RU2820927C2 - Detection and quantification of content of proteose peptones and/or content of beta-casein and nutritional composition with reduced content of beta-casein-derived proteose peptones - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for producing a milk whey protein fraction and a nutritional composition having a low content of proteose peptones. Method involves obtaining a fraction of milk whey protein, detection and quantitative determination of obtained from β-casein of proteose peptones in said fraction and selection of said fraction of milk whey protein containing not more than 10% by weight obtained from β-casein of proteosepeptones from the total amount of protein in said fraction. Detection and quantitative determination of said β-casein of proteose peptones in said fraction of milk whey protein includes the following stages: obtaining a milk-based product to be analysed, performing analysis of said product by liquid chromatography and mass spectrometry, detecting and/or quantifying said products obtained from β-casein proteosepeptones and/or β-casein in said product by detecting compounds with given mass/charge values m/z or deconvolution of one or more mass spectra to calculate monoisotopic masses. Alternatively, a method for producing a milk whey protein fraction having a reduced content obtained from β-casein of proteose peptones, involves obtaining a fraction of milk whey protein and reduction of content obtained from β-casein of proteose peptones by gel filtration in said fraction of milk whey protein to concentration of at most 10% by weight of the total amount of protein in the fraction of milk whey protein. Method of producing a nutritional composition involves adding a fraction of milk whey protein having a reduced content of obtained from β-casein of proteosepeptones, obtained according to the above methods.

EFFECT: invention enables to obtain nutritional compositions with low content of proteose peptones, which can be used to prevent and/or relieve abdominal pain in an infant.

3 cl, 30 dwg, 6 tbl, 12 ex

Description

The present invention relates to a method for the detection and quantification of β-casein-derived proteose peptones, such as PP-5, PP8s and PP8f, and/or β-casein. The present invention also relates to a nutritional composition and formula for newborns with a reduced content of β-casein-derived proteose peptones, such as PP-5, PP8s and PP8f.

Prerequisites for creating the invention

Milk from dairy cows is considered an ideal naturally occurring food product, providing an important source of nutrients including high-quality proteins, carbohydrates and several micronutrients. Milk proteins are predominantly breech proteins, and the remaining proteins belong to the group of whey proteins, the main representatives of which are beta-lactoglobulin and alpha-lactalbumin. Among caseins, β-casein is the second most abundant protein in cow's milk and has an excellent nutritional amino acid balance. However, various mutations in the bovine β-casein gene have resulted in several genetic variants, the most common being the so-called A1 and A2 variants.

The A1 and A2 β-casein variants differ in the position of amino acid 67 of the secreted β-casein protein, that is, as a result of a single nucleotide difference, the A1 β-casein variant contains histidine (His), and the A2 β-casein variant contains proline (Pro). This is illustrated in FIG. 1.

The A2 variant of β-casein is more similar to human β-casein in terms of intestinal breakdown. The A1 β-casein variant has been suggested to be associated with cow's milk intolerance, and a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 diet but not the A2 diet (Ho et al, 2014).

The difference in segregation observed between variants A1 and A2 may be due to a specific polymorphism, as described above. The polymorphism results in a key conformational change in the secondary structure of the β-casein protein. Gastrointestinal proteolytic cleavage of the A1 (but not A2) variant of β-casein results in the formation of β-casomorphin 7 (BCM-7), which is an exogenous opioid peptide (exorphin) that can activate opioid receptors in the body (EFSA Scientific Report, 2009). This is illustrated in FIG. 2.

Initially, all cows produced milk containing only the A2 variant of β-casein, but genetic variation resulted in mixed herds. Thus, cow herds commonly used for milk production typically produce a mixture of A1 and A2 variants of β-casein, but with different amounts of A1 compared to A2.

It has been shown that proteolysis of β-casein by plasmin leads to the formation of γ-caseins and protease peptones (fast PP8, slow PP8 and PP-5). Proteose peptones are not precipitated by caseins during acidification or enzymatic treatment with rennet and remain in the whey fraction (see Karamoko et al. 2013). Slow PP8s and PP-5 correspond to sequences 29-105/7 and 1-105/7, respectively, and both have position 67. Thus, whey products derived from milk containing A1 β-casein are likely to contain type A1 protease peptones . Proteose peptones are derived from the same gene and they arise through endogenous events, so they are also considered proteoforms of β-casein.

Native protein assays using reversed-phase high-performance liquid chromatography (RP-HPLC) or capillary electrophoresis (CEP) coupled with UV detection can efficiently separate most β-casein variants in raw milk samples (see, e.g., de Jong et al ., 1993, Visser et al., 1995, Bonfatti et al., 2008, Poulsen et al., 2016). However, in manufactured products, process conditions induce reactions between proteins and reducing sugars (Maillard reaction), resulting in the production of multiple proteoforms containing one or more sugar adducts (Fenaille et al., 2006). As a consequence, protein peaks split and expand, creating overlapping regions that do not allow analysis of individual proteoforms by UV detection (Vallejo-Cordoba, 1997, Fengetal., 2017).

Human breast milk (HBM) is known to contain the A2 form of human beta-casein. By definition, it represents the gold standard for infant nutrition. Thus, there is a need to provide (synthetic) nutritional compositions that optimally mimic the composition of HBM.

Due to the differences observed in humans in the degradation of the A1 and A2 variants of bovine β-casein, it would be preferable to be able to detect and quantify the content of β-casein and its proteoforms in a simple and efficient manner. There is currently no simple and effective way to characterize and quantify individual milk proteins and their proteoforms, such as proteose peptones, in finished products.

In addition, there is a need to provide a nutritional composition that contains a relatively low amount or proportion of β-casein A1 or β-casein-derived proteose peptones, or which may not contain any of its forms.

There is also a need to provide a nutritional composition that contains whey fractions that do not contain or have a reduced amount of β-casein-derived proteose peptones.

Summary of the invention

The present invention relates to an improved method for the efficient and simple detection of β-casein and its proteoforms, in particular its β-casein-derived proteose peptones, as well as a method for reducing the content of β-casein-derived proteose peptones.

It is a further object of the present invention to provide a nutritional composition, such as an infant formula, that can prevent abdominal pain and/or improve abnormal stool consistency in an infant.

Thus, one aspect of the invention relates to a method for the detection and quantitation of β-casein-derived protease peptones and/or β-casein, the method comprising the following steps:

(i) obtaining the milk-based product to be analyzed;

(ii) performing analysis of said product by liquid chromatography and mass spectrometry;

(iii) detecting and/or quantifying said β-casein-derived protease peptones and/or β-casein in said product by detecting compounds with target mass/charge values or deconvolving one or more mass 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 β-casein-derived proteose peptones, said method comprising the following steps

(i) obtaining a whey protein fraction;

(ii) detecting and quantifying said β-casein-derived protease peptones in said whey protein fraction as described herein; And

(iii) selecting said whey protein fraction containing no more than 10% by weight of β-casein-derived proteose peptones of the total amount of protein in said whey protein fraction, to form the 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 β-casein-derived proteose peptones, the method comprising the following steps

(i) obtaining a whey protein fraction;

(ii) reducing the content of β-casein-derived proteose peptones in said whey protein fraction to a concentration of not more than 10% by weight of the total protein content in the whey protein fraction to form a depleted whey protein fraction.

A fourth aspect of the invention relates to a method for producing a nutritional composition having a reduced content of β-casein-derived protease peptones, the method comprising the following steps

(i) obtaining a selected whey protein fraction or a depleted whey protein fraction as described herein;

(ii) producing said nutritional composition with a reduced content of β-casein-derived proteose peptones from said selected whey protein fractions, said depleted whey protein fractions, or a mixture thereof.

A fifth aspect of the invention relates to a whey protein fraction having a reduced content of β-casein-derived proteose peptones obtained by the method described herein.

A sixth aspect of the invention relates to a nutritional composition comprising said whey protein fraction having a reduced content of β-casein-derived proteose peptones prepared by the method described herein.

A seventh aspect of the invention relates to the nutritional composition described herein for use as a medicine.

An eighth aspect of the invention relates to an infant formula described herein for use in treating, preventing and/or relieving abdominal pain in an infant, and/or reducing bowel discomfort in said infant.

A ninth aspect of the invention relates to an infant formula described herein for use in the treatment, prevention and/or relief of lactose intolerance in an infant.

A tenth aspect of the invention relates to the use of the infant formula described herein to improve stool consistency.

Brief description of graphic materials

In fig. 1 shows a partial sequence alignment between the sequence of beta-casein A2 and beta-casein A1, belonging to amino acid sequences 59-71. The amino acid at position 67 is highlighted to demonstrate the difference between A1 beta-casein and A2 beta-casein in one amino acid being His and Pro, respectively;

in fig. 2 shows a partial sequence alignment between the sequence of beta-casein A2 and beta-casein A1 belonging to amino acid sequences 59-71. The difference in one amino acid between the two beta-caseins affects the ability to cleave the two chains, since A2 beta-casein is not cleaved due to the presence of Pro at position 67, while A1 beta-casein is cleaved on the N-terminal His side obtaining the possibility of forming a sequence seven amino acids long, i.e. beta-casomorphin-7 (BCM-7);

in fig. Figure 3 shows the steps of the LC-HRMS method: (a) sample preparation, (b) deconvolution of monoisotopic mass spectra for native β-casein, and (c) diagrammatic representation of the native region of β-casein as circles and arrows (circle sizes reflect signal intensities). ;

in fig. Figure 4 shows the overall fingerprint and native region of β-casein. Major milk proteins were separated by (a) the overall identification fingerprint of the raw milk sample. Various proteoforms (b) of native β-casein were detected. A1, A2 and B represent genetic variants, the designations in the superscript correspond to the number of lactose adducts (L);

in fig. 5 shows examples of a common fingerprint in samples of raw milk, human breast milk, infant formula (IF), skimmed milk powder (SMP), and whey;

in fig. Figure 6 shows the circle and arrow diagrams (native β-casein region) of seven samples of A2 infant formula that were either untreated or supplemented with 5% A1 β-casein (5 g native A1 β-casein/100 g total native β-casein). casein);

in fig. Figure 7 shows how glycation affects the MS data. (a) The circles and arrows (native β-casein region) show increased lactosylation in the solid state glycation experiment. (b) The percentage of non-glycated signal (black bar) decreasing over time, (c) shows that the total β-casein signal decreases with increasing glycation and (d) has a linear relationship with the percentage of non-glycated signal. Plotting the dependence of signal reduction on the percentage of non-glycated signal (e) allowed us to calculate the glycation correction factor;

in fig. Figure 8 shows the characterization of standard β-casein and the construction of a β-casein calibration curve. Analysis of standard β-casein showed that about 15% of the total signal could not be attributed to native β-casein(a). A linear calibration curve was generated using the normalized (to SMP, which was corrected for glycation) MS signal of native β-casein and corrected amounts of β-casein (b). Cross-mixing experiments between skim milk samples A1|A1 and A2|A2 showed that the relationship remained linear even in the milk matrix (c). During the cross-mixing experiment, protein content was kept constant;

in fig. Figure 9 shows the quantification of native β-casein in infant formulas. β-casein was quantified in seven samples of infant formula (a), with each sample analyzed five times in triplicate. Theoretical values are based on formulation and overall milk protein composition. An additional set of infant formulas (both A2-based and skim milk powder containing multiple genetic variants) were analyzed in triplicate over one day (b). The shaded bars indicate the most likely guesses since the whey % of these samples was not listed on the boxes;

in fig. Figure 10 shows the result of LC-HRMS analysis of native proteins in various whey protein concentrates (WPC). The native protein region is indicated by a circle with a continuous perimeter, and the β-casein protease peptones are included in the region indicated by a circle with a broken perimeter;

in fig. 11 is a close-up view of the area highlighted in FIG. 10 circle with a broken perimeter, demonstrating the content of proteose peptones. Black indicates β-casein A2-derived proteose peptones, gray indicates β-casein A1-derived proteose peptones, and white indicates unidentified compounds;

in fig. 12 shows the result of LC-HRMS analysis of native proteins in various finished products, focusing on the region containing proteose peptones;

in fig. 13 shows the correlation between the measured PP5 signal (for both PP5 A1 and PP5 A2) versus the percentage of SMP A2 in the sample. During the cross-mixing experiment, protein content was kept constant;

in fig. 14 schematically shows native β-casein and the products formed by the cleavage of β-casein. Horizontal arrows indicate the position of tryptic peptides with the common peptides Tot1 and Tot2, as well as the specific peptides A1/2N, A1/2S and A1/2T. Tot1 and Tot2 are located at the C-terminus of β-casein, where the A1 and A2 sequences are identical and are absent in protease peptones. Peptides A1/2N, A1/2S and A1/2T include an amino acid at position 67, which is different between A1 and A2;

in fig. 15 shows LC-MS trypsin digestion of various finished products, including skim milk powder and several infant formulas, for A1S peptide;

in fig. 16 shows LC-MS trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the A1N peptide;

in fig. 17 shows the LC-MS result of the trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the A1T peptide;

in fig. 18 shows LC-MS trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the A2S peptide;

in fig. 19 shows LC-MS trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the A2N peptide;

in fig. 20 shows the LC-MS result of the trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the A2T peptide;

in fig. 21 shows the result of trypsin digestion of various finished products, including skim milk powder and several infant formulas, against the Tot1 peptide;

in fig. 22 shows the result of trypsin digestion of various finished products, including skim milk powder and several infant formulas, for the Tot2 peptide;

in fig. 23 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of Tot1 peptide by LC-MS;

in fig. 24 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of Tot2 peptide by LC-MS;

in fig. 25 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of A1S peptide by LC-MS;

in fig. 26 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of the A1N peptide by LC-MS;

in fig. 27 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of A1T peptide by LC-MS;

in fig. 28 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of A2S peptide by LC-MS;

in fig. 29 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of the A2N peptide by LC-MS;

in fig. 30 shows the result of trypsin digestion of skim milk powder, lactose and various whey protein concentrates (WPC) with measurement of A2T peptide by LC-MS.

The present invention will be described in more detail below.

Detailed Description of the Invention

Definitions

Before describing the present invention in more detail, the following terms and conventions will be defined.

The term "LC" means "liquid chromatography", which is known to one skilled in the art. It should be emphasized that LC also includes HPLC, that is, high-performance liquid chromatography, and UHPLC, that is, ultra-performance liquid chromatography.

The term "MS" means "mass spectrometry", which is known to one skilled in the art. It should be emphasized that MS also includes MSVR, i.e. high resolution mass spectrometry.

The term “compounds with target mass/charge values” means that the MS is configured to measure compounds that have only mass/charge values specifically defined by the user when measuring a given sample.

The term “A1/A1 cows” means cows with the homozygous A1A1 genotype. Milk obtained from A1/A1 cows is designated as “A1 milk”.

The term “A2/A2 cows” means cows with the homozygous A2A2 genotype. Milk obtained from A2/A2 cows is referred to as “A2 milk”.

The term “A1/A2 cows” means cows with the heterozygous A1A2 genotype. Milk obtained from A1/A2 cows is designated as “A1/A2 milk”.

The term "A1 whey" means whey obtained substantially from A1 milk.

The term "A2 whey" means whey obtained substantially from A2 milk.

The term “A1/A2 whey” means whey obtained from A1/A2 milk or from a mixture of A1 whey and A2 whey, or obtained from a mixture of A1 milk and A2 milk, A1 milk and A1/A2 milk, A2 milk and milk A1/A2 or milk A1, milk A2 and milk A1/A2.

The term “A2 β-casein” means the A2 variant of bovine β-casein having the amino acid sequence according to SEQ ID NO. 1 (secreted form of protein). In the present context, other variants that include a proline at position 67 may be included in A2 β-casein.

The term “A1 β-casein” means the A1 variant of bovine β-casein having the amino acid sequence according to SEQ ID NO. 2 (secreted form of protein). SEQ ID NO. 1 and SEQ ID NO. The 2 differ from each other only in that β-casein A1 contains histidine at position 67, while β-casein A2 contains proline at position 67. In the present context, other variants that include histidine may be included in β-casein A1 in position 67.

The term “native β-casein” means a protein that is not degraded except for the removal of a signal sequence, for example, the protein described by SEQ ID NO. 1 and 2.

The term "β-casomorphin 7", also described as "BCM-7", means a peptide having the amino acid sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile.

The term "WPC" stands for "Whey Protein Concentrate". In this context, WPC includes traditional WPC as defined by the United States Pharmacopoeia (USP), as well as lactose-reduced whey and mineral-reduced whey, i.e. protein levels may be as low as 10% w/w.

The term “WPI” means “whey protein isolate” and is a whey protein concentrate with a whey protein content on a dry matter basis of at least 90% w/w.

The term "whey protein fraction" means a composition containing whey proteins, for example, WPC and/or WPI.

The term "standard SMP" refers to "standard skim milk powder" which is derived from milk produced by mixed herds of cows and thus contains a variety of β-casein variants, including A1 β-casein and A2 β-casein.

The term "SMP A2" means "A2 skim milk powder" which contains only A2 β-casein and not A1 β-casein.

The term "proteoform" refers to highly related protein molecules derived from all the combinatorial sources of variation that underlie the production of products derived from a single gene. This includes products that differ due to genetic variations, alternative splicing of RNA transcripts, and post-translational modifications.

The term "proteose peptone" is similar to the term used in Swaisgood's 1982 publication, i.e. The term "proteose peptone" means those proteins/peptides that remain in solution after milk is heated to 95°C for 20 minutes and then acidified with 12% trichloroacetic acid to a pH of 4.7.

The term “β-casein-derived protease peptones” means protease peptones derived only from β-casein, such as PP-5, fast PP-8 and slow PP-8.

The term “protease peptone 5”, “PP5” or “PP-5” refers to residues 1-105 and 1-107 derived from β-casein.

The term "fast proteose peptone 8", "PP8f" or "fast PP8" refers to residues 1-28 derived from β-casein. Fast PP8 may also be called "bcas4P 1-28".

The term "slow protease peptone 8", "PP8s" or "slow PP8" refers to residues 29-105 and 29-107 derived from β-casein. Slow PP8 may also be called "bcas1P 29-105" and "bcas1P 29-107".

The term "infant" means a child under 12 months of age; in one embodiment of the invention, the term "infant" may be expanded to include children of any age up to and including 18 months, or any age up to and including 24 months.

The term "infant formula" as used herein means a food product that is specifically intended for consumption by infants during the first months of life (e.g., 0 to 12 months, 0 to 10 months, 0 to 8 months, 0 to 6 months, 0 to 4 months) and which in itself satisfies the nutritional needs of this category of persons (Article 2(c) of the European Commission Directive 91/321/EEC 2006/141/EC of 22 December 2006 on formulas for newborns and formulas for breastfed children). It also refers to a nutritional composition intended for use by infants, as defined in the Codex Alimentarius (Codex STAN 72-1981) and Infant Specialties (including the article Food for Special Medical Purpose). The expression “infant formula” covers both “infant formula” and “follow-up formula” or “weaning formula”.

The term “follow-up formula” or “infant formula” means formula given from the 6th month onwards. It constitutes the main liquid element in the gradually increasing variety of diets for this category of people.

The term "powder" in the present context means a dry bulk solid consisting of a large number of very small particles that can be freely poured when shaken or tilted. The powder may contain water in amounts not exceeding 10%, for example not exceeding 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1% or 0. 5%.

The term “nutritional composition” means a composition that is fed to a subject. Such nutritional composition is typically administered orally or intravenously. It may include a lipid or fat source, a carbohydrate source, and/or a protein source. The nutritional composition of the present invention may be in solid form (eg, powder form) or liquid form.

In a specific 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 a specific embodiment, the nutritional composition of the present invention is an “artificial nutritional composition.” The expression “artificial nutritional composition” means a mixture produced by chemical and/or biological means that may be chemically identical to the naturally occurring mixture found in mammalian milk (i.e., the artificial nutritional composition is not breast milk).

The term “probiotic bacteria” means preparations of microbial cells or components of microbial cells that have a beneficial effect on the health or well-being of the host. The definition of probiotic bacteria is given in the publication Salminen S et al. 1999.

The term "prebiotic" means a selectively fermented ingredient that provides specific changes in both the composition and/or activity of the gastrointestinal microflora, beneficially affecting the well-being and health of the host. Prebiotics are discussed in Roberfroid MB 2007.

Detection and/or quantitation of β-casein-derived protease peptones and/or β-casein

The invention relates, in accordance with a first aspect, to a method for the detection and/or quantification of β-casein-derived protease peptones and/or β-casein, said method comprising the following steps:

(i) obtaining the milk-based product to be analyzed;

(ii) performing analysis of said product by liquid chromatography and mass spectrometry;

(iii) detecting and/or quantifying said β-casein-derived protease peptones and/or β-casein in said product by detecting compounds with target mass/charge values or deconvolving one or more mass spectra to calculate monoisotopic masses.

The milk-based product may be selected from a list of prepared products such as infant formula, maternal nutrition, adult nutritional supplements or dairy products such as milk, milk powder, liquid whey, whey powder, caseinates, WPC, WPI .

In one embodiment, the milk-based product to be analyzed is an infant formula or whey protein fraction.

The milk-based product to be analyzed may be a powder or a liquid. In one embodiment, the product is a powder that is dissolved before analyzing the product. The powder is preferably dissolved by dispersing the powder in water. However, the powder can also be dissolved in other liquids such as a reducing agent as defined below, buffer solutions such as ammonium bicarbonate, TRIS, trisodium citrate, HEPES, TEAB (triethylammonium bicarbonate) or denaturing buffer solutions as defined below, and their combinations.

In one embodiment, the powder is dissolved in a reducing agent and a buffer solution, and optionally a denaturing buffer is added sequentially. In a further embodiment, the powder is dissolved in a reducing agent and a denaturing buffer, and optionally the buffer solution is added sequentially. In yet another further embodiment, the powder is dissolved in a buffer solution and a denaturing buffer, and optionally a reducing agent is added sequentially.

In one embodiment, the powders are dispersed in a liquid to a concentration of no more than 10% w/v. protein, for example not more than 9.5% w/v. protein in liquid, for example not more than 9% w/v. protein in liquid, for example, no more than 8.5% w/v. protein in liquid, for example not more than 8% w/v. protein in liquid, for example not more than 7.5% w/v. protein in liquid, for example not more than 7% w/v. protein in liquid, for example not more than 6.5% w/v. protein in liquid, for example not more than 6% w/v. protein in liquid, for example not more than 5.5% w/v. protein in liquid, for example not more than 5% w/v. protein in liquid, for example not more than 4.5% w/v. protein in liquid, for example not more than 4% w/v. protein in liquid, for example not more than 3.5% w/v. protein in liquid, for example no more than 3% w/v. protein in liquid, for example not more than 2.5% w/v. protein in liquid, for example not more than 2% w/v. protein in liquid, for example not more than 1.5% w/v. protein in liquid, for example not more than 1% w/v. protein in liquid, for example not more than 0.5% w/v. protein in liquid.

After dissolving the samples, or in an alternative embodiment, to dissolve the samples, they are denatured and reduced using denaturing and reducing buffers such as urea, thiourea, guanidine hydrochloride, DTT, beta-mercaptoethanol, TCEP.

In one embodiment, the sample is dissolved in water and further diluted in a mixture of trisodium citrate, guanidine hydrochloride and DTT.

In another embodiment, the sample is dissolved in Tris and urea and diluted in ammonium bicarbonate and DTT.

Preferably, the samples are also cleaned before analysis. Purification can be accomplished by centrifugation. However, it can also be done through filtering.

In one embodiment, the product to be analyzed is analyzed using a native protein assay, in which liquid chromatography followed by mass spectrometry (LC-MS), such as liquid chromatography-high resolution mass spectrometry (LC-HRMS), is applied to native proteins. The use of native protein analysis allows the differentiation of different β-casein proteoforms and genetic variants of full-length β-casein, including A1 and A2 variants. Thus, measuring the native protein in products provides a complete identification fingerprint of the product's protein profile. Also, using the method of analyzing the native protein, it is possible to differentiate different genetic variants of proteose peptones.

In another embodiment, the specified product is analyzed using a peptide assay, including the step of enzymatic cleavage of the specified product before its analysis. Thus, before LC-MS analysis, the product is subjected to enzymatic digestion, for example by digestion of the product with trypsin or GluC (GluC endoproteinase [straphylococcuc aureus V8 protease]).

Enzymatic digestion can be used to determine the presence of β-casein A1 or the protease peptones β-casein A1 and/or A2 in the product. However, due to the peculiarities of the method, which includes the breakdown of proteins as such, it is impossible to distinguish between proteose peptones and full-length β-casein, and only genetic variants A1 and A2 can be differentiated. This can be done by detecting sequences spanning amino acid 67, which is different in variants A1 and A2. Thus, a complete identification fingerprint of the protein profile will not be obtained, but the presence of the A1 variant and possible precursors for BCM-7 will be detected.

In a further embodiment, said enzymatic digestion is performed by trypsin digestion or GluC digestion.

In one embodiment, the liquid chromatography is high performance liquid chromatography. In a further embodiment, the liquid chromatography is ultra-performance liquid chromatography. In yet another further embodiment, the liquid chromatography is nanoliquid chromatography.

For this measurement, standard liquid chromatography settings known to one skilled in the art can be used.

In a preferred embodiment, for native protein analysis, samples can be separated on a C4 column using a gradient of 0.5 ml/min. Preferably, a buffer containing trifluoroacetic acid is used. However, the gradient may vary from 0.1 ml/min to 1 ml/min, for example from 0.1 ml/min to 0.5 ml/min. In an alternative embodiment, buffers such as water/methanol or water/acetonitrile, optionally including formic acid, difluoroacetic acid and/or trifluoroacetic acid, can be used for LC separation.

In a preferred embodiment, for peptide analysis, samples can be separated on a C18 column using a 75 μl/min gradient. Preferably, a buffer containing formic acid is used. However, the gradient can vary from 0.1 μl/min to 100 μl/min, for example from 0.4 μl/min to 85 μl/min. In an alternative embodiment, buffers such as water/methanol or water/acetonitrile, optionally including formic acid, difluoroacetic acid and/or trifluoroacetic acid, can be used for LC separation.

MS chromatograms can be recorded on instruments such as the Thermo Orbitrap Elite or Thermo Q-Exactive HF.

In one embodiment, MS chromatograms are recorded on a Thermo Orbitrap Elite (heater temperature: 60°C, shield gas: 20, auxiliary gas: 5, purge gas: 0, spray voltage: 3.8 kV, capillary temperature: 320°C , RF level of S-lens: 60%, mass number range from 400 to 2000 mass/charge, resolution 240,000, target AGC value: 1e6).

In another embodiment, MS chromatograms are recorded on a Thermo Q-Exactive HF (heater temperature: 100°C, shield gas: 53, auxiliary gas: 14, purge gas: 3, spray voltage: 3.5 kV, capillary temperature: 320 °C, S-lens RF level: 70%, mass number range: 400 to 2000 mass/charge, resolution: 240,000, AGC target: 1e6, maximum IT: 200 ms).

In one embodiment, said mass spectrometry analysis is a high resolution mass spectrometry analysis.

In one embodiment, HRMS detection is preferably performed at a resolution greater than 100,000, such as 120,000 to 240,000.

In accordance with one embodiment, the detection and/or quantitation of specified β-casein-derived proteose peptones is accomplished by detecting compounds with predetermined mass/charge values, as specified. In this case, the presence and quantity of specific peptide sequences can be detected. This is possible because enzymatic digestion results in the formation of specific peptides as a result of specific digestion.

In one embodiment, the peptide method uses non-targeting (information-dependent detection - DDA). In another embodiment, the peptide method uses a non-targeting (information independent detection - DIA) approach.

In a further embodiment, the peptide method uses a PRM (parallel reaction monitoring) or MRM (multiple reaction monitoring) method. Precursors can be preferentially selected by the first quadrupole and fragmented, and MS2 data can be recorded.

In one embodiment, the selection of the precursor can be made in accordance with the list of peptides presented in SEQ ID 3-10.

In another embodiment, the mass spectrometry data obtained after LC-MS measurement for each sample is deconvoluted to calculate monoisotopic masses. Thus, in one embodiment, detection and/or quantitation of said β-casein-derived protease peptones in said product is performed by deconvolving one or more mass spectra to calculate monoisotopic masses.

Deconvolution can be performed using commercially available software such as Thermo BioPharma Finder.

In one embodiment, deconvolution is performed using a sliding window algorithm. In a further embodiment, deconvolution is performed using a fixed window algorithm.

In one preferred embodiment, deconvolution was performed using Thermo BioPharma Finder 1.0 software using the Xtract algorithm (S/N threshold: 3, relative abundance threshold: 1%, match factor: 80%, residual threshold: 25%, overlap ,charge states: 5 to 50, minimum intensity: 1, expected intensity error: 3, mass/charge: 600 to 2000, minimum number of charge states detected: 3) and sliding windows (time: 5 to 20 min, width target average spectrum: 0.1 min, target average spectrum offset: 50%, fusion tolerance: 1.5 Yes, maximum RT interval: 0.5 min, minimum number of detected intervals: 3, XIC).

Monoisotopic masses after deconvolution were compared with a protein database containing the major milk protein components αS1-CN, αS2-CN, β-CN, κ-CN, γ-CN, α-lactalbumin, β-lactoglobulin, CGMP, and β-casein protease peptones. To identify most of the signals, combinatorial addition of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation, and pyroglutamic acid) was examined.

One way to quantify β-casein-derived protease peptones and/or β-casein is to compare signal intensities obtained by measurements against a standard curve obtained by measuring known amounts of different protease peptones and/or β-casein. In this manner, the absolute amount of proteose peptones and/or β-casein in a given sample can be calculated and quantified.

In an alternative embodiment, the signal intensity of the proteose peptone of interest can be expressed in relative terms, such as a percentage of the total protein in the sample or a percentage of the β-casein content.

The compounds to be detected and/or quantified are β-casein-derived protease peptones and/or β-casein. In one embodiment, the proteose peptones are β-casein A1-derived proteose peptones. In a further embodiment, the β-casein-derived protease peptones are fast PP8, slow PP8 and/or PP-5. In yet another further embodiment, the β-casein A1-derived protease peptones are fast PP8, slow PP8 and/or PP-5.

In one embodiment, the β-casein to be detected and/or quantified is A1 β-casein.

Whey protein fraction with reduced levels of β-casein-derived protease peptones (selected whey protein fraction)

In a second aspect, the present invention relates to a method for producing a whey protein fraction having a reduced content of β-casein-derived proteose peptones, the method comprising the following steps:

(i) obtaining a whey protein fraction;

(ii) detecting and quantifying said β-casein-derived protease peptones in said whey protein fraction as described herein; And

(iii) selecting said whey protein fraction containing no more than 10% by weight of β-casein-derived proteose peptones of the total amount of protein in said whey protein fraction, to form the selected whey protein fraction.

Available whey protein fractions intended for use in finished products such as nutritional compositions, including infant formulas, often contain A1 whey and/or A1A2 whey according to standard manufacturing methods. However, this has not previously been considered a problem, for example in the production of A2 infant formula, since A1 β-casein is precipitated during the whey production process and is therefore not part of the whey protein fraction. However, as shown in the examples, β-casein A1-derived protease peptones are found in whey protein fractions such as WPC.

In one embodiment, the whey protein fraction is based on A1 whey and/or Al A2 whey

Due to the advantages of the above-described method for detecting and quantifying β-casein variants and proteoforms, the content of β-casein-derived proteose peptones can be easily detected and quantified. In this way, whey protein fractions can be divided into whey protein fractions containing different amounts of proteozo peptones. Therefore, it is possible to select whey protein fractions that contain no more than 10% by weight. proteose peptones from the total amount of proteins in the whey protein fraction.

In one embodiment, the content of β-casein-derived proteose peptones in the selected whey protein fraction, for example, the content of β-casein-derived proteose peptones is no more than 9.5% by weight, for example no more than 9% by weight, preferably no more than 8. 5% by weight, for example not more than 8% by weight, more preferably not more than 7.5% by weight, for example not more than 7% by weight, even more preferably not more than 6.5% by weight, for example not more than 6% by weight by weight, even more preferably not more than 5.5% by weight, for example not more than 5% by weight, most preferably not more than 4.5% by weight, for example not more than 4% by weight, not more than 3.5% by weight, for example not more than 3% by weight, preferably not more than 2.5% by weight, for example not more than 2% by weight, more preferably not more than 1.5% by weight, for example not more than 1% by weight, even more preferably not more 0.75% by weight, for example not more than 0.50% by weight, even more preferably not more than 0.25% by weight, for example not more than 0.10% by weight, most preferably not more than 0.05% by weight, for example, not more than 0.01% by weight of the total protein content of the whey protein fraction.

The total amount of protein in the whey protein fraction is preferably measured using Kjeldahl assays (ISO 8968-1:2014).

In one embodiment, the proteose peptones are fast PP8, slow PP8 and/or PP-5. In a further embodiment, the protease peptones are slow PP8 and/or PP-5.

In one embodiment, the content of slow PP8 and/or PP-5 in the selected whey protein fraction is no more than 9.5% by weight, for example no more than 9% by weight, preferably no more than 8.5%) by weight, e.g. more than 8% by weight, more preferably not more than 7.5% by weight, for example not more than 7% by weight, even more preferably not more than 6.5% by weight, for example not more than 6% by weight, even more preferably not more than 5.5% by weight, for example not more than 5% by weight, most preferably not more than 4.5% by weight, for example not more than 4% by weight, not more than 3.5% by weight, for example not more than 3% by weight , preferably not more than 2.5% by weight, for example not more than 2% by weight, more preferably not more than 1.5% by weight, for example not more than 1% by weight, even more preferably not more than 0.75% by weight, for example not more than 0.50% by weight, even more preferably not more than 0.25% by weight, for example not more than 0.10% by weight, most preferably not more than 0.05% by weight, for example not more than 0.01% by weight of the total protein content in the whey protein fraction.

In yet another further embodiment, the proteose peptone is PP-5.

Whey protein fraction with reduced content of proteose peptones (depleted whey protein fraction)

In a third aspect, the present invention relates to a method for producing a whey protein fraction having a reduced content of β-casein-derived proteose peptones, the method comprising the following steps:

(i) obtaining a whey protein fraction;

(ii) reducing the content of β-casein-derived proteose peptones in said whey protein fraction to a concentration of not more than 10% by weight of the total protein content in the whey protein fraction to form a depleted whey protein fraction.

In a further aspect, the present invention relates to a method for producing a whey protein fraction having a reduced content of β-casein-derived protease peptones, the method comprising the following steps:

(i) obtaining a whey protein fraction having an initial content of proteose peptones;

(ii) reducing the content of β-casein-derived proteose peptones in said whey protein fraction to a concentration that is at least 5 times less than the initial content in said whey protein fraction, producing a depleted whey protein fraction, e.g. 8 times less, for example, at least 10 times less, for example, at least 15 times less than the initial content of whey protein in the specified fraction.

In one embodiment, said whey protein fraction is based on A1 whey and/or A1 whey A2.

As an example, the expression "at least 5 times less" should be understood to mean that if, for example, the initial content was 5 μg/mg, then a reduction of 5 (five times) would mean that the depleted whey protein fraction contains no more than 1 µg/mg, and similarly, if the signal intensity is 500,000,000 for the initial content, then the signal intensity will be no more than 100,000,000 for the depleted whey protein fraction.

A reduction of at least 5-fold, 8-fold, 10-fold, or 15-fold can be measured by the signal intensity obtained by the detection and quantitation method described herein.

In one embodiment, a whey protein fraction is tested using a method described herein to detect and quantify proteose peptone content to determine whether the proteose peptone content needs to be reduced or not before using the whey protein fractions.

The content of β-casein-derived protease peptones in the whey protein fraction is reduced by no more than 10% by weight of the total protein content in the whey protein fraction.

The total amount of protein in the whey protein fraction is preferably measured using Kjeldahl assays (ISO 8968-1:2014).

In one embodiment, proteose peptones are reduced by gel filtration. However, protease peptones can also be reduced using ion exchange chromatography, affinity chromatography or membrane separation.

In a further embodiment, the final proteose peptone content of the depleted whey protein fraction can be detected and quantified in the manner described above.

In one embodiment, the protease peptone content of the β-casein-derived lean whey protein fraction is no more than 9.5% by weight, for example no more than 9% by weight, preferably no more than 8.5% by weight, for example no more than 8% by weight, more preferably not more than 7.5% by weight, for example not more than 7% by weight, even more preferably not more than 6.5% by weight, for example not more than 6% by weight, even more preferably not more than 5.5 %" by weight, for example not more than 5% by weight, most preferably not more than 4.5% by weight, for example not more than 4% by weight, not more than 3.5% by weight, for example not more than 3% by weight, preferably not more than 2.5% by weight, for example not more than 2% by weight, more preferably not more than 1.5% by weight, for example not more than 1% by weight, even more preferably not more than 0.75% by weight, for example not more than 0.50% by weight, even more preferably not more than 0.25% by weight, for example not more than 0.10% by weight, most preferably not more than 0.05% by weight, for example not more than 0.01% by weight of the total protein content in the whey protein fraction.

In one embodiment, the proteose peptones are fast PP8, slow PP8 and/or PP-5.

In a further embodiment, the protease peptones are slow PP8 and/or PP-5.

In one embodiment, the content of slow PP8 and/or PP-5 in the lean whey protein fraction is no more than 9.5% by weight, such as no more than 9% by weight, preferably no more than 8.5% by weight, e.g. 8% by weight, more preferably not more than 7.5% by weight, for example not more than 7% by weight, even more preferably not more than 6.5% by weight, for example not more than 6% by weight, even more preferably not more than 5 .5% by weight, for example not more than 5% by weight, most preferably not more than 4.5% by weight, for example not more than 4% by weight, not more than 3.5% by weight, for example not more than 3% by weight, preferably not more than 2.5% by weight, for example not more than 2% by weight, more preferably not more than 1.5% by weight, for example not more than 1% by weight, even more preferably not more than 0.75% by weight, for example not more than 0.50% by weight, even more preferably not more than 0.25% by weight, for example not more than 0.10% by weight, most preferably not more than 0.05% by weight, for example not more than 0.01% by weight of the total protein content in the whey protein fraction.

In yet another further embodiment, the proteose peptone is PP-5.

Nutritional composition, e.g., infant formula with reduced proteose peptone content

In a fourth aspect, the invention relates to a method for producing a nutritional composition having a reduced content of β-casein-derived protease peptones, the method comprising the following steps:

(i) obtaining a selected whey protein fraction or a depleted whey protein fraction as described herein;

(ii) obtaining said nutritional composition with a reduced content of proteose peptones from said selected whey protein fractions, said depleted whey protein fractions, or a mixture thereof.

In a fifth aspect, the invention relates to a whey protein fraction having a reduced content of β-casein-derived proteose peptones obtained by the method described herein.

In a sixth aspect, the invention relates to a nutritional composition comprising said whey protein fraction having a reduced content of β-casein-derived proteose peptones described herein or prepared by a method described herein.

In a further embodiment, the content of β-casein-derived proteose peptones in a nutritional composition, such as an infant formula, is no more than 9% by weight of the total protein content of the nutritional composition.

The total amount of protein in the whey protein fraction is preferably measured using Kjeldahl assays (ISO 8968-1:2014).

In yet another further embodiment, the content of β-casein-derived protease peptones in the nutritional composition is no more than 8.5% by weight, more preferably no more than 8% by weight, for example no more than 7.5% by weight, even more preferably no more 7% by weight, for example not more than 6.5% by weight, even more preferably not more than 6% by weight, for example not more than 5.5% by weight, most preferably not more than 5% by weight, for example not more than 4.5 % by weight, for example not more than 4% by weight, not more than 3.5% by weight, more preferably not more than 3% by weight, for example not more than 2.5% by weight, even more preferably not more than 2% by weight, for example not more than 1.5% by weight, even more preferably not more than 1% by weight, for example not more than 0.75% by weight, most preferably not more than 0.50% by weight, for example not more than 0.25% by weight , for example not more than 0.1% by weight, for example not more than 0.05% by weight, for example not more than 0.01% by weight of the total protein content in the whey protein fraction.

The nutritional composition of the invention may be intended for any mammal, such as, for example, humans, and domestic animals, such as cats and dogs. In a preferred embodiment, the mammal is a human.

Examples of nutritional compositions are infant formulas, maternal foods, adult nutritional supplements, or dairy products.

In one embodiment, the nutritional composition is an infant formula.

The general composition of a nutritional composition, such as an infant formula for use in accordance with the present invention, may optionally contain substances that may provide beneficial effects, such as probiotic bacteria, fiber, lactoferrin, nucleotides, nucleosides and/or the like, in amounts such as those typically found in infant feeding compositions.

The probiotic bacteria may be selected from the group consisting of lactobacilli such as Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus reuteri, and bifidobacteria such as Bifidobacterium lactis, Bifidobacterium breve and Bifidobacterium longum.

A nutritional composition, such as an infant formula, may optionally further contain prebiotics, such as non-digestible carbohydrates, which promote the growth of probiotic bacteria in the intestine.

In a preferred embodiment, the nutritional composition, such as an infant formula, contains prebiotics selected from the group consisting of fructooligosaccharides (FOS), raftilose, inulin, raftilin, lactulose, 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.

Thus, a preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising vitamins.

Some vitamins and minerals have minimum requirements. 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, molybdenum, taurine and L-carnitine. Minerals are usually added in the form of salts.

If desired, the nutritional composition, for example, infant formula, may contain emulsifiers and stabilizers such as soy lecithin, citric acid esters of mono- and diglycerides, and the like. This is especially true when the composition is presented in liquid form.

A preferred embodiment relates to a nutritional composition, such as an infant formula, wherein at least one carbohydrate source is selected from the group consisting of lactose, corn syrup solids, fructose, glucose, maltodextrins, glucose syrups, sucrose, trehalose, galactose , maltose, honey powders, starch, oligosaccharides, raftilin and raftilose.

Another preferred embodiment relates to a nutritional composition, such as infant formula, further comprising anhydrous milk fat.

Another preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising long chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and/or arachidonic acid (ARA).

The nutritional composition, such as infant formula, may further contain flavoring agents such as, but not limited to, vanillin.

A particular preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising fructooligosaccharides such as raftilin and/or raftilose.

The nutritional composition, such as infant formula, may be a liquid mixture or powder that must be reconstituted before use. A preferred embodiment is an infant formula that is a powder.

In a preferred embodiment, the nutritional composition is a liquid composition prepared by powder reconstitution.

The present invention further provides a nutritional composition, such as the infant formula of the present invention, for use as complementary food for infants in combination with human breast milk.

In certain embodiments, the infant is 0-12 months of age, preferably 0-6 months of age.

Therapeutic and non-therapeutic uses of a nutritional composition or formula for newborns with a reduced content of proteose peptones.

In a seventh aspect of the present invention, the nutritional composition described herein can be used as a medicine.

In an eighth aspect of the present invention, the infant formula described herein relates to use in the treatment, prevention and/or relief of abdominal pain in an infant.

In a ninth aspect of the present invention, the infant formula described herein relates to use in the treatment, prevention and/or alleviation of lactose intolerance in an infant.

A tenth aspect of the present invention relates to the use of the infant formula described herein to improve stool consistency.

BCM-7 is known to affect opioid receptors and relate to stool consistency and abdominal pain. Thus, an infant formula containing no or only a very limited amount of BCM-7 precursors, i.e. proteose peptones PP-5 and slow PP8 derived from β-casein A1 will result in the formation of less BCM-7 upon infant formula consumption and therefore less adverse effects on stool consistency and abdominal pain.

Additionally, it is believed that administering an infant formula that contains fewer BCM-7 precursor molecules and therefore produces less BCM-7 will have less of an effect on opioid receptors, preventing lactose intolerance later in that infant's life. There is growing evidence that events that occur early in development can trigger disease, even if the disease may not manifest itself until later in life.

In this context, treatment should be understood as a situation in which a disorder has already developed, as opposed to prevention, which occurs before the disorder develops. By relief is meant a situation in which a disorder has already developed, but is subjected to a treatment that does not lead to the disappearance of the disorder, but simply improves the health of the subject suffering from said disease.

In another aspect, the present invention relates to a method of treating, preventing and/or relieving abdominal pain in an infant.

In a further aspect, the present invention provides a method for treating, preventing and/or alleviating lactose intolerance in an infant.

It should be noted that the embodiments and features described in the context of one aspect of the present invention are also applicable to other aspects of the invention.

All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

The present invention will be further described in detail in the following non-limiting examples.

Examples

Materials and methods Chemical reagents and samples

Guanidine hydrochloride (RDD001), sodium citrate dihydrate (S1804), DL-dithiothreitol (43819) and β-casein standard (C6905) were purchased from Sigma-Aldrich (St. Louis, MI, USA), TFA (1.08178. 0050) from VWR International (Radnor, PA, USA) and LC-MS water (1.15333.1000) and acetonitrile (1.00029.1000) from Merck (Darmstadt, Germany).

Milk samples

Raw milk was collected from Teagasc Moorepark dairy farm (Fermoy, Co., Cork, Ireland) from cows with the A1/A1 genetic profile, defatted, pasteurized using a Microthermics apparatus (UHT/HTST Electric Model 25 HV Hybrid, Liquid Technologies, Wexford, Ireland), kept at a temperature of 85°C for 23 s, followed by 2-stage homogenization [GEA Niro Soavi S.p.A. Type: NS2006H (Non-Aseptic)] at a total homogenization pressure of 2500 psi. inch. The sample was spray dried to obtain a skim milk powder sample referred to as SMP A1|A1. Commercially available SMP A1/A2 and A2/A2 were purchased from Europe and the USA, buffalo SMP was purchased from India, and pasteurized pooled human breast milk (991-01-P) was purchased from Lee Biosolutions (Maryland Heights, NY). Missouri, USA).

Different batches received from the same or different manufacturers are numbered A, B, C, etc.

Finished whey protein products and concentrates

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 in the experiments were designated either IF1-IF22 (see table 1), or IFa-IFj. In this regard, it should be noted that IFa=IF1, IFb=IF2, IFc=IF3, IFd=IF4, IFe=IF5, IFf=IF6 and IFg=IF7.

Three different commercially available batches (labeled A, B and C) of WPC were tested. WPC35 is a whey protein concentrate with a protein content of 35% w/w.

Two different commercially available batches of WPC28 (labeled A and B) were tested. WPC28 is a demineralized WPC with a protein content of 28% w/w.

WPC80 is a commercially available whey protein concentrate containing 80% w/w protein, enriched with alpha-lactalbumin.

Receiving a sample

The powders were dispersed to achieve a concentration of 3.5% w/v. protein in water using a volumetric flask, stirred for at least 30 min at room temperature, denatured with 4 volumes of denaturing buffer (guanidine hydrochloride 7.5 M, sodium citrate 6.25 mM, DTT 23 mM), incubated at room temperature for 30 min and cleared by centrifugation at 16,000 g for 10 min.

Trypsin digestion of native proteins

Powder samples were dispersed to achieve a concentration of 3.5% w/v. protein in Tris 50 mM + 6 M urea by mixing on an orbital shaker for one hour.

200 μl of sample was diluted with 1000 μl of ammonium bicarbonate 100 mM and vortexed. 10 μl of DTT (45 mM) was added to 100 μl of the diluted solution and incubated at 60°C for 30 minutes. The sample was then cooled to room temperature, after which the sample was rapidly rotated and 10 μL of iodoacetamide (100 mM) was added. The sample was then incubated for 30 minutes at room temperature in the dark, after which 6 μl of trypsin (0.2 μg/μl) was added and incubated overnight at 37°C. Trypsin digestion was stopped by adding 6 μl of 10% formic acid. The digested sample was centrifuged at 14,000 g for 10 minutes, after which the liquid phase was transferred to an injection vial and determined by LC-MS.

LC-MS Analysis

Cleared samples were analyzed by LC-MS based on existing methods (Bonfatti et al., 2008, Frederiksen et al., 2011).

For native protein analysis, samples were separated on a C4 column (Acquity UPLC Protein BEH C4, 300 A, 1.7 μm, 2.1 mm × 150 mm) using the following gradient at 0.5 ml/min (Table 2):

Buffer A: 0.1% trifluoroacetic acid (TFA) in water, Buffer B: 0.1% TFA in acetonitrile:water 90:10

The MS signal was recorded from 4 to 20 min in Full MS mode on a Thermo Q-Exactive HF device (heater temperature: 100°C, shielding gas: 53, auxiliary gas: 14, purge gas: 3, spray voltage: 3.5 kV , capillary temperature: 320°C, S-lens RF level: 70%, mass number range: 400 to 2000 mass/charge, resolution: 240,000, AGC target value: 1e6, maximum IT: 200 ms).

For peptide analysis, i.e. Trypsin digestion or GluC digestion, the peptides were separated on a C18 column (Acquity UPLC BEN C18, 130 A, 1.7 μm, 1.0 × 150 mm) using the following gradient at 75 μl/min (Table 3):

The MS signal was recorded from 3.5 to 35 min in PRM mode on a Thermo Q-Exactive HF device (heater temperature: 30°C, shielding gas: 8, auxiliary gas: 0, purge gas: 0, spray voltage: 3.6 kV, capillary temperature: 320°C, S-lens RF level: 55, default charge: 2, MS2 resolution: 30,000, AGC target: 1e5, max IT: 100 ms, isolation window: 1.5 mass/charge , insulation bias: 0.5 mass/charge).

The inclusion list is shown in Table 4:

Data deconvolution

Raw data files were deconvoluted with Thermo BioPharma Finder 1.0 software using the Xtract algorithm (S/N threshold: 3, relative abundance threshold: 1%, match rate: 80%, residual threshold: 25%, overlaps, charge states: 5 to 50, minimum intensity: 1, expected intensity error: 3, mass/charge: 600 to 2000, minimum number of charge states detected: 3) and sliding windows (time: 5 to 20 min, target width average spectrum: 0.1 min, target average spectrum offset: 50%, fusion tolerance: 1.5 Yes, maximum RT interval: 0.5 min, minimum number of detected intervals: 3, XIC). Monoisotopic masses, total signal intensity values, and peak apex RT values were exported as CSV files and used in the following steps.

Limit of detection experiments were also performed by deconvolving the raw data files using a fixed window centered around the β-casein peak. The following parameters were used: S/N Threshold: 1, Relative Abundance Threshold: 1%, Match Ratio: 25%, Residual Threshold: 25%, Overlaps, Charge States: 12 to 30, Minimum Intensity: 1, Expected Error intensity: 3, mass/charge: 800 to 2000, minimum number of charge states detected: 3, time: 11-13.5 min, relative intensity threshold: 1%. Monoisotopic masses and total signal intensity values were used for the following steps. Determination of proteoforms

Monoisotopic masses after deconvolution were compared with a protein database containing the major milk protein components αS1-CN, αS2-CN, β-CN, κ-CN, γ-CN, α-lactalbumin, β-lactoglobulin, CGMP and proteose peptones using in-house developed Nestle software (Protein Analyzer). To identify most of the signals, combinatorial addition of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation, and pyroglutamic acid) was examined. Incorrectly identified signals were corrected through manual verification. Total signal intensity values for the corresponding proteins or proteoforms (i.e., total β-casein, β-casein lactosylation states, β-casein genetic variants, etc.) were extracted. For clarity, only the A1, A2 and B genetic variants of β-casein are described in detail in the figures. β-casein I/H2 co-eluted with β-casein and was combined with β-casein A2. Both variants contain proline at position 67 and belong to type A2.

Protein glycation in solid state

The solid state glycation experiment was carried out based on Fenaille, 2003 (Fenaille et al., 2003). Briefly, 45 g of milk powder was incubated in a closed container saturated with dipotassium carbonate for 8-10 days until the moisture content reached 5.4% (initial value was 3.8%). Nine 2.1 g aliquots of the moistened powder were incubated in 25 ml glass tubes at 60°C in an oven for 45 minutes to 24 hours. Glycation was stopped by transferring the tubes to ice for 5 minutes. Then 20 g of water was added to obtain a 3.5% (w/v) protein solution, which was stirred at room temperature for 1 h 30 on a roller mixer. Solutions were stored at 4°C overnight until completion of the experiment and warmed for 2 h at RT before analysis. Samples were prepared as described above, except that the incubation step lasted 10 min at 60°C in a thermal mixer (650 rpm). MS chromatograms were recorded on a Thermo Orbitrap Elite (heater temperature: 60°C, shielding gas: 20, auxiliary gas: 5, purge gas: 0, spray voltage:

3.8 kV, capillary temperature: 320°C, S-lens RF level: 60%, mass number range: 400 to 2000 mass/charge, resolution: 240,000, target AGC value: 1e6). Quantitative determination of β-casein

A sample of skim milk powder (SMP) was injected multiple times in each assay run to normalize signals across experiments.

An external calibration curve was generated using a β-casein standard corrected for protein purity (see text). β-casein signal intensity values were normalized to the average β-casein signal of SMP samples (corrected for glycation, see above). The calibration curve was forced to 0 (y=0.0524 ⋅ x, where x is expressed in μg of injected β-casein and y is dimensionless).

For each sample, β-casein intensity values were first corrected for glycation and normalized to the average β-casein signal in the SMP samples (corrected for glycation). The amount of β-casein injected was then calculated from an external calibration curve. This calculation can be done for total β-casein, for a given variant such as A1 β-casein, or for a set of variants (results for A2 β-casein also include the related I/H2 variant).

The effectiveness of the method was assessed by two operators on seven samples of infant formula (stages 1-4, prepared using type A2 skim milk powder), using three repeated injections on five different days.

Example 1: LC-MS Method, Data Output, Deconvolution and Imaging To analyze native proteins, infant formula and skim milk powder samples were dispersed in water to achieve a protein concentration of 3.5% (v/w) and denatured in 6 M guanidine. , reduced with DTT and clarified by centrifugation. Native proteins were separated by ultraperformance liquid chromatography (UHPLC) on a C4 column using a water/ACN gradient with 0.1% TFA and analyzed by high-resolution mass spectrometry (Fig. 3A). To obtain monoisotopic masses of proteins, chromatograms are deconvoluted sequentially using the sliding window algorithm and the Xtract algorithm (Fig. 3B). Finally, the proteoforms are visualized in circles and arrows, with the x-axis representing retention time, the y-axis representing monoisotopic size, and the area of the circles representing signal intensity (Figure 3C).

Example 2: General ID Fingerprint

In fig. 4A shows the result of the method with cow's raw milk and shows where the main milk proteins (αS1-, αS2-, β-, κ- and γ-casein, α-lac and β-lg) are found. Zooming in on the β-casein region (Figure 4 B) allows detailed analysis of the different β-casein proteoforms, including genetic variants and protein modification (method-induced lactosylated adducts are detected in all skim milk powder samples). The mass accuracy is shown in Table 5 for the raw milk sample. The 1 Da difference may be due to a misidentified monoisotopic signal propagating during deconvolution, as a 1.5 Da pooling tolerance is applied between sliding windows to avoid splitting the signals into two masses separated by 1 Da. The method can be used to profile a wide range of raw materials and finished products (Fig. 5). Interestingly, this method readily detects casein glycomacropeptide (CGMP), the C-terminal portion of κ-casein cleaved by rennet during cheese making, a polypeptide that is frankly difficult to detect by gel staining or UV profiling.

Example 3: Detection of β-casein A1 in infant formulas containing β-casein A2

Seven A2 β-casein infant formulas were fortified with A1|A1 SMP to achieve 5% A1 β-casein (5 g A1 β-casein per 100 g total β-casein). At this concentration, β-casein A1 signal was detected in all cases (Fig. 6). Example 4: Protein Quantification and Glycation

Protein quantification requires not only a suitable standard to construct a calibration curve, but also that different proteoforms exhibit similar signal responses. This is necessary to apply the calibration curve to unknown samples, given that the standard and samples are likely to have different proteoform distributions. In an alternative embodiment, the method should provide a method for adjusting the distribution of proteoforms.

To test whether protein lactosylation affects MS signal intensity, glycation was induced in a skim milk powder sample by heating as described in Materials and Methods (Figure 7A). The increase in glycated β-casein content (Fig. 7 B) was accompanied by a decrease in the total β-casein signal (Fig. 1C). The plot of the total β-casein signal versus the percentage of non-glycated β-casein signal was linear (Fig. 7D) and was converted to a fold reduction in signal (Fig. 7E) of the form y=1/(ax+b), where a and b correspond to 0.878 and 0.122, respectively. Thus, for β-casein, the glycation correction factor (GCF) can be calculated based on the percentage of unglycated β-casein (%UG _β ) using Equation 1.

Multiplying the measured β-casein signal by GCF results in the corrected β-casein signal (Pcas _corr ), which corresponds to the expected signal if all β-casein were non-glycated (Equation 2).

Example 5 Quantitative determination of β-casein

Quantification was performed using a sliding window algorithm. An external calibration curve was generated using commercial purified β-casein. This standard also contained αS1-casein, κ-casein, and various unidentified peptides and protein fragments, most of which corresponded to degradation products of β-casein (Fig. 8A). The proportion of the total signal attributed to native β-casein in the seven most concentrated calibration samples reached 85.6 ± 0.4%, and the concentration of standard β-casein was corrected using this purity factor. For each concentration, the total β-casein signal was normalized to the average (and corrected for glycation) signal of SMP samples that were injected in the same batch (and in all subsequent quantitation experiments to account for variations in MS parameters), and the normalized values were plotted on graph as a function of the amount of β-casein loaded onto the column. The calibration curve (Fig. 8B) showed good linearity (R ² =0.996) with random distribution of residuals. From this calibration curve, the amount of β-casein injected into the column (in μg) can be calculated using Equation 3:

where Pcas _corr , _norm is the corrected β-casein signal in the sample (Equation 2) divided by the average of the adjusted signals of skim milk powder samples injected in the same batch (Equation 4).

Quantification of β-casein variants in the milk matrix was further assessed by mixing skim milk powder containing only the A1 variant with skim milk powder containing only the A2 type (Fig. 8C). The response to β-casein A2 showed good linearity (R ² =0.996), indicating a negligible matrix effect. The measured and expected A1/A2 ratios showed a very strong linear relationship (%A2 _calc =1.05 ⋅ %A2 _exp , R ² =0.996). This means that the response rates for both variants are very similar and that the calibration curve is also suitable for quantifying individual genetic variants. Quantification of β-casein in two SMP samples resulted in an average value of 28.3 g β-casein/100 g protein, which corresponds to the value of 27% described in the Handbook of milk composition (Swaisgood, 1995) (Table 6).

Example 6. Efficiency of the method (quantitative determination of β-casein)

The effectiveness of the method was assessed on seven infant formulas containing only β-casein A2. Each sample was prepared five different times and analyzed using three replicate injections. The amount of β-casein measured was essentially in agreement (83%-136%) with the theoretical values calculated by multiplying the protein content by the casein content and 33%, which is the generally accepted proportion of β-casein to total casein (Swaisgood, 1995) (Fig. 9A).

The method was further tested on another series of commercially available infant formulas (either A2 based or formulated with standard SMP) and the results were shown to be substantially consistent with the theoretical values (Fig. 9B, for % whey shaded values based on assumptions). Differences between measured and theoretical β-casein content were greatest in samples with the lowest β-casein content. This may be due to less abundant proteoforms becoming too similar to noise to be measured effectively, particularly in samples with many genetic variants such as IF8 and IF13.

Example 7 Detection of A1-Specific Proteose Peptones in Whey Protein Concentrate by Native Protein Assay

Six different batches of whey protein concentrates were analyzed by native protein assay as described in Methods and Materials using LC-HRMS.

Analyzed raw materials:

• Three batches of WPC35

• Two batches of WPC28

• One batch of WPC80 (enriched with α-lac)

In fig. Figure 10 shows individual analyzes of six different whey protein concentrates.

No traces of β-casein were detected in any of the WPCs tested, as no signals were detected within the solid circle (i.e., the circle in the upper right corner).

In contrast, all WPCs tested exhibited signals corresponding to proteose peptones, detected as signals within a circle with a dotted perimeter (i.e., the circle in the lower left corner).

The analysis further revealed that all ingredients contained CGMP (casein glycomacropeptide), which is a casein-derived peptide identified in the lower left corner of each of the diagrams. Directly below the circle with the dotted perimeter.

In fig. 11 is a close-up view of the area within the circle with the dotted perimeter shown in FIG. 10. This close-up shows that signals consistent with the expected masses for a wide range of protease peptones were detected in all ingredients. Black indicates β-casein A2-derived proteose peptones, gray indicates β-casein A1-derived proteose peptones, and white indicates unidentified compounds.

Some of these peptones are: PP5 A1 1-105, PP5 A1 1-107, PP5 A2 1-105, PP5 A2 1-107, bcas1P A1 29-105 (i.e. PP8s A1 29-105), bcas1P A1 29-107 (i.e. PP8s A1 29-107), bcas1p A2 29-105 (i.e. PP8s A2 29-105) and bcas1P A2 29-107 (PP8s A2 29-107).

Gamma-casein was not detected in the tested WPC.

WPC80 did not contain slow PP8 (29-105/7). Probably due to the ultrafiltration step of the WPC80 production process, which likely removes the slow PP8.

As in fig. 10 and in FIG. 11 several signals (shown in white) both within and outside the dotted circle in FIG. 10 relate to the identification of the remaining peptides.

Accordingly, β-casein protease peptones A1 (and A2) were detected in all whey raw materials tested. Thus, they will be present in the finished products and are very likely to be responsible for the strong A1 signal obtained in all finished products tested.

Example 8 Detection of A1-specific proteose peptones in finished products using native protein analysis

Various finished products representing different infant formulas from different brands were analyzed for the detection of specific proteose peptones. All infant formulas are considered A2 end products.

The finished products were dissolved and analyzed by LC-HRMS as described in the Materials and Methods section above.

In fig. 12 shows that proteose peptones were detected in all batches tested, even though the proteose peptone profile varied between finished products.

β-casein A1-derived proteose peptones were found in all infant formulas tested, even those based on milk containing A2 β-casein.

Example 9 Quantitative determination of A1- and A2-specific protease peptones using intact protein assay

The relative quantitation of PP5 A1 and PP5 A2 in the milk matrix was assessed by mixing skim milk powder containing only the A1 variant with skim milk powder containing only the A2 type (Fig. 13). The responses for PP5 A2 and PP5 A1 showed good dose dependence with quadratic regression (R ² =0.999 and R ² =0.995, respectively), indicating a negligible matrix effect. This means that the response rates for both variants are very similar and that the calibration curve is also suitable for quantifying individual genetic variants.

In conclusion, it is shown that certain protease peptones can be quantified by native protein assays, even in a matrix.

Example 10 Detection of A1-specific proteose peptones in finished products using trypsin digestion

The various finished products are either standard skim milk powder (ie standard SMP), skim milk powder containing only the A2 β-casein variant, or various infant formulas from different brands and for different age groups. All infant formulas are considered A2 end products.

The finished products were dispersed to a concentration of 3.5% (w/v) protein in Tris 50 mM + 6 M urea and mixed on an orbital shaker for one hour. 200 μL of sample was diluted with 1000 μL of 100 mM ammonium bicarbonate and vortexed. 10 μL of DTT (45 mM) was added to 100 μL of the diluted solution. Samples were incubated at 60°C for 30 minutes, cooled to RT, and quickly centrifuged to collect droplets formed after evaporation. 10 μl of iodoacetamide 100 mM (in ammonium bicarbonate 100 mM) was added; samples were incubated for 30 minutes at room temperature in the dark. 6 μl of 0.2 μg/μl trypsin was added and samples were incubated overnight at 37°C. To stop the digestion, 6 μl of 10% formic acid was added, the solutions were centrifuged at 14,000 g for 10 minutes; the liquid phase was transferred to an injection vial and analyzed by LC-MS as described in the Materials and Methods section above.

The assay focuses on the detection of the A1S peptide, i.e. An A1-specific peptide having the position shown in FIG. 14. As can be seen from the position of the A1S peptide, it can be distinguished from the A2S peptide because it covers the region of β-casein including amino acid 67.

In fig. 15 shows that no A1S was detected in the SMP A2 samples. A1S was detected in one sample of SMP A2, which was subsequently found to erroneously contain A1 β-casein. However, the peptide is significantly expressed in standard SMP. Surprisingly, it was found that a strong signal for the A1S peptide was detected in all infant formulas tested. Similar results were obtained for peptides A1N (Fig. 16) and A1T (Fig. 17).

As expected, peptides A2S (FIG. 18), A2N (FIG. 19), A2T (FIG. 20), Tot 1 (FIG. 21), and Tot 2 (FIG. 22) showed strong signals in all regions tested.

Example 11 Detection of A1-specific proteose peptones in whey protein concentrate by trypsin digestion

Different batches of whey protein concentrates were analyzed together with skim milk powder, both standard and skim milk powder containing only the A2 βcasein variant.

Analyzed raw materials (RM):

• Three batches of WPC35

• Two batches of WPC28

• One batch of WPC80 (enriched with α-lac)

• Two different commercially available lactose ingredients

Products were dissolved and trypsin digested prior to LC-MS analysis as described in the Materials and Methods section above.

The assays are focused on detecting the Tot1 and Tot2 peptides, i.e. peptides common to β-casein A1 and A2, peptide A1S, i.e. A1-specific peptide, A1N peptide, i.e. A1-specific peptide, A1T peptide, i.e. A1-specific peptide, A2N peptide, i.e. A2-specific peptide, A2T peptide, i.e. A2-specific peptide, and A2S peptide, i.e. A2-specific peptide. The position of the peptides is shown in Fig. 14.

In fig. 23 and 24 present the results obtained when determining the amount of peptides Tot1 and Tot2, respectively. It is a common peptide (for A1 and A2) and is also a peptide present only in native β-casein and γ-casein. The total β-casein peptide signal was shown to be significantly reduced in all whey ingredients and absent from lactose ingredients, but present in SMP and SMP A2 as expected.

This indicates that, as expected, very low amounts of native β-casein or γ-casein are present in RM (~1%) compared to SMP even when equivalent protein content is used.

In fig. 25 shows the results obtained by determining the amount of A1S peptide. As expected, the A1S peptide signal is strongest in SMP but absent in SMP A2. Additionally, lactose samples do not show any signal. For whey protein samples, the amount was surprisingly found to be approximately one-quarter of that observed in SMP.

Similar results were obtained when samples were tested for the presence of two other A1-specific peptides A1N and A1T (Fig. 26 and Fig. 27).

In fig. 28 shows the results obtained when determining the amount of A2S.

The A2S (A2-specific) peptide signal was shown to be strongest for SMP A2, but weaker for SMP, as expected. As expected, lactose samples do not show any signal. For whey protein samples, the amount was surprisingly found to be approximately one-quarter of that observed in SMP.

Similar results were obtained when samples were tested for the presence of two other A2-specific peptides, A2N and A2T (FIGS. 29 and 30).

Accordingly, based on the above results, it can be concluded that in all whey RM analyzed, the signal intensity of total β-casein peptide is highly reduced (~100-fold) compared to SMP. In addition, A1- and A2-specific peptides are reduced only ~4-5 times compared to SMP. Thus, selective enrichment of A1- and A2-specific peptides is observed.

Thus, when applying the trypsin digestion method and identifying the various peptides, it would appear that the whey ingredients still contain ~20% β-casein fragments (compared to SMP), which may at least partially represent known protease peptones such as PP5 and PP8s.

Example 12. Reduction of protease peptones in whey protein fractions

To obtain a depleted whey protein fraction, three batches of WPC35, which were found to contain proteose peptones in Example 9, were subjected to gel filtration.

Three batches of WPC35 were further purified by gel filtration on a (550 x 22 mm) Sephadex G-75 column prepared and equilibrated with a volatile 0.1 M NH ₄ HCO ₃ buffer (pH 8.0-8.5) .

0.5 g-0.7 g of each batch was dissolved in 5-7 ml of buffer (with the addition of a few ml of 1 M NaOH to neutralize residual traces of TCA and facilitate dissolution) and applied to the column. The flow rate was adjusted to 0.5 mL/min and 5 mL fractions were collected.

Fractions were analyzed by the detection and quantitation method described herein, i.e. LC-HRMS, in accordance with the section “Materials and Methods”. Fractions without protease peptones PP5 and slow PP8 were combined to form the depleted whey protein fraction.

References

Bonfatti, V. et al. J Chromatogr A, 2008; 1195(1-2): 101-106. de Jong et al. J Chromatogr A, 1993; 652(1):207-213.

EFSA Scientific Report, an EFSA scientific report prepared by the DATEX working group on the potential health effects of b-casomorphins and related peptides. 2009; 231; 1-107.

Fenaille, F. et al. Rapid Commun Mass Spectrom, 2003; 17(13):1483-1492.

Fenaille, F. et al. International Dairy Journal, 2006; 16(7):728-739.

Feng, P. et al. JAO AC Int, 2017; 100(2):510-521.

Frederiksen, P. D. et al. J Dairy Sci 2011; 94(10):4787-4799.

Ho, S. et al. Eur. J. Clin. Nutr. 2014, 68, 994-1000.

Karamoko, G. et al. Biotechnologie, Agronomie, Societe et Environnement, 2013; 17(2):373-382.

Poulsen, N. A et al. Acta Agriculturae Scandinavica, Section A - Animal Science, 2016; 66(4): 190-198.

Roberfroid MB. J Nutr. 2007; 137:830S.

Salminen S et al. Trend Food Sci. Technol., 1999; 10 107-110.

Swaisgood, H. E. Handbook of Milk Composition. R.G. Jensen, ed. Academic Press, San Diego 1995; 464-468.

Swaisgood. Developments in Dairy Chemistry. Fox (Ed), Proteins, vol. 1, 1982; 63-110.

Vallejo-Cordoba, B. J Capillary Electrophor, 1997; 4(5):219-224.

Visser, S. et al. J Chromatogr A, 1995; 711(1): 141-150.

Features of the invention

1. A method for detecting and/or quantifying β-casein-derived protease peptones and/or β-casein, said method comprising the following steps

(i) obtaining the milk-based product to be analyzed;

(ii) performing analysis of said product by liquid chromatography and mass spectrometry;

(iii) detecting and/or quantifying said β-casein-derived protease peptones and/or β-casein in said product by detecting compounds with target mass/charge values or deconvolving one or more mass spectra to calculate monoisotopic masses.

2. The method according to feature 1, in which said proteose peptones are proteose peptones obtained from β-casein A1.

3. The method according to feature 2, in which said β-casein-derived proteose peptones are fast PP8, slow PP8 and/or PP-5, for example, the A1 β-casein-derived proteose peptones are fast PP8, slow PP8 and/or PP -5.

4. A method according to any of the preceding characteristics, wherein said product is an infant formula or a whey protein fraction.

5. A method according to any of the preceding features, wherein the product is a powder, wherein said powder is dissolved prior to analysis of said product.

6. A method according to any of the preceding characteristics, wherein said product is analyzed using a native protein assay.

7. The method of feature 6, wherein said mass spectrometric analysis is a high resolution mass spectrometric analysis.

8. A method according to any one of features 6-7, wherein the detection and/or quantification of said β-casein-derived proteose peptones in said product is performed by deconvolution of one or more mass spectra to calculate monoisotopic masses.

9. The method according to feature 8, in which the specified deconvolution is performed using a sliding window algorithm.

10. The method according to any one of features 1-5, in which the specified product is analyzed using a peptide analysis, including the step of enzymatic cleavage of the specified product before its analysis.

11. The method of feature 10, wherein said enzymatic digestion is performed by trypsin digestion or GluC digestion.

12. A method according to any one of features 10-11, wherein the detection and/or quantification of said β-casein-derived protease peptones is performed by detecting compounds with predetermined mass/charge values.

13. A method for obtaining a whey protein fraction having a reduced content of proteose peptones derived from β-casein, the method comprising the following steps:

(i) obtaining a whey protein fraction;

(ii) detecting and quantifying said β-casein-derived protease peptones in said whey protein fraction as described in any of Features 1-12; And

(iii) selecting said whey protein fraction containing no more than 10% by weight of β-casein-derived proteose peptones of the total amount of protein in said whey protein fraction, to form the selected whey protein fraction.

14. A method for obtaining a whey protein fraction having a reduced content of protease peptones derived from β-casein, wherein said method includes the following steps:

(i) obtaining a whey protein fraction;

(ii) reducing the content of β-casein-derived proteose peptones in said whey protein fraction to a concentration of not more than 10% by weight of the total protein content in the whey protein fraction to form a depleted whey protein fraction.

15. The method of feature 14, further comprising the step before and/or after step (ii) of detecting and quantifying said β-casein-derived proteose peptones in said whey protein fraction as described in any of features 1-12.

16. The method according to any of the characteristics 14-15, in which the content of proteose peptones is reduced by means of gel filtration.

17. A method according to any one of features 13-16, wherein the content of β-casein-derived protease peptones in said selected or depleted whey protein fraction is not more than 9.5% by weight, for example not more than 9% by weight, preferably not more than 8.5% by weight, for example not more than 8% by weight, more preferably not more than 7.5% by weight, for example not more than 7% by weight, even more preferably not more than 6.5% by weight, for example not more than 6 % by weight, even more preferably not more than 5.5% by weight, for example not more than 5% by weight, most preferably not more than 4.5% by weight, for example not more than 4% by weight, not more than 3.5% by weight by weight, for example not more than 3% by weight, preferably not more than 2.5% by weight, for example not more than 2% by weight, more preferably not more than 1.5% by weight, for example not more than 1% by weight, even more preferably not more than 0.75% by weight, for example not more than 0.50% by weight, even more preferably not more than 0.25% by weight, for example not more than 0.10% by weight, most preferably not more than 0.05% by weight weight, for example not more than 0.01% by weight of the total protein content in the whey protein fraction.

18. A method according to any one of features 13-17, wherein said proteose peptones are fast PP8, slow PP8 and/or PP-5.

19. A method for producing a nutritional composition having a reduced content of β-casein-derived proteose peptones, the method comprising the following steps:

(i) obtaining a selected whey protein fraction or a depleted whey protein fraction as described in any of features 13-18;

(ii) obtaining said nutritional composition with a reduced content of proteose peptones from said selected whey protein fractions, said depleted whey protein fractions, or a mixture thereof.

20. A whey protein fraction having a reduced content of β-casein-derived proteose peptones, obtained by a method according to any of the characteristics 14-18.

21. A nutritional composition containing the specified fraction of whey protein, having a reduced content of proteose peptones derived from β-casein, according to feature 20 or obtained by a method according to feature 19.

22. Nutritional composition according to criterion 21, which is a mixture for newborns.

23. A nutritional composition according to any of the characteristics 21-22, in which the content of these proteose peptones is no more than 9% by weight of the total protein content in the nutritional composition.

24. Nutritional composition according to any of the characteristics 21-23 for use as a medicine.

25. A mixture for newborns according to any of the characteristics 22-23 for use in the treatment, prevention and/or relief of abdominal pain in an infant.

26. An infant formula according to any of the characteristics 22-23 for use in the treatment, prevention and/or alleviation of lactose intolerance in an infant.

27. Use of formula for newborns for any of the signs 22-23 to improve stool consistency.

List of sequences

SEQ ID NO. 1 (amino acid sequence of β-casein A2):

SEQ ID NO. 2 (amino acid sequence of β-casein A1):

SEQ ID NO. 3 (amino acid sequence A1N): AQTQSLVYPF PGPIHN

SEQ ID NO. 4 (amino acid sequence A2N): AQTQSLVYPF PGPIPN SEQ ID NO. 5 (amino acid sequence of Tot2): AVPYPQR

SEQ ID NO. 6 (A1S amino acid sequence): IHPFAQTQSL VYPFPGPIHN

SEQ ID NO. 7 (AIT amino acid sequence): IHPFAQTQSL VYPFPGPIHN SLPQNIPPLT QTPVVVPPFL QPEVMGVSK

SEQ ID NO. 8 (A2S amino acid sequence): IHPFAQTQSL VYPFPGPIPN

SEQ ID NO. 9 (A2T amino acid sequence): IHPFAQTQSL VYPFPGPIPN SLPQNIPPLT QTPVVVPPFL QPEVMGVSK

SEQ ID NO. 10 (amino acid sequence of Tot1): VLPVPQK

--->

LIST OF SEQUENCES

<110> Société des Produits Nestlé S.A.

<120> DETECTION AND QUANTIFICATION OF CONTENTS

PROTEOSOPEPTONES AND/OR BETA-CASEIN CONTENT AND NUTRIENT

COMPOSITION WITH A REDUCED CONTENT OF BETA-CASEIN PRODUCTS

PROTEOSOPEPTONES

<130> 16547

<150>EP18214628.2

<151> 2018-12-20

<160> 10

<170> BiSSAP 1.3.6

<210> 1

<211> 209

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of beta-casein A2

<400> 1

Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu

1 5 10 15

Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys

20 25 30

Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys

35 40 45

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

50 55 60

Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

65 70 75 80

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

85 90 95

Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu Met Pro Phe Pro

100 105 110

Lys Tyr Pro Val Glu Pro Phe Thr Glu Ser Gln Ser Leu Thr Leu Thr

115 120 125

Asp Val Glu Asn Leu His Leu Pro Leu Pro Leu Leu Gln Ser Trp Met

130 135 140

His Gln Pro His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln

145 150 155 160

Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys

165 170 175

Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu

180 185 190

Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile

195 200 205

Val

<210> 2

<211> 209

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of beta-casein A1

<400> 2

Arg Glu Leu Glu Glu Leu Asn Val Pro Gly Glu Ile Val Glu Ser Leu

1 5 10 15

Ser Ser Ser Glu Glu Ser Ile Thr Arg Ile Asn Lys Lys Ile Glu Lys

20 25 30

Phe Gln Ser Glu Glu Gln Gln Gln Thr Glu Asp Glu Leu Gln Asp Lys

35 40 45

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

50 55 60

Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

65 70 75 80

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

85 90 95

Lys Val Lys Glu Ala Met Ala Pro Lys His Lys Glu Met Pro Phe Pro

100 105 110

Lys Tyr Pro Val Glu Pro Phe Thr Glu Ser Gln Ser Leu Thr Leu Thr

115 120 125

Asp Val Glu Asn Leu His Leu Pro Leu Pro Leu Leu Gln Ser Trp Met

130 135 140

His Gln Pro His Gln Pro Leu Pro Pro Thr Val Met Phe Pro Pro Gln

145 150 155 160

Ser Val Leu Ser Leu Ser Gln Ser Lys Val Leu Pro Val Pro Gln Lys

165 170 175

Ala Val Pro Tyr Pro Gln Arg Asp Met Pro Ile Gln Ala Phe Leu Leu

180 185 190

Tyr Gln Glu Pro Val Leu Gly Pro Val Arg Gly Pro Phe Pro Ile Ile

195 200 205

Val

<210> 3

<211> 16

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence A1N

<400> 3

Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly Pro Ile His Asn

1 5 10 15

<210> 4

<211> 16

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence A2N

<400> 4

Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly Pro Ile Pro Asn

1 5 10 15

<210> 5

<211> 7

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of Tot2

<400> 5

Ala Val Pro Tyr Pro Gln Arg

15

<210> 6

<211> 20

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of A1S

<400> 6

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile His Asn

20

<210> 7

<211> 49

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of A1T

<400> 7

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile His Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

<210> 8

<211> 20

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of A2S

<400> 8

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile Pro Asn

20

<210> 9

<211> 49

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence A2T

<400> 9

Ile His Pro Phe Ala Gln Thr Gln Ser Leu Val Tyr Pro Phe Pro Gly

1 5 10 15

Pro Ile Pro Asn Ser Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr

20 25 30

Pro Val Val Val Pro Pro Phe Leu Gln Pro Glu Val Met Gly Val Ser

35 40 45

Lys

<210> 10

<211> 7

<212>PRT

<213> Unknown

<220>

<223> amino acid sequence of Tot1

<400> 10

Val Leu Pro Val Pro Gln Lys

15

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Claims (15)

1. A method for obtaining a whey protein fraction having a reduced content of protease peptones derived from β-casein, the method comprising the following steps: (i) obtaining a whey protein fraction; (ii) detecting and quantifying said β-casein-derived protease peptones in said whey protein fraction; And (iii) selecting said whey protein fraction containing no more than 10% by weight of β-casein-derived proteose peptones of the total amount of protein in said whey protein fraction, to form the selected whey protein fraction, wherein (ii) detecting and quantifying said β-casein-derived proteose peptones in said whey protein fraction comprises the steps of: (a) obtaining the milk-based product to be analyzed; (b) performing analysis of said product by liquid chromatography and mass spectrometry; (c) detecting and/or quantifying said β-casein-derived protease peptones and/or β-casein in said product by detecting compounds with target mass/charge m/z values or deconvolving one or more mass spectra to calculate monoisotopic masses. 2. A method for obtaining a whey protein fraction having a reduced content of protease peptones derived from β-casein, the method comprising the following steps: (i) obtaining a whey protein fraction; (ii) reducing the content of β-casein-derived proteose peptones in said whey protein fraction to a concentration of at most 10% by weight of the total amount of protein in the whey protein fraction, to form whey protein fractions with reduced proteose peptones, in which the content of proteose peptones is reduced by gel filtration. 3. A method for producing a nutritional composition having a reduced content of β-casein-derived proteose peptones, the method comprising the following steps: (i) obtaining a whey protein fraction having a reduced content of β-casein-derived protease peptones according to claim 1 or 2; (ii) obtaining said nutritional composition with a reduced content of protease peptones from β-casein from said fraction with a reduced whey protein content.