Fig. 7C), showing that the total β-casein signal decreases with increasing glycation and (Fig. 7D) had a linear relationship with the percentage of non-glycated signal. Graphing the signal reduction as a function of the percentage of non-glycated signal (Fig. 7E) allowed the calculation of a glycation correction factor of 20; Figures 8A and 8B show a characterization of the β-casein standard and the establishment of the β-casein calibration curve. Analysis of the β-casein standard revealed that approximately 15% of the total signal could not be attributed to intact β-casein (Fig. 8A). A linear calibration curve was established using the MS signal of normalized intact β-casein (to SMP, which was selected for Qlieaeion) and adjusted amounts of Haseina (Fig. 8B). Cross-mixing experiments between skim milk samples A11 and A21A2 indicated that the relationship remained linear even in a dairy matrix (c). The protein content remained constant during the cross-mixing experiment. Figures 9A and 9B show the quantification of intact β-casein in infant formulas. β-Casein was quantified in seven infant formula samples (Fig. 9A), where each sample was analyzed in triplicate five times. Theoretical values ​​are based on the recipe and generic milk protein composition. An additional set of infant formulas (based on A2 or skimmed milk powder containing multiple genetic variants) was analyzed in triplicate on a single day (Fig. 9B). The shaded bars represent the best guesses since the whey percentage of those samples was not indicated in the boxes; Figure 10 shows the results of LC-HRMS analysis of intact proteins in different whey protein concentrates (WPC). The intact protein region is marked by the circle with a solid perimeter, while proteose β-casein peptones are included in the region marked by the circle with a dashed perimeter. Figure 11 shows a close-up of the area shown in Figure 1 by the circle with the dashed perimeter, which represents the proteose peptone content. Black represents proteose peptones derived from β-casein A2, gray represents proteose peptones derived from β-casein A1, and white represents unassigned compounds. Figure 12 shows the result of the LC-HRMS analysis of intact proteins in different finished products focusing on the area containing parateosá zs peptones, Figure 13 shows the correlation between the PP5 signal measured for PP5 A1 and PP5 A2 in relation to the percentage of SMP A2 in the sample. The protein content was kept constant during the cross-mixing experiment; Figure 14 shows a schematic view of intact β-casein and the products generated after cleavage of β-casein. The position of the tryptic peptides is shown by horizontal arrows with the common peptides Tot1 and TotS, as well as the specific peptides A1 / 2N, A1 / 2S, and A1 / 2T. Tot1 and Tot2 are at the C-terminus of β-casein where the sequence is identical for A1 and A2 and are not present in proteose peptones. The peptides A1 / 2N, A1 / 2S, and A1 / 2T include the amino acid at position 67, which differs between A1 and A2. Figure 15 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed milk powder and various infant formulas with respect to the A1S peptide; Figure 16 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed milk powder and various infant formulas with respect to the A1N peptide; Figure 17 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed milk powder and various infant formulas with respect to the A1T peptide; Figure 18 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed milk powder and various infant formulas with respect to the A2S peptide; Figure 19 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed date powder and various infant formulas with respect to the A2N peptide; Figure 20 shows the result obtained by LC-MS of a tryptic digestion of different finished products including skimmed milk powder and various infant formulas with respect to the A2T peptide; Figure 21 shows the result of a three-fold digestion of different finished products including skimmed milk powder and various infant formulas with respect to the Tet1 peptide; Figure 22 shows the result of a triplicate digestion of different finished products including skimmed milk powder and various infant formulas with respect to the Tot2 peptide; Figure 23 shows the result of a triplicate digestion in skimmed milk powder, lactose and different whey protein concentrates (WPG) that measures the amount of Total peptide using LC-MS; Figure 24 shows the result of a tryptic digestion in skimmed milk powder, lactose and different whey protein concentrates 15 (WPG) that measures the amount of peptide Tot2 using LQ-MS; Figure 25 shows the result of a triplicate digestion of skimmed milk powder, lactose and different whey protein concentrates (WPG) measuring the amount of AI S peptide using LC-MS; Figure 26 shows the result of a triplicate digestion of skimmed milk powder, lactose, and different whey protein concentrates (WPG) measuring the amount of peptide A1N using LC-MS; Figure 27 shows the result of a triplicate digestion of skimmed milk powder, lactose, and different whey protein concentrates (WPG) measuring the amount of peptide A1T using LC-MS; Figure 28 shows the result of a triptych digestion of skimmed milk powder, lactose and different whey protein concentrates (WPC) measuring the amount of peptide A2S using LC-MS; Figure 29 shows the result of a tryptic digestion of skimmed milk powder, lactose and different whey protein concentrates 5 (WPC) measuring the amount of peptide A2N using LC-MS: Figure 30 shows the result of a tryptic digestion of skimmed milk powder, lactose and different whey protein concentrates (WPC) measuring the amount of peptide A2T using LC-MS; The present invention will now be described in greater detail in the following description. DETAILED DESCRIPTION OF THE 1^^ Definitions Before analyzing the present invention in greater detail, the following terms and conventions will first be defined: The term “LC” refers to “liquid chromatography,” as it is known to those skilled in the technique. It should be noted that LC also includes HPLC, or high-performance liquid chromatography, and UPLC, or ultra-high-performance liquid chromatography. The term “MS” refers to “mass spectroscopy,” as it is known to those skilled in the technique. It should be noted that MS also includes HRMS, that is, high-resolution mass spectrometry. The term “composites of defined m / z values” means that the MS is configured to measure compounds that have only m / z values ​​specifically defined by the user when measured in a given sample. The term “AI / AI cows” refers to cows with the homozygous genotype A1A1. Milk obtained from A1 / A1 cows is called “AI milk”. The term “A2 / A2 cows” refers to cows with the homozygous genotype A2A2. Milk obtained from A2 / A2 cows is called “A2 milk”. The term “A1 / A2 cows” refers to cows with the heterozygous genotype A1A2. The milk obtained from A1 / A2 cows is called “A1 / A2 milk”. The term “Al whey” refers to whey produced essentially from Al milk. The term “A2 whey” refers to whey produced essentially from A2 milk. The term “A1 / A2 whey” refers to whey produced from A1 / A2 milk or that is a mixture of A1 whey and A2 whey or that is produced from a mixture of A1 milk and A2 milk, A1 milk and A1 / A2 milk, A2 and A1 / A2 milk or A1 milk, A2 milk and A1 / A2. The term “β-casein A2” refers to the A2 variant of bovine β-casein having the amino acid sequence according to sequence ident. no. 1 (secreted form of protein). In the present context, other variants, including a proline at position 67, may be included in β-casein A2. The term “β-casein A1” refers to the A1 variant of bovine β-casein that has the amino acid sequence according to sequence number 2 (secreted form of protein). Sequence number 1 and sequence number 2 differ from each other in that β-casein A1 contains a histidine at position 67, while β-casein A2 contains a proline at position 67. In the present context, other variants, which include a histidine at position 67, may be included in β-casein A1. The term “intact β-casein” refers to the protein, which is not cleaved except by removal of the signal sequence, e.g., a protein as described by the sequence with idents 1 and 2, The term β-casomorphin 7” also described as BCM-7” refers to the 5 peptide that has the amino acid sequence Tyr'Pro-Phe-Pro-Gly-Pro-lle, The term "WPG" refers to whey protein concentrate. In this context, WPG includes traditional WPG as defined by the USP, as well as lactose-reduced and mineral-reduced whey protein, meaning the protein level can be as low as 10% w / w. The term WPI” refers to whey protein isolate” and is a whey protein concentrate that has a whey protein content on a dry basis of not less than 90% by weight. The term "whey protein fraction" refers to a composition comprising whey proteins, e.g., WPG and / or WPI. The term "standard SMP" refers to "standard skimmed milk powder" and is derived from milk obtained from mixed herds of cows and therefore comprises multiple variants of β-casein including β-casein A1 and β-casein A2. The term SMP A2” refers to skimmed milk powder A2” and comprises only β-casein A2 and not β-casein A1. The term "proteoform" refers to closely related protein molecules that arise from all combinatorial sources of variation resulting from products originating from a single gene. This includes products that differ due to genetic variations, alternatively spliced ​​RNA transcripts, and post-translational modifications. The term “proteose peptone” is the same as that used in Swaisgood, 1982, that is, the term “proteose peptone” refers to those proteins / peptides that remain in solution after the milk has been heated to 95°C for 20 minutes and then acidified to pH 4.7 with 12% trichloroacetic acid. The term “β-caselin-derived proteose peptones” refers to proteose peptones derived from β-casein alone such as PP-5, PP-8 fast and PP-8 slow. The term "pMeose peptone 5", "PP5" or "PP-5" refers to residues 1-105 and 1-107 derived from β-casein. The term “fast proteose peptone 8”, “PPSf” or “fast PP8”* refers to residues 1-28 derived from β-casein. Fast PP8 may also be called “bcasePI-Zr”. The term “slow proteose peptone S”, “PP8s” or “slow PP8” refers to residues 29-105 and 29-107 derived from p-casein. Slow PP8 may also be called “beasl P 29-105” and “bcás1 P 29-107”. The term 'infant' refers to a child under 12 months of age; in one embodiment of the invention, the term 'infant' can be extended to include children of any age up to and including 18 months, or of any age up to and including 24 months. The term “infant formula”, as used in this description, refers to a food substance intended for particular nutritional uses by infants during the first months of life (such as 0 to 12 months, 0 to 10 months, 0 to 8 months, 0 to 6 months, or 0 to 4 months) and which, by itself, satisfies the nutritional requirements of this category of person (Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formula and follow-on formula). It also refers to a nutritional composition intended for infants and as defined in the Codex STAN 72:1981 and Infant Specialities (ind. Food for special medical purposes). The expression 'infant formula' encompasses both 'starter infant formula' and 'follow-up formula' or 'maintenance formula'. The term “follow-up formula” or “maintenance formula” refers to a formula that is given from the 6th month onward. This constitutes the main liquid component in the progressively diversified diet of this age group. The term “powder” in the present context means a dry bulk solid W composed of a large number of very fine particles that can flow freely when agitated or tilted. The powder may contain water in amounts not exceeding 10%, such as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5%. The term “nutritional composition” means a composition that nourishes a subject. Usually, this nutritional composition is administered orally or intravenously. It may include a source of lipid or fat, a source of carbohydrate, and / or a source of protein. The nutritional composition of the present invention may be in solid form (e.g., powder) or in liquid form. In one particular embodiment, the composition of the present invention is a hypoallergenic nutritional composition. The expression “hypoallergenic nutritional composition” means a nutritional composition that is unlikely to cause allergic reactions. In one particular embodiment, the nutritional composition of the present invention is a “synthetic nutritional composition.” The expression “synthetic nutritional composition” means a mixture obtained by chemical and / or biological means, which may be chemically identical to the naturally occurring mixture in mammalian milk (i.e., the synthetic nutritional composition is not breast milk). “Probiotic bacteria” means preparations of microbial cells or components of microbial cells with a beneficial effect on the health or well-being of the host. A definition of probiotic bacteria is provided in Salmínen S et al, 1999; “Prebiotic” means a selectively fermented ingredient that allows specific changes in the composition and / or activity of the gastrointestinal microbiota, conferring benefits to the host's health and well-being. Prebiotics are described in Roberford MB 2007; Detection and / or quantification of proteose ketones derived from β-casein v / o S-casein The invention relates in a first aspect to a method for determining and / or quantifying proteose peptones derived from β-casein and / or β-casein, said method comprising the steps of (i) providing a dairy-based product for analysis; (ii) subject said product using liquid chromatography-mass spectrometry analysis; (iii) determine and / or quantify said proteose peptones derived from β-casein and / or β-casein in said product by detecting compounds of 20 defined m / z values ​​or by deconvolving one or more mass spectrometry spectra to calculate monoisotopic masses. The dairy-based product may be selected from a list of finished products such as infant formula, maternal nutrition, adult nutritional products or dairy products such as milk, milk powder, liquid whey, whey powder, caseinates, WPC, WPI. 5.10 In one embodiment, the dairy-based product to be analyzed is an infant formula or a whey protein fraction. The dairy-based product to be analyzed may be a powder or a liquid. In one embodiment, the product is a powder, which is dissolved before analysis. The powder is preferably dissolved by dispersing it in water. However, the powder may also be dissolved in other liquids such as a reducing agent as defined below, buffer solutions such as ammonium bicarbonate, Tris, trisadium citrate, HEPES, TEAS (triethylammonium bicarbonate), or denaturing regulators as defined below, and combinations thereof. In one embodiment, the powder is dissolved in a reducing agent and a buffer solution, and optionally, a denaturing regulator is subsequently added.In a further embodiment, the powder is dissolved in a reducing and denaturing regulator and, optionally, a buffer solution is subsequently added. Also in a further embodiment, the powder is dissolved in a buffer solution and a denaturing regulator and, optionally, a reducing agent is subsequently added. In one embodiment, the powders are dispersed to a concentration of at most 10% w / v protein in the liquid, such as at most 9.5% w / v protein in the liquid, at most 9% w / v protein in the liquid, such as at most 8.5% w / v protein in the liquid, at most 5% w / v protein in the liquid, as well as at most 7.5% w / v protein in the liquid, at most 7% w / v protein in the liquid, such as at most 6%.5% w / v protein in the liquid, as a maximum 6% w / v protein in the liquid, such as a maximum 5.5% w / v protein in the liquid, as a maximum 5% w / v protein in the liquid. 25 such as a maximum 4.5% w / v protein in the liquid, as a maximum 4% w / v. of protein in the liquid, such as a maximum of 3.5% w / v of protein in the liquid, such as a maximum of 3% w / v of protein in the liquid, such as a maximum of 2.5% w / v of protein in the liquid, such as a maximum of 2% w / v of protein in the liquid, such as a maximum of 1.5% w / v of protein in the liquid, such as a maximum of 1% w / v of protein in the liquid, such as a maximum of 0.5% w / v of protein in the liquid. After dissolution of the samples, or alternatively for the dissolution of the samples, they are denatured and reduced by means of denaturing and reducing regulators such as urea, guanidine HCl, DTT, beta-heap ethanol, TCEP, In one modality, the sample is dissolved in water and further diluted in a mixture of trisodium citrate, guanidine-HCl, and DTT. In another modality, the sample is dissolved in Tris and Urea and diluted in ammonium bicarbonate and DTT. Preferably, the samples are also clarified before being analyzed. This can be done by centrifugation. However, it can also be done by filtration. In one modality, the product to be analyzed is examined using intact protein analysis, where the proteins are preserved by subjecting them to liquid chromatography followed by mass spectrometry (LC-MS), such as liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The use of intact protein analysis makes it possible to distinguish between different proteoforms of α-casein and genetic variants of full-length β-casein, including the A1 and A2 variants. Therefore, measuring the protein content of the products makes it possible to obtain a complete protein profile of the product. Also, the different 2S genetic variants of proteose peptones can be distinguished using the intact protein analysis method. In another modality, the product is analyzed by peptide analysis, which includes an enzymatic digestion step before analysis. Therefore, before undergoing LC-MS analysis, the product is subjected to enzymatic digestion, e.g., by digesting it with trypsin or GluC (GluC Endqproteinase [Straphylococcus aureus Protease V81]). The use of enzymatic digestion makes it possible to determine whether β-casein A1 is present in a product or whether the proteose peptones of β-casein A1 and / or A2 are present. However, due to the method's configuration, which involves digesting proteins as whole units, it is not possible to distinguish between proteose peptones and full-length β-casein, but only between the A1 and A2 genetic variants. This can be achieved by detecting sequences covering amino acid 67, which differs between the A1 and A2 variants. Therefore, a complete protein fingerprint profile would not be obtained, but the presence of the A1 variant and potential precursors for 8CM-7 would be detected. In an additional modality, this enzymatic digestion is carried out by tryptic digestion or digestion with GluC. In one form, liquid chromatography is high-performance liquid chromatography. In another form, liquid chromatography is ultra-performance liquid chromatography. Yet another form, liquid chromatography is nanochromatography. In this measurement, the standard configuration can be used to run the liquid chromatography process as it is known to the person skilled in the technical aspects. In a preferred modality for intact protein analysis, samples can be separated on a C4 column using a gradient of 0.5 mi / min. Preferably, a buffer containing trifluoroacetic acid is used. However, the gradient can vary from 0.1 mJ / min to 1 ml / min, such as from 0.1 mJ / min to 0.5 ml / min. Alternatively, the following buffers can be used for LC separation, such as water / methanol or water / acetonitrile, possibly including formic acid, ditharoacetic acid, and / or trifluoroacetic acid. In a preferred modality for peptide analysis, samples can be separated on a single C18 column using a gradient at 75 µl / min. Preferably, a buffer containing formic acid is used. However, the gradient can vary from 0.1 µl / min to 100 µl / min, such as from 0.4 µl / min to 85 µl / min. 10 Alternatively, the following buffers can be used for LC separation, such as water / methanol or water / acetone phosphate, possibly including formic acid, difluoroacetic acid, and / or trifluoroacetic acid. MS chromatograms can be recorded on machines such as Thermo Orbitrap Elite or Thermo Q-Exactive HE, In one modality, MS chromatograms are recorded on a Thermo Qrbitrap Elite {heater temperature: 60°C, cover gas: 20, auxiliary gas: 5, sweep gas: 0, spray voltage: 3.8 kV, capillary temperature: 320°C, lens RF level S: 60%, mass range: 400 to 2000 m / s, resolution: 240,000, AGC objective: Te6} In another mode, MS chromatograms are recorded on a Thermo 20 Q-Exactive HF (heater temperature: 100 *C, cover gas: 53, aux gas: 14, scan gas: 3, spray voltage: 3.5 kV, capillary temperature: 320 °C, RF level of lens S: 70%, mass range: 400 to 2000 m / z, resolution: 240,000, AGC objective: 1e6, maximum IT: 200 ms). In one modality, said mass spectrometry analysis is a high-resolution mass spectrometry analysis. In one modality, HRMS acquisition is preferably performed at a resolution above 100,000, such as between 120,000 and 240,000. According to one modality, the determination and / or quantification of these proteose peptones derived from β-casein is performed by detecting compounds with five defined m / z values, as indicated. In this way, the presence and quantity of specific peptide sequences can be detected. This is possible because enzymatic digestion would lead to specific peptides due to a specific cleavage. In one modality, the peptide method uses a non-targeted (data-dependent acquisition - DDA) approach. In another modality, the peptide method uses a non-targeted (data-independent acquisition - DIA) approach. In an additional modality, the peptide method uses a PRM (parallel reaction monitors) or MRM (multiple reaction monitor) method.The precursors can be preferably selected from the first quadruple and fragmented and recorded in MS2 data. In one embodiment, the precursor selection can be performed according to the peptide list described by the SEO with ID numbers 3-10. According to another embodiment, the mass spectrometry data obtained after measurement using LC-MS for each sample are deconvolved to calculate monoisotopic masses. Therefore, in one embodiment, the determination and / or quantification of said β-casein-derived proteose peptones in said product is performed by deconvolving one or more mass spectrometry spectra to calculate monoisotopic masses. The deconvolution can be performed using commercially available software such as Thermo BioPharma Finder. 5 W 15 In one mode, deconvolution is performed using a sliding window algorithm. In another mode, deconvolution is performed using a fixed window algorithm. In a preferred mode, deoonvolution was performed using Thermo BioPharma Finder 1.0 software with the XWci algorithm (S / N threshold: 3, relative abundance threshold: 1%, adjustment factor: 80%, remaining threshold: 25%, overlaps: 5 to 50 charge states, minimum intensity: 1, expected intensity error: 3, m / z: 600 to 2000, minimum number of detected charge states: 3) and sliding windows (time: 5 to 20 min, target average spectrum width: 0.1 min, target average spectrum deviation: 50%, fusion tolerance: 1.5 Da, maximum RT gap: 0.5 min, minimum number of detected intervals: 3, XIC).The unconvoluted monolithic masses were compared with a protein database containing the major components of milk proteins qS1-CN, qS2-CN, β-CN, κ-CN, γ-CN, q-lactalbumin, β-lactoglobulin, CGMP, and β-casein proteose peptones. The combinatorial addition of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation, and pyroglutamic acid) was tested to identify the majority of the signals. One way to quantify β-casein-derived proteose peptones and / or β-casein is to compare the signal intensities obtained from the 20 measurements with a standard curve derived from measurements of known quantities of the different proteose peptones and / or β-casein. In this way, the absolute amount of proteose peptones and / or β-casein in a given sample can be calculated and the amount quantified.Alternatively, the signal intensity of the proteose peptone of interest 25 can be expressed in terms of relative terms such as a percentage of. the total amount of protein in the sample or as a percentage of the β~casein content. The compounds to be determined and / or quantified are proteose peptones derived from β-casein and / or β-casein. In one embodiment, the proteose peptones are proteose peptones derived from β-casein A1. In another embodiment, the proteose peptones derived from β-casein are fast PP8, slow PP8, and / or PP-5. In one modality, the β-casein that will be detected and / or quantified is βίο casein Al. peptones derived from β-casein (selected whey protein fraction) In a second aspect, this invention relates to a method for producing a whey protein fraction having a reduced content of 15 proteose peptones derived from β-casein, said method comprising the steps of (i) providing a whey protein fraction; («) determine and quantify said β-casein-derived proteose peptones in said whey protein fraction as described herein; and (H) select said whey protein fraction having at most 10% by weight of β-casein-derived proteose peptones based on the total protein in said whey protein fraction forming a selected whey protein fraction. The whey protein fractions available for use in 25 finished products, such as nutritional compositions including infant formulas, frequently comprise whey A1 and / or whey A1 / A2 due to standard production methods. However, this has not previously been considered a problem, e.g., for the manufacture of A2 infant formula, since β-casein A1 would precipitate during whey preparation and, therefore, would not be part of the whey protein fraction. However, as shown in the examples, proteose peptones derived from β-casein A1 are detected in whey protein fractions such as WPC. In one modality, whey protein traction is based on whey A1 and / or whey A1A2 Due to the advantages of the method described above for the determination and quantification of β-casein variants and proteoforms, the content of proteose peptones derived from β-cawfna can be easily detected and quantified. In this way, whey protein fractions can be separated into whey protein fractions containing different amounts of proteose peptones. Therefore, whey protein fractions can be selected that comprise a maximum of 10% by weight of proteose peptones of the total amount of protein in the whey protein fraction. In one embodiment, the selected whey protein fraction has a proteose peptone content derived from β-casein, such as a proteose peptone content derived from β-casein, of at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, or at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, even more preferably at most 5.5% by weight, or at most 5% by weight, with the highest preference being at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, or at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, or at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, even more preferably at most 0.25% by weight, such as at most 0.10% by weight, with the maximum preference at most 0.05% by weight, such as at most 0.01% by weight, based on the total protein in the whey protein fraction. The total amount of protein in the whey protein fraction is preferably measured by Kjeldahl analysis (ISO 8968-1:2014). In one form, protease peptones are rapid PP8, slow PP8 and / or PP-5. In an additional modality, proteose peptones are slow PP8 and / or PP-5. In one embodiment, the selected whey protein fraction has a slow PP8 and / or PP-5 content of at most 0.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, with greater preference at most 7.5% by weight, such as at most 7% by weight, even with greater preference at most 6.5% by weight, such as at most 6% by weight, even with greater preference at most 5.5% by weight, such as at most 5% by weight, with the maximum preference at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, even more preferably at most 0.25% by weight, such as at most 0.10% by weight, with the maximum preference as at most 0.05% by weight, and as at most 0.01% by weight, based on the total protein in the whey protein fraction. Even in an additional form, proteose peptone is PP-5. Whey protein fraction with reduced proteose peptone content (reduced whey protein fraction) In a third aspect, this invention relates to a method for producing a whey protein fraction having a reduced content of proteose peptones derived from β-casein, said method comprising the steps of (i) providing a whey protein fraction; (ii) reducing the proteose peptone content derived from β-casein in said whey protein fraction to a concentration of not more than 10% by weight based on the total protein in the whey protein fraction, forming a reduced whey protein fraction, In a further aspect, this invention relates to a method for producing a whey protein fraction having a reduced content of proteose peptones derived from p-casein, said method comprising the steps of (i) providing a whey protein fraction which has an initial content of proteose peptones; (ü) reducing the proteose peptone content derived from β-casein in said whey protein fraction to a concentration at least 5x less than the initial content of said whey protein fraction, obtaining a reduced whey protein fraction, such as at least 8x less, as at least 10x less, as at least 15x less than the initial content of said whey protein fraction. In one form, said whey protein fraction is based on A1 whey and / or A1 A2 whey. As an example, the at least 5x less should be understood as if, for example, the initial content is 5 yg / mg then a 5x (five times) reduction would mean that the reduced whey protein fraction contains at most 1 yg / mg and similarly if the signal intensity is 500,000,000 for the initial content then the signal intensity would be at most 100,000,000 for the reduced whey protein fraction. The reduction of at least 5x less, 8x less, 0x less or 15x less can be measured by means of the signal intensity obtained by the method for determining and quantifying as described in the present description. In one embodiment, the whey protein fraction is tested using the method as described herein to determine and quantify the proteose peptone content to decide whether the proteose peptone content needs to be reduced or not before the whey protein fractions are used. The content of β-casein-derived prateose peptones in the whey protein fraction is reduced to a maximum of 10% by weight based on the total protein in the whey protein fraction. The total amount of protein in the whey protein fraction is preferably measured by Kjeldahl analysis (ISO 8988-1:2014). In one method, the proteose peptone content is reduced by gel filtration. However,* the proteose peptone content can also be reduced by ion-exchange chromatography, affinity chromatography, or membrane separation. In an additional embodiment, the final proteose peptone content in the reduced whey protein fraction can be determined and quantified using the method described above, In one embodiment, the reduced whey protein fraction derived from β-casein has a proteose peptone content of at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, or at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, even more preferably at most 5.5% by weight, such as at most 5% by weight, with the highest preference at most 4.5% by weight, such as at most 4% by weight, or at most 3.5% by weight, preferably at most 3% by weight, or at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, or at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, even with greater preference at most 0.25% by weight, such as at most 0.10% by weight, with the highest preference at most 0.05% by weight, such as at most 0.1% by weight, based on total protein in the whey protein fraction. In one form, proteose peptones are PP8 fast, PP8 slow and / or PP-5. In one additional embodiment, the proteose peptones are slow PP8 and / or PP-5. In one embodiment, the reduced whey protein fraction has a slow PP8 and / or PP-5 content of at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.8% by weight, such as at most 7% by weight, more preferably at most 6.5% by weight, such as at most 6% by weight, more preferably at most 5.5% by weight, such as at most 5% by weight, more preferably at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight. weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, even with greater preference as a maximum of 0.25% by weight, such as as a maximum of 0.10% by weight, with the highest preference as a maximum of 0.05% by weight, such as as a maximum of 0.01% by weight, based on the total protein in the whey or milk protein fraction. Even in an additional form, the pre-strength peptone is PP-5. Nutritional composition such as an infant formula that has a reduced content of proteose peptones In a fourth aspect, the invention relates to a method for producing a nutritional composition having a reduced content of proteose peptones derived from β-casein, said method comprising the steps of (i) providing a selected whey protein fraction or a reduced whey protein fraction as described in the present description; (ii) prepare said nutritional composition with a reduced content of proteos® peptones from said selected whey protein fractions, said reduced whey protein fractions or a mixture thereof. In a fifth aspect, the invention relates to a whey protein fraction having a reduced content of proteose peptones derived from β-casein, obtainable by the method as described herein. In a sixth aspect, the invention relates to a nutritional composition comprising said whey protein fraction having a reduced content of proteose peptones derived from β-casein as described herein or obtainable by the method as described herein. In an additional modality, the proteose peptone content derived from β-casein of the nutritional composition tai as an infant formula is at most 9% by weight based on the total protein in the nutritional composition. The total amount of protein in the whey protein fraction is preferably measured by Kjeldahl analysis (ISO 8968-1:2014). Even in an additional formulation, the content of proteose peptones derived from β-casein in the nutritional composition is at most 8.5% by weight, with a higher preference for a maximum of 8% by weight, such as at most 7.5% by weight, with an even higher preference for a maximum of 7% by weight, such as at most 6.5% by weight, with an even higher preference for a maximum of 6% by weight, and as a maximum 5.5% by weight, with the highest preference being at most 5% by weight, such as at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, with the highest preference being at most 3% by weight, such as at most 2.5% by weight, with the highest preference being at most 2% by weight, such as at most 1.5% by weight, with the highest preference being at most 1% by weight, such as at most 0.75% by weight, with the maximum preference being at most 0.50% by weight, such as at most 0.25% by weight, such as at most 0.1% by weight, such as at most 0.05% by weight, such as at most 0.01% by weight, based on the total protein in the whey protein fraction. The nutritional composition of the invention can be intended for any mammal, such as humans, and pets such as cats and dogs. In a preferred embodiment, the mammal is a human. Examples of nutritional compositions are infant formula, maternal nutrition, nutritional products for adults, or dairy products. In one form, the nutritional composition is that of an infant formula. The general composition of a nutritional composition such as an infant formula for use according to the present invention may optionally contain substances that may have a beneficial effect such as probiotic bacteria, fiber, lactoferrin, nucleotides, nucleosides and / or the like in amounts such as those commonly found in nutritional compositions for infant feeding. Probiotic bacteria can be selected from the group consisting of Lactobacilli such as Lactobacillus rhamnosus, Lactobacillus paracasel and Lactobacillus reuteri and Bifidobacterium such as Bifidobacterium icictis, Bifidobacterium breve and Bifidobacterium longum. The nutritional composition, such as infant formula, may also optionally include prebiotics, such as non-digestible carbohydrates that promote the growth of probiotic bacteria in the gut. In a preferred embodiment, the nutritional composition, such as infant formula, comprises prebiotics selected from the group consisting of 25 fructooligosaccharides (POS), raftylose, indin, raftilin, lactdpsa, cow's milk oligosaccharides (CMOS), and galactooligosaccharides (GOS). The nutritional composition may also contain all the vitamins and minerals considered essential in the daily diet in nutritionally significant amounts. Therefore, a preferred modality relates to a nutritional composition, such as an infant formula, which also includes vitamins. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins, and other nutrients optionally present in the nutritional composition include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride, potassium, sodium, selenium, chromium, dibdenum, taurine, and L-carnitine. Minerals are usually added in the form of salts. If necessary, the nutritional composition, such as infant formula, 15 may contain emulsifiers and stabilizers such as soy lecithin, citric acid esters of mono- and diglycerides, and the like. This is especially the case if the composition is provided in liquid form. A preferred modality relates to a nutritional composition, such as an infant formula, where at least one carbohydrate source is selected from the group consisting of lactose, corn syrup solids, fructose, glucose, maltodextrins, dried glucose syrups, sucrose, trehatose, galactose, maltose, honey powders, starch, oligosaccharides, raftiliin, and raftylose. Another preferred modality relates to a nutritional composition, such as an infant formula, which also includes anhydrous tea fat, Yet another preferred modality relates to a nutritional composition, such as an infant formula, which also includes LOPUFA tetes such as DHA, EPA, OPA and / or ARA. The nutritional composition, such as infant formula, may also include flavorings such as, but not limited to, vanillin. One particular preferred modality relates to a nutritional composition, such as an infant formula, which further comprises fructooligosaccharides such as raftiliha and / or raftilose, The nutritional composition, such as infant formula, can be a liquid formula or a powder that must be reconstituted before use. A preferred form is an infant formula that is a powder. In a preferred embodiment, the nutritional composition is a liquid composition that has been prepared by reconstituting a powder. The present invention further provides a nutritional composition, such as an infant formula, according to the present invention for use as 1S complementary feeding of infants in association with human breast milk. According to particular modalities, the infant is between 0-12 months old, preferably between 0-6 months old. Therapeutic and non-therapeutic uses of a nutritional composition or infant formula that has a reduced content of proteose peptones. In a seventh aspect of the present invention, the nutritional composition as described herein can be used as a medicament. In an eighth aspect of the present invention, the infant formula as described herein relates to its use in the treatment, prevention and / or improvement of abdominal pain in an infant, In a ninth aspect of the present invention, infant formula as described A fifth aspect of the present invention relates to its use in the treatment, prevention, and / or improvement of lactose intolerance in an infant. A tenth aspect of the present invention relates to the use of the infant formula as described herein to improve stool consistency. BCM-7 is known to affect opioid receptors and is related to stool consistency and abdominal pain. Therefore, an infant formula comprising none or only a very limited amount of BCM-7 precursors, namely slow proteose peptones PP-5 and PP-8 derived from gamma-assein I, would result in less BCM-7 being formed during consumption of the infant formula and, therefore, less adverse influence on stool consistency and abdominal pain.Furthermore, it is believed that administering an infant formula containing fewer 8CM-7 precursor molecules, and consequently resulting in less BCM-7 being formed, will have less influence on the opioid receptors affected, thus preventing lactose intolerance later in the infant's life. There is growing evidence that early life events can precipitate a disease, even though the disease may only manifest later in life. In this context, treatment should be understood as a situation in which a disorder is established, as opposed to prevention, which takes place before a disorder is established. Improvement should be understood as a situation in which a disorder is established but is not treated in a way that results in the disappearance of the disorder; rather, the health conditions of the individual suffering from the disease are simply improved.In another aspect, the present invention relates to a method for. To treat, prevent and / or improve abdominal pain in an infant. In an additional aspect, the present invention relates to a method for treating, preventing and / or improving lactose intolerance in an infant, It should be noted that the modalities and characteristics described in section 5 of one of the aspects of the present invention also apply to the other aspects of the present invention. All references to patents and non-patents cited in this application are incorporated herein by reference in their entirety. The invention will now be described in additional detail in the following 10 non-limiting examples. Examples Materials and methods Chemical reagents and samples Guanidine hydrochloride (RDDÓ01), trisodium nitrate dihydrate (SI 804), DL-dithiothreitol (43819) and β-casein standard (C690S) were acquired from Sigma-Aldrich (St. Louis, MI, USA), TEA (1,08178.00^ International (Radnor, PA, USA) and LC-MS grade water (1,15333.1000) and acetonitrile (1,00029,1000) from Merck (Darmstadt, Germany). Milk samples Raw milk was collected from the Teagasc Moorepark dairy farm (Fermoy, Co. Cork, Ireland) from cows genetically profiled as A1 / A1, defatted, and pasteurized using a Microthermics unit (UHT / HTST electric model 25HV hybrid, Liquid Technologies, Wexford, Ireland) heated to 85°C and held for 23 seconds, followed by homogenization [GEA Ñire Soavi SpA Type: NS2006H (non-aseptic)] using two-stage homogenization at a total homogenization pressure of 2500 psi. The sample was spray-dried to produce a skimmed milk powder sample designated SMP A1|A1. SMP A1 / A2 and A2 / A2 were acquired commercially available from Europe and the USA, buffalo SMP was acquired from India, and pooled human donor pasteurized maternal milk (991-01-P) was acquired from Lee Biosolutiens (Maryland Heights, MO USA). The different batches, whether from the same or different manufacturers, are numbered A, B, C, etc. Terminated and canned products of whey protein Commercially available powdered infant formulas suitable for infants (0-12 months) and children (1 year and older) were obtained from different manufacturers and for different age groups. The different samples are denoted either IF14F22 (see Table 1) or IFa-IF1 in the experiments. In this regard, it should be noted that IFa-IF1, IFb-IF2, IFc-IF3, IFd-IF4, IFe-IF5, IFMF6, and IFg-IF7. Table 1 ID Type Stage % of tip % of homemade* % of β-eaeein** IF1 A2 1 10.2 35 1.19 IF2 A2 2 15.3 60 3.06 IF3 A2 3 13.9 ¡60 278 IF4 M 4 17.0 60 3.40 IF5 A2 1 10.4 30 1.04 ÍF6 A2 2 15.0 50 2.50 ¡FZ ¡A2 3 15.0 60 3.00 ÍFS ÍA1 / Á2 1 10.6 40 1.41 IF9 A1 / Á2 2 14.9 40® 199 ÍF1O A1 / A2 3 15.3 70® 3.57 iFlI A1 / A2 3 22.2 78 5.77 !F12 A1 / A2 1 11.Q 40 147 IF13 AÍ / A2 1 10.2 35 1.25 IF14 AVA2 2 15.3 60 3.02 IF15 Α1.Ά2 3 15.0 60 3.07 IF1S AVA2 4 ito 60 3.48 ΡΓ7 A2 1 9.9 30 1.01 IF18 A2 2 10.4 30 1.03 IF19 A2 3 12.8 50 1.94 IF20 A2 1 10.1 35 1 32 IF21 A2 2 11.7 60 2.41 IF22 A2 3 15.2 60 3.12 * as indicated &n te etiquete (except in §, which are based on assumptions), ~ esteteada ai Multiply the protein content by the casein content and divide by 3 (not indicated in Swaisyecd, 1995) Three different commercially available batches (denoted A, B, and C) of WPG were tested. WPC35 is a whey protein concentrate with 35% w / w protein. Two different commercially available batches of WPC28 (denoted A and B) were tested. WPC28 is demineralized WPG with 28% w / w protein. WPC8Q is a whey protein concentrate available with 80% w / w protein that is enriched with alpha-lactalbumin. Sample preparation The powders were dispersed at 3.5% (w / v) protein in water using a volumetric flask, shaken for at least 30 min at room temperature, denatured with 4 volumes of denaturing regulator (guanidine HCI 7.5 M, trisodium citrate 6.25 mM, DTT 23 mM), incubated at room temperature for 30 min and cleared by centrifugation at 1600 g for 10 min. Trypsin digestion of intact proteins Sample powders were dispersed at 3.5% (w / v) protein in 50 mM Tris + 6 M urea by mixing on an orbital shaker for one hour. The 200 µl sample was diluted with 1000 µl of 100 mM ammonium bicarbonate and vortexed. 10 µl of DTT (45 mM) was added to 0 µl of the diluted solution and incubated at 60°C for 30 minutes. The sample was then cooled to room temperature before rapid centrifugation and the addition of 10 µl of 5-iodoacetamide (100 mM). The sample was then incubated for 30 minutes at room temperature in the dark before adding 6 µl of trypsin (0.2 pg / µL) and incubating overnight at 37 °C. Tryptic digestion was stopped by adding 6 µl of 10% formic acid. The digested sample was centrifuged at 14,000 g for 10 minutes before transferring the liquid phase to a bottle for injection and determination by LC-MS. Analysis using LC-MS The cleared samples were analyzed by LC-MS based on existing methods (Bonfatti et al., 2008, Frederiksén et al., 2011). For intact protein analysis, samples were separated on a C4 column (Acquity UPLC Prctein BEH C4. 3QOÁ, 1.7 ym, 2.1 mm x 150 mm) using the following 1S gradient at 0.5 ml / min (Table 2): Tabia 2 Time (mine) Ϊ0 4 7 22 Ϊ23 Í24 i 25 ¡30% off» 8 i 15 35 42.5 52.5 |® |80 hs |l5 Regatta? A: Added riftwoaeéticQ (TFA) at 0.1% in water, regulator &: TEA at 0.1% in 90:10 of acetate:water The MS signal was recorded from 4 to 20 min in Fuel MS mode on a Thermo Q-Exactive HF (heater temperature 100°C, cover gas: 53, auxiliary gas: 14, sweep gas: 3, spray voltage: 3.5 kV, capillary temperature 320°C, RF level of the latent S: 70%, mass range: 400 to 2000 m / z, resolution: 240,000, ACC target: 1e0). IT maximum: 200 ms). For peptide analysis, i.e., triplicate digestion or GluC digestion, the peptides were separated on a C18 column (Acguity UPLG BEH C18, 130 A, 1.7 gm, 1.0 x 150 mm) using the following gradient at 75 μI / min (Table 3): Table 3 Time (mine) 0 30 hi 33 35 |45 Regulator speed 8 2 60 1100 100 2 ¡ 2 The MS signal was recorded from 3.5 to 35 min in PPM mode on a Thermo QExactive HF (heater temperature: 30 “G, cover gas: 8, aux gas: 0, sweep gas: 0, spray voltage: 3.6 kV, capillary temperature: 320 *C, S lens RF level: 55, default load: 2, MS2 resolution: 30Ό00, AGC target: 1e5, maximum IT: 100 ms, isolation window: 1.5 m / Z, isolation deviation: 0.5 m / z). The inclusion list was as shown in Table 4: Table 4 Name Mass [fn / z] Formula [M] Sec. with mim of idem. A1N 884.95449 AQTqSLWPFPGPíHN 3 A2N 864.95141 AQTQSLVYPFPGPIPN 4 Toi2 415.72960 AypypqR 5 AIS 755.06013 IHPFAQTQSL VYPFPGR HN 6 A1T 1072.17772 iHPFAQTQSLVYprpGpíHNSLPGNtePLTQIPVWPPFLQPEVMWSK 7 A2S 1112.08349 1HPFAQTQSLVYPFPGPIPNSLPQNIPPLTaTPVWPPFLQPEVMGVSK 9 Tetl 390T5254 VLPVPQK 5 Data Deconvolution The raw data files were deconvolved using Thermo BioPharma Finder 1.0 software using the Xtract algorithm (S / N threshold: 3, relative abundance threshold: 1%, adjustment factor: 80%, remaining threshold: 25%, overlaps, charge states: § to 50, minimum intensity: 1, expected intensity error: 3, m / z: 600 to 2000, minimum number of charge states detected: 3) and sliding windows (time - 5 to 20 min, target average spectrum width: 0.1 min, target average spectrum deviation: 50%, fusion tolerance: 1.5 Da, maximum RT gap: 0.5 min, minimum number of intervals detected: 3, XIC). The monoisotopic masses, total signal intensities, and apex RTs were exported as osv files and used for the following stages.Limit of detection experiments were also performed by deconvolving the raw data files using a fixed window centered around the β-casein peak. The parameters were as follows: S / N threshold: 1, relative abundance threshold: 1%, adjustment factor: 25%, remaining threshold: 25%, overlaps, charge states: 12 to 30, minimum intensity: 1, expected intensity error: 3, m / z: 800 to 2000, minimum number of charge states detected: 3, time: 11 to 13.5 min, relative intensity threshold: 1%. The monoisotopic masses and the total signal intensities were used for the following steps. Proteoform attribution: The deconvolved monoisotopic masses were compared with a protein database containing the major components of milk proteins (osi-CN). <iS2~CN, β-CN, k-CN, y-CN, a-lactoalbúmina, β-lactoglobulina, CGMP y proteosa peptonas usando un software desarrollado por Nestié (Proteín Ánalyzer).With. 5. The combinatorial addition of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation, and pyroglutamic acid) was tested to identify most of the signals. Misattributed signals were corrected by manual verification. Total signal intensities were extracted for the relevant proteins or proteoforms (i.e., total β-casein, lactosilael states of β-casein, β-casein genetic variants, etc.). For clarity, only the β-casein genetic variants I1, A2, and B are detailed in the Figures. β-casein I / H2 co-elutes with β-casein and is fused with β-casein A2. Both variants have a prefin at position 67 and belong to the A2 type. 10 15 Solid-state protein glycation The solid-state glycation experiment was based on Fenaille, 2003 (Fenaille et al, 2003). Briefly, 45 g of milk powder was incubated in a dipotassium carbonate-saturated chamber for 8–10 days to reach 5.4% moisture (initial % was 3.8%). Nine 2.1 g aliquots of the humidified powder were incubated in 25 mL glass tubes at 60 °C in an oven for 45 min to 24 h. Glycation was stopped by transferring the tubes to ice for 5 min. Then, 20 g of water was added to obtain a 3.5% (w / v) protein solution, which was mixed at room temperature for 1 h 30 min in a roller mixer. The solutions were stored at 4 °C overnight until the end of the experiment and warmed for 2 h 20–25 min before analysis. The samples were prepared as described above except that the incubation step was 10 min at 60 °C in a thermomixer (650 rpm). MS chromatograms were recorded on a Thermo Qrbitrap Elite (heater temperature: BO X, cover gas).20, auxiliary gas: 5, sweep gas: 0, spray voltage: 3.8 kVs, capillary temperature 320 aCt, lens RF level S: 60%, mass range: 400 to 2000 m / z, resolution: 240,000, objective AGC: 1 e6). Quantification of O-casein A sample of skimmed milk powder (SMP) was injected several times throughout each analytical series to normalize the signals in the experiments. An external calibration curve was established using a β-casein standard adjusted for protein purity (see text). The β-casein signal intensities were normalized to the average β-casein signal from the SMP samples (corrected for glycation, see above). The calibration curve was forced to 0 (y ~ 0.0524¾ where x is expressed in pg of injected β-casein and y is dimensionless). For each sample, the β-casein intensity values ​​were first corrected for glycation and normalized to the average β-casein signal from the SMP samples (corrected for glycation). The amount of injected β-casein was then calculated using the external calibration curve. This calculation can be performed for total β-casein, for a given variant, e.g., β-casein A1, or for a set of variants (the results for β-casein A2 also include the related variant VH2). The performance of the method was evaluated by two operators on seven samples of infant formula (stages 1-4, manufactured using skimmed milk powder of type A2) using triplicate injections on five different days. Example 1 - LC-MS method, data output, deconvolution and visualization To analyze intact proteins, infant formula and skim milk powder samples were dispersed in water at 3.5% (v / w) protein, denatured in 6 M guanidine, reduced with DTT, and clarified by centrifugation. Intact proteins were separated by UPLC on a C4 column using a water / ACN gradient with 0.1% TFA and analyzed by High-resolution mass spectrometry (Figure 3A). The chromatograms are sequentially deconvolved using sliding windows and the Xtract algorithm to obtain the monoisotopic masses of the proteins (Figure 3B). Finally, the proteoforms are visualized in bubble plots, with their retention time on the X-axis, the monoisotopic size on the Y-axis, and the signal intensity as the bubble area (Figure 3C). Example 2 - Global fingerprints Figure 4A illustrates the result of the method with raw cow's milk and highlights where the main milk proteins are detected (gSH, oS2-, β-, κ- and γ-casein, α-lac, and β-lg). Zooming in on the β-casein region (Figure 4B) allows for a detailed analysis of the various proteoforms of picasels, including genetic variants and protein modification (process-induced lactosylated adducts are detectable in all skimmed milk powder samples).The mass accuracy is shown in Table 5 for the raw milk sample. A difference of 1 Da may be due to a misattributed monoisotopic signal propagating along the deconvolution, since a melting tolerance of 1.5 Da is applied between the sliding windows to avoid splitting the signals into two masses separated by 1 Da. The method can be used to profile a wide variety of raw materials and finished products (Figure 5). Interestingly, this method readily detects casein glycomacropeptide (CGMP), the C-terminal portion of K-casein released by the rennet enzyme during the cheesemaking process, a polypeptide notoriously difficult to detect by gel staining or UV profiling. Table 5: Mass accuracy in the raw jackfruit milk sample. esperada detectada Amasa eéppm β-caseína Al (5P) 24008.16 24007.00 -1.16 -48.51 β-caseína Α2 (5P) 23963.16 23967,10 -1.06 -44.09 aSI-caseína Í8P} 23600.21 23599.64 -0.57 24.18 aS2 -caseína (IIP) 25212.98 ¿5212.12 -0.86 -34.17 e-íac 14176,81 14176.67 006 4.44 18355,46 18355.54 0.08 4.09 β-lgS 18269.42: 18269.49 0.07 3.78 κ-casein A 19025.53 19025.62 0.09 4.85 κ-casein B 18993.58 18993.73 0.15 7.86 γ2-casein 1161623 11816.25 0.00 -0.42 γS-casein 11551.10 11551.13 0.03 2.60 Example 3 - Detection of β-oasein in children of β-casein A2 If infant formulas of β-casein A2 are enriched with SMP Al |Al at 5% of β-casein A1 (Sg of β-casein Al in 100 g of total β-casein).: At this concentration, the signal of β-casein A1 is detected in all houses (Figure 6). Item 4 - Cuantlficación v alicacion de proteínas Protein quantization requires not only a suitable standard for establishing a calibration curve, but also that different proteoforms exhibit similar signal responses. This is necessary for applying the calibration curve to unknown samples, since the standard and samples are likely to have different proteoform distributions. Alternatively, the method must provide a way to correct for proteoform distribution. To explore whether protein lactosylation influences MS signal intensity, glycation was induced in a skimmed milk powder sample by heating as described in the Materials and Methods section (Figure 7A). The increase in glycated β-casein (Figure 7B) was accompanied by a decrease in the total β-casein signal (Figure 7C). The plot of the total β-casein signal against the percentage of non-glycated β-casein signal followed a linear relationship (Figure 7D) that became a signal reduction in times (Figure 7E) of the form y - 1 / (ax+b), with a and b corresponding to 0.878 and 0.122, respectively. For β-casein, a glycation correction factor (GCF) can therefore be calculated from the percentage of β-casein n© glycated (^UGíj) using Equation 1. 878 x + 0422 (Equation 1) Multiplying the β-casein signal measured by the GCF gives the corrected β-casein signal that corresponds to the expected signal if all the β-casein were non-glycated (Equation 2). (Equation 2) Example 5 - Quantification of β-casein Quantification was assessed using the sliding windows algorithm. 20 An external calibration curve was established using commercially purified β-casein. This standard also contained qSI-casein, κ-casein, and several unidentified peptides and protein fragments, most of which matched β-casein cleavage products (Figure 8A). The fraction of the total signal attributable to intact β-casein in the seven most concentrated calibration samples reached 85.6 ± 0.4%, and the concentration of the standard β-casein was adjusted with this purity factor. For each concentration, the total β-casein signal was normalized to that of the average signal (and corrected for glycation) from the SMP samples injected in the same run (and in all subsequent quantification experiments to account for variations in MS performance), and the normalized values ​​were plotted against the amount of β-casein loaded onto the column.The calibration curve (Figure 88) showed good linearity (R2~ 0.996) with a random distribution of the residuals. The amount of β-casein injected into the column (in pg) can be calculated from this calibration curve using Equation 3;. 10-®^= fEcuaetóna» where is the corrected signal for β-casein in the sample (Equation 2) divided by the average of the corrected signals for the skimmed milk powder samples injected in the same series (Equation 4), Mcaswr.sana “ -- (Ecuactén.4). jW The quantification of β-casein variants in a dairy matrix was further evaluated by mixing skimmed milk powder containing only variant A1 with skimmed milk powder containing only type A2 (Figure 8C). The response for β-casein A2 showed good linearity (R2 - 0.996), indicating that the effect of the matrix was negligible. The measured and expected A1 / A2 ratios showed a very strong linear correlation (%A2^ ” 1.05 * %A2®φ, R2= 0.996). This means that the response factors of both variants are very close and suggests that the calibration curve is also suitable for quantifying individual genetic variants. The quantification of β-casein in the two BMP samples gave an average value of 28.3 g of β-casein / 100 g of protein, in line with the value of 27% described in Handbook of Milk Composition (Swaisgood, 1995) (Table 6). Table 6: Quantification of β-casein MS Signal ¡A2iA2 Al ¡Al SMP(n=3) norms to SMP A2|A2 Al JAI pg of β-casein in column A2iA2 AliAl Repl ¡WW6O 311884671 336558582 Rep2 :374319489 319123478 336538582 Average ¡382138173 315504074 336558582 1.18 0 93 1Λ1 0.95 1.14 0.94 22.1 17.7 21.2 18.1 217 17.9 pg of protein in the column β-casein content 79 0 70.0 31 % 26% Example 6 - Method performance (quantification of β-casein) The performance of the method was evaluated in seven infant formulas containing only β-casein L2. Each sample was prepared five different times and analyzed by injection in triplicate. The measured amount of β-casein was generally in line (83%-136%) with the theoretical values ​​calculated by multiplying the protein content by the casein content and by 33%, which represents the generally accepted proportion of β-casein in total casein (Swaísgood, 1995) (Figure 9A). The method was further tested on a range of other commercially available infant formulas (either A2-based or manufactured with standard SMP) and was shown to be generally in line with theoretical values ​​(Figure 9B, whey % is based on assumptions for shaded values). The differences between measured and theoretical β-casein content were greatest for the samples with the lowest β-casein content. This could be due to the fact that less abundant proteoforms are too close to noise to be measured effectively, particularly in samples with multiple genetic variants such as IF8 and IF13. Example 7 - Detection of specific Al proteose peptones in whey protein concentrate by intact protein analysis The six different batches of whey protein concentrates were analyzed using intact protein analysis as described in the methods and materials section using LC-HRMS. Raw material analyzed: • Three batches of WPC35 • Two batches of WPC28 • One batch of WPC80 (enriched with oHac) Figure 10 illustrates the separate analyses of the six different whey protein concentrates. None of the tested WPCs showed traces of β-casein as no signals were detected within the circle with the continuous perimeter (i.e., the circle in the 20 upper right corner). Conversely, all the WPCs tested showed signals that corresponded to the proteose peptones detected as signals within the circle with the discontinuous perimeter (i.e., the circles in the inner left corner). Furthermore, the analysis showed that all ingredients contained CGMP 2S (casein glycomacropeptide), a peptide derived from casein, is identified in the lower left corner of each diagram, just below the circle with the dashed outline. Figure 11 is a close-up of the area within the circle with the dashed perimeter shown in Figure 10. This close-up demonstrates that signals corresponding to the expected masses for a wide variety of proteose peptones were found in all ingredients. Black represents proteose peptones derived from β-caselin A2, gray represents proteose peptones derived from β-casein A1, and white represents unassigned compounds. Some of these peptones are: PP5 A1 1-105, PP5 A1 1-107, PP5 A2 110 105, PP5 A2 1-107, bcasl P A1 29-105 (i.e. PP8s Al 29-105), bcasl P Al 29-107 (i.e. PP8s Al 29-107), bcasl p A2 29-105 (i.e. PP8s A2 29-105) and bcasl P A2 29107 (PPSs A2 29-107). Gamma-casein was not detected in the tested WPG. WPC80 did not contain slow PP8 (29-105 / 7). This was probably due to the 15th ultrafiltration stage in the WPC80 preparation process, which likely removed slow PP8. In Figure 10 and Figure 11, several signals (shown in white) inside and outside the circle with the dashed perimeter in Figure 10 relate to the remaining peptide identification, Consequently, proteose peptones of β-casein Al (and A2) in all the tested whey raw material. Therefore, they will be present in the finished products and are very likely responsible for the high Al signal detected in all the tested finished products. Example 8 - Detection of Al-specific proteose peptones in finished products by intact protein analysis Different finished products, including various infant formulas from different brands, were analyzed for the detection of specific proteose peptones. All infant formulas are considered A2 finished products. The finished products were dissolved and analyzed by LC-HRMS as described in the materials and methods section above. Figure 12 shows that proteose peptones were found in all tested batches although the proteose peptone profile differed between the finished products. Proteopeptones derived from p-casein A1 are found in all tested infant formulas even though they are based on milk with β-casein A2, Example 9 - Quantification of proteose peptones specific to A1 and A2 by The relative quantification of PP5 Al and PP6 A2 in a dairy matrix was evaluated by mixing a skimmed milk powder containing only the Al variant with a skimmed milk powder containing only the A2 type (Figure 13). The responses for PP5 A2 and PP5 A1 showed a good dose response with quadratic regression (R2 ~ 0.999 and R2 = 0.99S, respectively), indicating that the effect of the The 2Q matrix was negligible. This means that the response factors of both variants are very close and suggests that the 1a calibration curve is also suitable for quantifying individual genetic variants. In conclusion, it is shown that specific proteose peptones can be quantified using intact protein analysis even in a matrix. Example 10 ~ Detection of A1-specific proteose peptones in finished products by tryptic digestion The different finished products are standard skimmed milk powder (i.e., standard SMP), skimmed milk powder containing only the A2 version of β-casein, or different infant formulas of different brands and for different age groups. All infant formulas are considered A2 finished products. The finished products were dispersed at 3.5% (w / v) protein in 50 mM Tris + 6 M urea and mixed on an orbital shaker for one hour. 10,200 µL of sample was diluted with 1000 µL of 100 mM ammonium bicarbonate and vortexed. To 100 µL of the diluted solution, 0 µL of 45 mM DTT was added. The samples were incubated at 60°C for 30 minutes, cooled to room temperature, and rapidly centrifuged to collect evaporation droplets. 10 µL of 100 mM iodoacetamide (in 100 mM sodium bicarbonate) was added; the samples were incubated for 30 minutes at room temperature in the dark. 6 µL of 0.2 pg / µL trypsin was added, and the samples were incubated overnight at 37°C. 6 µL of 10% formic acid was added to stop digestion, and the solutions were centrifuged at 14,000 g for 10 minutes. The liquid phase was transferred to a flask for injection and analyzed by LC-MS as described in the Materials and Methods section above. The analysis focused on the detection of the AIS peptide, i.e., an Al-specific peptide that has a position as illustrated in Figure 14. As is evident from the position of the AIS peptide, it can be distinguished from the A2S peptide since it covers the area of ​​β-casein that includes amino acid 67. Figure 15 illustrates that AIS is not detected in the SMP A2 samples. AIS was detected in one SMP A2 sample, which was subsequently found to contain β⁰ casein I₁. However, the peptide is significantly expressed in standard BMP. Surprisingly, a strong signal for the AIS peptide was detected in all infant formulas tested. Similar results were obtained for the A1N (Figure 16) and A1T (Figure 17) peptides. As expected, the peptides A2S (Figure 18), A2N (Figure 19), A2T (Figure 20), Tot 1 (Figure 21) and Tot 2 (Figure 22) showed strong signals in all tested areas. Example 11 - Detection of specific Al oxides in concentrate of Ib whey protein through triple digestion Different batches of whey protein concentrates were analyzed together with skimmed milk powder, both skimmed milk powder and standard, which contained only the A2 version of β-casein. Raw materials (RM) analyzed: "Three totes of WPC35 • Two batches of WPC28 • One batch of WPC80 (enriched with a-lac) • Two different commercially available lactose ingredients The products were dissolved and subjected to triplicate digestion before being analyzed by LC-MS as described in the materials and methods section above. The analyses focused on the detection of peptides Tot1 and Tot2, i.e., common to β-casein for A1 and A2, peptide AIS, i.e., a peptide specific to A1, peptide A1N, i.e., a peptide specific to A1, peptide A1T, i.e., a peptide specific to A2, peptide A2N, i.e., a peptide specific to A2, peptide A2T, i.e., a peptide specific to A2, and peptide A2S, i.e., a peptide specific to A2. The position of the peptides is illustrated in Figure 14. Figures 23 and 24 illustrate the results obtained when determining the amount of peptide Tot1 and Tot2, respectively. It is a common peptide (A1 and A2) but also a peptide present only in intact β-casein and γ-casein. It is shown that the signal for the common β-casein peptide is severely reduced in all whey ingredients, and is absent in lactose ingredients but present in SMP and SMP A2, as would be expected. This indicates that very little intact β-caserin or γ-casein is present in the RM (~1%) compared to the SMP even when an equivalent protein content was used, as expected. Figure 25 illustrates the results obtained when determining the amount of peptide A1S. It shows that the signal for peptide A1S is stronger for SMP but absent for SMP A2, as expected. The lactose samples also showed no signal. For the whey protein samples, a surprising amount was determined, approximately one-quarter of the amount observed in SMP. Similar results were obtained when the samples were tested for the presence of two other A1-specific peptides, A1N and A1T (Figure 26 and Figure 27). Figure 28 illustrates the results obtained when determining the amount of A2S. It shows that the signal for the A2S peptide (specific to A2) is stronger for SMP A2 but weaker for SMP ia, as expected. The lactose samples showed no signal, also as expected. For the whey protein samples, a surprising amount of approximately one-quarter of the amount observed in SMP was determined. Similar results were obtained when the samples were tested for the presence of two other A2-specific peptides, A2N and A2T (Figures 29 and 30). Consequently, it can be concluded from the above results that in all the analyzed whey MRI samples, the signal intensity of the common β-casein peptide is severely reduced (~100x) compared to SMP. Also, the Al and A2-specific peptides are reduced by only -4-δχ compared to SMP. Therefore, there is a selective enrichment of the Al and A2-specific peptides. Therefore, using the tryptic digestion method and determining the different peptides, it would appear that whey ingredients still contain -20% β-casein fragments (compared to SMP), which may be at least partly proteose peptones known as PP5 and PP8s. Example 12 - Reduction of proteose peptones in whey protein fractions To obtain a reduced whey protein fraction, three batches of WPC35 in which the presence of proteose peptones was determined in Example 9, are subjected to gel filtration, The three batches of WPC35 are further purified by gel filtration on a Sephadex 0-75 column (550 x 22 mm) prepared and balanced with volatile NHsHCOs 04 M buffer (pH 8.0-8.5). The 0.5 g–0.7 g of each batch are dissolved in 5–7 ml of buffer (with the addition of a few ml of 1 M NaOH to neutralize the residual traces of TGA and aid dissolution) and applied to the column. The flow rate is set to 0.5 ml / min and 5 ml fractions are collected. The fractions are analyzed using the detection and quantification method described herein, i.e., LC-HRMS, in accordance with the Materials and Methods section. The proteose-free peptone fractions PP5 and PP8 are pooled to form a reduced whey protein fraction. References Bonfatti, V. etaí. JCñromaiogr A, 2008; 1195(1-2);101 -106. de Jong et al JChromatogr A, 1993; 652(1):207-213. EFSA Scfeni& Repo / t Scientífío Report of EFSÁ prepared by a DATEX 10 Working Group on trie potential health impact of b-casomorphins and related peptides. 20091231 ;1 -107. Fenaille, F. et al. Rapld Commun Mass Spectrom. 2003: 17(13):1483-1492. Fenailie, F, et al International Dairy Journal, 2006; 16(7):728-739. Feng; P. et al J AOÁO Int, 2017; 100(2):510-521. Frederíksen, PD et al. J Dairy Scí, 2011; 94(10):4787-4799. He, S. etal Eur. J. Clin. Rutr. 2014, 68, 994-1000. Karamoko, G, et al 8 / ofedmo / og / e, Agronomía, Société et Environnement, 2013; 17(2):373-382. Poulsen, N. A et al. Acta Agricultura® ScandínaWca, Seotion A—Animal Science, 2018; 66(4):190-198. Roberfroid MB. J Nuir. 2007; 137: 830S. Salminen S et al TrendFood Sci. Technol, 1999;1O 107-110. Swaísgood, Η. E. Handbook of Milk Con^osition. RG Jensen, ed. Academíe Press, San Diego 1995; 464-468. Swaisgoad, Déve / opmenfs í Dairy Chemístry. Fox (Ed), Proteins, vol. 1, 1982; 63-110. Valiejo-Cordoba, B. j Cap / fery Efectrophor, 1097; 4(5):219-224. Visser, S. el al 7 Cftramatogr A 1995; 711(1):141-150. Articles 1. A method for determining and / or quantifying proteose peptones derived from β-casein and / or β-casein, said method comprising the steps of (i) providing a dairy-based product for analysis; (ii) subject said product using liquid chromatography - mass spectrometry analysis; (uí) determine and / or quantify said proteose peptones derived from β-casein and / or β-casein in said product by detecting compounds of defined m / z values ​​or by deconvolving one or more mass spectrometry spectra to calculate monoisotopic masses, 1§ 2. B method in accordance with article 1, wherein said proteose peptones are proteose peptones derived from β-casein Ai. 3. The method according to article 2, where said proteose peptones derived from β-casein are fast PP8, slow PP8 and / or PP-5, such as proteose peptones derived from β-casein A1 are PP8 2P fast, PP8 slow and / or PP-5. 4. The method in accordance with any of the above articles, wherein said product is an infant formula or a whey protein fraction. 5. The method in accordance with any of the above articles, where the product is a powder, said powder is dissolved before heating said product. 6. The method in accordance with any of the above articles, wherein said product is analyzed by intact protein analysis. 7. The method in accordance with Article 6, wherein said mass spectrometry analysis is a high-resolution mass spectrometry analysis. 8. The method in accordance with any of Articles 6-7, wherein determining and / or measuring said proteose peptones derived from β-casein in said product is carried out by deconvolving one or more mass spectrometry spectra to calculate monoisotopic masses. 9. The method in accordance with Article 8, where said deconjecture is performed by means of a sliding window algorithm. 10. The method in accordance with any of Articles 1-5, wherein said product is analyzed by peptide analysis comprising an enzymatic digestion step of said product prior to analysis, 11. The method in accordance with Article 10, wherein said enzymatic digestion is carried out by tryptic digestion or digestion with GluC. 12. The method according to any of the articles T0Ί1, where determining and / or quantifying said proteose peptones derived from β~casein is carried out by detecting compounds of defined m / z values, 13. A method for producing a whey protein fraction having a reduced content of proteose peptones derived from 3-casein, said method comprising the steps of (i) providing a whey protein fraction; (II) determining and quantifying said β-casein-derived proteose peptones in said whey protein fraction as described in any of Articles 1-12; and (iii) selecting said whey protein fraction having at most 10% by weight of β-casein-derived proteose peptones based on the total protein in said whey protein fraction forming a selected whey protein fraction. 14. A method for producing a whey protein fraction having a reduced content of proteose peptones derived from β-casein, said method comprising the steps of (i) providing a whey protein fraction; (ü) reducing the proteose peptone content derived from β-casein in said whey protein fraction to a concentration of at most 10% by weight based on the total protein in the whey protein fraction forming a reduced whey protein fraction. 15. The method in accordance with Article 14 further comprising the step before and / or after step (ii) of determining and quantifying said proteose peptones derived from β-casein in said whey protein fraction as described in any of Articles 1-12. 2S 16. The method in accordance with any of Articles 14-15, wherein the proteose peptone content is reduced by gel filtration. 17. The method in accordance with any of Articles 13-16. wherein said selected or reduced whey protein fraction has a proteose peptone content derived from β-casein of at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, even more preferably at most 5.5% by weight, such as at most 5% by weight, with the highest preference at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as a maximum of 0.50% by weight, even with greater preference as a maximum of 0.25% by weight, such as as a maximum of 0.10% by weight, with the highest preference as a maximum of 0.05% by weight, such as as a maximum of 0.01% by weight, based on the total protein in the whey protein fraction. 18. The method in accordance with any of Articles 13-17, wherein said proteose peptones are fast PP8, slow PP8 and / or PP-5. 19. A method for producing a nutrient composition having a reduced content of proteose peptones derived from β-casein, said method comprises the steps of (i) providing a selected whey protein fraction or a reduced whey protein fraction as described in any of Articles 13-18; (ii) preparing said nutritional composition with a reduced proteose peptone content from said selected whey protein fractions, said reduced whey protein fractions, or a mixture thereof. 20. A whey protein fraction having a reduced proteose peptone content derived from β-casein, obtainable by the method according to any of Articles 14-18. 21. A nutritional composition comprising said whey protein fraction having a reduced proteose peptone content derived from β-casein according to Article 20 or obtainable by the method according to Article 19. 22.23. A nutritional composition in accordance with Article 21, wherein said nutritional composition is an infant formula. 24. A nutritional composition in accordance with any of Articles 20, 21, and 22, wherein said proteose peptone content is at most 9% by weight based on the total protein in the nutritional composition. 25. A nutritional composition in accordance with any of Articles 21 and 23 for use as a medicament. 26. An infant formula in accordance with any of Articles 22 and 23 for use in the treatment, prevention, and / or relief of pain. ©2 abdominal in an infant. 26. An infant formula is in accordance with any of Articles 22-23 for use in the treatment, prevention and / or improvement of lactose intolerance in an infant. 27. Use of infant formula in accordance with any of the articles 22-23 to improve stool consistency. List of sequences Seo, with no., of ident, 1 (amino acid sequence of β-casein Á2): RELELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDK1H PFAQTQSLVY PFPGPÍPNSL PQNÍPPLW PVWPPFLQP EVMGVSKVKE AMAPKHKEMP FPOPVEPFT ESQSLTL1W ENLHLPLPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRQMPIQAF LLYQEPVLGP VRGPPPIIV Ssc, With identity number, 2 (amino acid sequence of β-casein Al k RELEELNVPG ElVESLSSSE ESÍTRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVY PFPGPIHNSL PQNIPPLTQT PVWPPFLQP EVMGVSKVKE AMAPKHKEMP FPKYPVEPFT ESQSLTLTDV ENLHLPLPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGP VRGPPPIIV Dry. with no. of ident. 3 (amino acid sequence of A1N): AQTQSLVYPF PGPIHN Sea with number, of ident 4 (amino acid sequence of A2N): AQTQSLVYPF PGPIPN Dry. with no. of ident. 5 (amino acid sequence of Tot2): AVPYPQR Sequence, with identity number 6 (AIS amino acid sequence): IHPFAQTQSL VYPFPGPIHN Seo, with ident number 7 (amino acid sequence of AID: IHPFAQTQSL VYPFPGPIHN SLPQNIPPLT QTPVWPPFL QPEVMGVSK Sequence number 8 of ident (A2S amino acid sequence): IHPFAQTQSL VYPFPGPIPN Seo with ident number 9 (amino acid sequence of A2TX) IHPFAQTQSL VYPFPGRPN SLPQNIPPLT QTPVWPPFL QPEVMGVSK Seo, with ident number 10 (Toti amino acid sequence): VLPVPQK

Claims

1. A method for determining and / or quantifying proteose peptones derived from β-casein and / or β-casein, the method comprising the steps of: (i) providing a dairy-based product for analysis; (ii) subjecting the product to liquid chromatography-mass spectrometry analysis; (iii) determining and / or quantifying the proteose peptones derived from β-casein and / or β-casein in the product by detecting compounds of defined m / z values ​​or by deconvolving one or more mass spectra to calculate monoisotopic masses.

2. The method according to claim 1, characterized in that the proteose peptones derived from β-casein are fast PP8, slow PP8 and / or PP-5, such as the proteose peptones derived from β-casein A1 are fast PP8, slow PP8 and / or PP-5, 15 3. The method according to any of the preceding claims, characterized in that the product is analyzed by intact protein analysis, 4. The method according to any of claims 1-2, characterized in that the product is analyzed by peptide analysis comprising 2b an enzymatic digestion step of the product prior to analysis.

5. A method for producing a whey protein fraction having a reduced content of β-casein-derived proteose peptones, the method comprising the steps of (i) providing a whey protein fraction; (ii) determining and quantifying the β-casein-derived proteose peptones in the whey protein fraction as described in any one of claims 1-4; and (iii) selecting the whey protein fraction having at most 10 wt% of β-casein-derived proteose peptones based on the total protein in the whey protein fraction forming a selected whey protein fraction.

6. A method for producing a whey protein fraction having a reduced content of proteose peptones derived from β-casein. The method comprises the steps of (i) providing a whey protein fraction; (ii) reducing the content of proteose peptones derived from β-casein in the whey protein fraction to a concentration of at most 10% by weight based on the total protein in the whey protein fraction, forming a reduced whey protein fraction.

7. The method according to any of claim 6, characterized in that the proteose peptone content is reduced by filtration of gel. 20 8. A method for producing a nutritional composition having a reduced content of proteose peptones derived from β-casein, the method comprising the steps of (i) providing a selected whey protein fraction or a reduced whey protein fraction as described in any one of claims 566; (ii) preparing the nutritional composition with a reduced proteose peptone content from the selected whey protein fractions, the reduced whey protein fractions, or a mixture thereof.

9. A whey protein fraction having a reduced content of proteose peptones derived from β-casein, obtainable by the method according to any of claims 5-7.

10. A nutritional composition comprising the whey protein fraction having a reduced content of proteose peptones derived from β-casein according to claim 9 or obtainable by the method according to claim 8, 11. The nutritional composition according to claim 10, characterized in that the nutritional composition is an infant formula. 15 12. A nutritional composition according to any of claims 10-11 for use as a medicament.

13. An infant formula according to any of claim 11 for use in the treatment, prevention and / or improvement of abdominal pain in an infant, and / or the improvement of bowel comfort in the infant, 20 14. An infant formula according to any of claim 11 for use in the treatment, prevention and / or improvement of lactose intolerance in an infant.

15. Use of infant formula according to any of claims 11 to improve stool consistency. Abstract The present invention relates to a method for determining and quantifying proteose peptones derived from β-casein and / or β-casein, said method comprising the steps of (I) providing a dairy product for analysis; (II) subjecting said product to liquid chromatography-mass spectrometry analysis; (III) determining and / or quantifying said proteose peptones derived from β-casein and / or β-casein in said product by detecting compounds of defined m / z values ​​or by deconstructing the mass spectrometry spectra to calculate monoisotopic W masses. The present invention also relates to nutriclonal compositions having a reduced content of proteose peptones derived from β-casein and the uses thereof for, e.g. e.g., to treat, prevent and / or improve abdominal pain in an infant.