Determining and quantifying peptone content and / or beta-casein content and nutritional compositions with reduced beta-casein derived peptone content

Abstract

The present invention relates to nutritional compositions for determining and quantifying peptone content and / or beta-casein content and having reduced beta-casein derived peptone content. The invention also relates to a method for determining and quantifying beta-casein-derived peptones and / or beta-casein, comprising the steps of: (i) providing a milk-based product to be analyzed; (ii) subjecting the product to analysis using liquid chromatography-mass spectrometry; (iii) the single isotope mass is calculated by detecting compounds having a defined m / z value or deconvolution mass spectrograms to determine and / or quantify the beta-casein derived peptone and / or beta-casein in the product. The present invention also relates to nutritional compositions having reduced beta-casein derived peptone content and their use for, for example, treating, preventing and / or ameliorating abdominal pain in infants.

Description

[0001] This application is filed on November 28, 2019, and the name of the invention is “Determining and Quantifying Peptone content and / or β-casein content and having reduced β-casein derived Divisional application of Chinese application No. 201980083331.7 entitled "Nutritional composition with high peptone content". Technical Field

[0002] The present invention relates to a method for determining and quantifying β-casein derived The present invention also relates to nutritional compositions and infant formulas comprising reduced β-casein derived proteins. Peptone (such as PP-5, PP8s and PP8f) content. Background Art

[0003] Cow's milk is considered nature's perfect food, providing a valuable source of nutrients, including high-quality protein, carbohydrates, and selected micronutrients. Milk proteins are primarily caseins, with the remaining proteins classified as whey proteins, primarily β-lactoglobulin and α-lactalbumin. Among the caseins, β-casein is the second most abundant protein in cow's milk and has an excellent amino acid balance. However, different mutations in the bovine β-casein gene have led to the development of several genetic variants, the most common of which are the so-called A1 and A2 variants.

[0004] The A1 and A2 variants of β-casein differ at amino acid position 67 of the secreted β-casein due to a single nucleotide difference: the β-casein A1 variant has histidine (His) at this position, and the β-casein A2 variant has proline (Pro) at this position. Figure 1 This situation is shown.

[0005] The β-casein A2 variant is more similar to human β-casein in terms of digestion. The β-casein A1 variant has been suggested to be involved in cow milk intolerance, and a statistically significant positive correlation was observed between abdominal pain and stool consistency when subjects were fed an A1 diet rather than an A2 diet (Ho et al., 2014).

[0006] The observed differences in digestion between the A1 and A2 variants may be due to this specific polymorphism, which results in a major conformational change in the secondary structure of β-casein. Gastrointestinal proteolytic digestion of the A1 (but not the A2) variant of β-casein leads to the production of β-casomorphin 7 (BCM-7), an exogenous opioid peptide (exorphin) that activates opioid receptors systemically (EFSA Scientific Report, 2009). Figure 2 This situation is shown.

[0007] Originally, all dairy cows produced milk containing only the A2 variant of beta-casein, but genetic variation has led to the creation of mixed herds. As a result, herds commonly used for milk production typically produce a mixture of the A1 and A2 variants of beta-casein, but with varying amounts of A1 compared to A2.

[0008] Studies have shown that plasmin hydrolyzes β-casein to produce γ-casein and Peptones (PP8 Fast, PP8 Slow and PP-5). During acidification or enzymatic treatment with chymosin, Peptones do not precipitate with caseins and are retained in the whey fraction (reviewed in Karamoko et al., 2013). PP-8 and PP-5 correspond to the 29-105 / 7 sequence and the 1-105 / 7 sequence, respectively, and both include position 67. Therefore, whey products derived from milk containing A1 β-casein will likely contain A1 type Peptone. Peptones are derived from the same gene and are produced by endogenous events, so they are also considered to be protein forms of β-casein.

[0009] Intact protein analysis using reversed-phase high-performance liquid chromatography (RP-HPLC) or capillary electrophoresis (CE) with UV detection can effectively separate most β-casein variants from raw milk samples (for examples, see de Jong et al., 1993; Visser et al., 1995; Bonfatti et al., 2008; Poulsen et al., 2016). However, in finished products, processing conditions cause proteins to react with reducing sugars (Maillard reactions), resulting in the generation of multiple protein forms containing one or more sugar adducts (Fenaille et al., 2006). As a result, protein peaks split and broaden, resulting in overlap that hinders analysis of individual protein forms using UV detection (Vallejo-Cordoba, 1997; Feng et al., 2017).

[0010] Human breast milk (HBM) is known to contain the A2 form of human beta-casein. By definition, it represents the gold standard in infant nutrition. Therefore, there is a need to provide (synthetic) nutritional compositions that mimic the composition of HBM as closely as possible.

[0011] Due to the observed differences in human digestion of the A1 and A2 variants of bovine β-casein, it would be advantageous to be able to detect and quantify β-casein and its protein forms in a simple and efficient manner. Currently, there is no simple and efficient method to characterize and quantify the various milk proteins and their protein forms (such as peptone).

[0012] In addition, it is necessary to provide an A1 β-casein or β-casein derived The nutritional composition may contain a relatively small amount or proportion of peptone, or may not contain these substances in any form.

[0013] It is also necessary to provide a nutritional composition comprising a whey fraction in which beta-casein derived Peptone has been removed or reduced. Summary of the Invention

[0014] One object of the present invention relates to a method for the efficient and simple detection of β-casein and its protein forms (especially its β-casein-derived Improved method for reducing β-casein derived Method for determination of peptone content.

[0015] Another object of the present invention is to provide a nutritional composition, such as an infant formula, that can prevent colic and / or improve stool consistency in infants.

[0016] Therefore, one aspect of the present invention relates to a method for determining and / or quantifying β-casein derived A method for producing peptone and / or β-casein, said method comprising the steps of:

[0017] (i) providing a dairy-based product to be analyzed;

[0018] (ii) subjecting the product to analysis using liquid chromatography-mass spectrometry;

[0019] (iii) determining and / or quantifying the β-casein derived compounds in the product by detecting compounds with defined m / z values ​​or calculating monoisotopic masses by deconvoluting one or more mass spectra. Peptone and / or β-casein.

[0020] A second aspect of the present invention relates to a method for producing a β-casein-derived A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps:

[0021] (i) providing a whey protein fraction;

[0022] (ii) determining and quantifying the β-casein derived protein in the whey protein fraction as described herein Peptone; and

[0023] (iii) selecting a whey protein fraction having at most 10% by weight of β-casein derived proteins based on the total protein in the whey protein fraction The whey protein fraction is purified by the peptone to form a selected whey protein fraction.

[0024] A third aspect of the present invention relates to a method for producing a β-casein-derived A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps:

[0025] (i) providing a whey protein fraction;

[0026] (ii) based on the total protein in the whey protein fraction, the β-casein-derived The peptone content is reduced to a concentration of at most 10% by weight, thereby forming a whey protein fraction with a reduced content.

[0027] A fourth aspect of the present invention relates to a method for producing a β-casein-derived A method for preparing a nutritional composition having a high peptone content, the method comprising the steps of:

[0028] (i) providing a selected whey protein fraction or a whey protein fraction having a reduced content as described herein;

[0029] (ii) preparing a reduced β-casein derived protein from said selected whey protein fraction, said reduced whey protein fraction or a mixture thereof Nutritional composition with peptone content.

[0030] A fifth aspect of the present invention relates to a protein derived from a protein having reduced β-casein A peptone-rich whey protein fraction obtainable by a method as described herein.

[0031] A sixth aspect of the present invention relates to a nutritional composition comprising a whey protein fraction having a reduced content of β-casein derived proteins as described herein. Peptone may be obtained by the methods described herein.

[0032] A seventh aspect of the present invention relates to a nutritional composition as described herein for use as a medicament.

[0033] An eighth aspect of the present invention relates to an infant formula as described herein, which is used to treat, prevent and / or improve colic in infants and / or improve intestinal comfort in the infants.

[0034] A ninth aspect of the present invention relates to an infant formula as described herein, which is used for treating, preventing and / or improving lactose intolerance in infants.

[0035] A tenth aspect of the present invention relates to the use of an infant formula as described herein for improving stool consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A partial sequence alignment of the A2β-casein sequence and the A1β-casein sequence is shown for amino acids 59 to 71. Amino acid position 67 is highlighted to show that the one amino acid difference between A1β-casein and A2β-casein is His and Pro, respectively;

[0037] Figure 2 A partial sequence alignment of the A2 and A1 β-casein sequences for amino acids 59-71 is shown. The one-amino acid difference between the two β-caseins affects the cleavage ability of the two chains, as the A2 β-casein is not cleaved due to the presence of a Pro residue at position 67, while the A1 β-casein is cleaved at the N-terminus of the His residue, potentially forming a seven-amino acid sequence, β-casomorphin-7 (BCM-7).

[0038] Figure 3 A description of the steps of the LC-HRMS method is shown: (A) sample preparation, (B) deconvoluted spectrum of the monoisotopic mass of intact β-casein, and (C) bubble plot representation of the intact β-casein region (bubble size indicates signal intensity).

[0039] Figure 4 Global fingerprint analysis and intact β-casein regions are shown. Major milk proteins were isolated from the global fingerprint of a raw milk sample in (A). Different proteoforms of intact β-casein were detected in (B). A1, A2, and B are genetic variants, and the superscript symbols correspond to the number of lactose (L) adducts.

[0040] Figure 5 Examples of global fingerprint analysis of raw milk, human breast milk, infant formula (IF), skim milk powder (SMP), and whey samples are shown;

[0041] Figure 6 Shown are bubble plots (intact β-casein area) for seven A2 infant formula samples that were either untreated or spiked with 5% β-casein A1 (5 g intact β-casein A1 / 100 g total intact β-casein);

[0042] Figure 7 Figure 3 shows how glycation affects MS readings. (A) Bubble plot (intact β-casein region) shows increasing lactosylation in a solid-state glycation experiment. (B) The percentage of unglycated signal (black bars) decreases over time, and (C) shows that the total β-casein signal decreases with increasing glycation and (D) is linearly related to the percentage of unglycated signal. The signal reduction is plotted as a function of the percentage of unglycated signal, allowing calculation of a glycation correction factor (E).

[0043] Figure 8 Characterization of β-casein standards and development of a β-casein calibration curve are shown. Analysis of the β-casein standard showed that approximately 15% of the total signal could not be attributed to intact β-casein (A). A linear calibration curve was established using the normalized intact β-casein MS signal (SMP corrected for glycation) and the adjusted β-casein content (B). Cross-mixing experiments between A1|A1 and A2|A2 skim milk samples showed that this relationship remained linear even in a dairy matrix (C). Protein content was kept constant during the cross-mixing experiments;

[0044] Figure 9 Quantification of intact β-casein in infant formula is shown. β-casein was quantified in seven infant formula samples (A), where each sample was analyzed five times in triplicate. Theoretical values ​​are based on the formula and general milk protein composition. Another set of infant formulas (based on A2 or on skim milk powder containing various genetic variants) was analyzed in triplicate on one day (B). The shaded bars represent best estimates, as the whey % is not shown on the boxes for those samples.

[0045] Figure 10 The results of LC-HRMS analysis of intact proteins in different whey protein concentrates (WPC) are shown. The intact protein region is marked by a circle with a solid line edge, β-casein Peptone is included in the area marked by the circle with a dashed edge;

[0046] Figure 11 Shown Figure 10 The shown image is an enlarged view of the area marked by the dotted circle, which shows the The content of peptone. Black indicates β-casein A2 derived Peptone, gray indicates β-casein A1 derived peptone, and white indicates unassigned compounds;

[0047] Figure 12 The results of LC-HRMS analysis of intact proteins in different finished products are shown, with an emphasis on Peptone area.

[0048] Figure 13 The relationship between the measured PP5 signals for PP5 A1 and PP5 A2 with respect to the percentage of A2SMP in the samples is shown. The protein content was kept constant in the cross-mixing experiments;

[0049] Figure 14 Schematic diagram showing intact β-casein and the products produced by cleavage of β-casein. The locations of tryptic peptides are indicated by horizontal arrows with the common peptides Tot1 and Tot2 and the specific peptides A1 / 2N, A1 / 2S, and A1 / 2T. Tot1 and Tot2 are located at the C-terminus of β-casein, while the sequences of A1 and A2 are identical and the sequence is not present in In peptone, peptides A1 / 2N, A1 / 2S, and A1 / 2T include amino acid 67, which differs between A1 and A2;

[0050] Figure 15 Shown are the results obtained by LC-MS for the peptide A1S of tryptic digests of different finished products including skim milk powder and several infant formulas;

[0051] Figure 16 Shown are the results obtained by LC-MS for the peptide A1N of tryptic digests of different finished products including skimmed milk powder and several infant formulas;

[0052] Figure 17 Shown are the results obtained by LC-MS for the peptide A1T of tryptic digests of different finished products including skimmed milk powder and several infant formulas;

[0053] Figure 18 Shown are the results obtained by LC-MS for the peptide A2S from tryptic digests of different finished products including skim milk powder and several infant formulas;

[0054] Figure 19 Shown are the results obtained by LC-MS for tryptic digests of different finished products including skim milk powder and several infant formulas for peptide A2N;

[0055] Figure 20Shown are the results obtained by LC-MS for the peptide A2T from tryptic digests of different finished products including skim milk powder and several infant formulas;

[0056] Figure 21 Results for peptide Tot1 are shown for tryptic digests of different finished products including skimmed milk powder and several infant formulas;

[0057] Figure 22 Results for peptide Tot2 are shown for tryptic digests of different finished products including skimmed milk powder and several infant formulas;

[0058] Figure 23 Shown are the Tot1 peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0059] Figure 24 Shown are the Tot2 peptide contents of tryptic digests of skim milk powder, lactose and different whey protein concentrates (WPC) measured using LC-MS;

[0060] Figure 25 Shown are the AS1 peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0061] Figure 26 Shown are the A1N peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0062] Figure 27 Shown are the A1T peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0063] Figure 28 Shown are the A2S peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0064] Figure 29 Shown are the A2N peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0065] Figure 30 Shown are the A2T peptide contents of tryptic digests of skim milk powder, lactose, and different whey protein concentrates (WPC) measured using LC-MS;

[0066] The present invention will now be described in more detail hereinafter. DETAILED DESCRIPTION

[0067] definition

[0068] Before discussing the present invention in further detail, the following terms and conventions are first defined.

[0069] The term "LC" refers to "liquid chromatography" as known to those skilled in the art. It should be emphasized that LC also includes HPLC (ie, high performance liquid chromatography) and UPLC (ie, ultra-performance liquid chromatography).

[0070] The term "MS" refers to "mass spectrometry" as known to those skilled in the art. It should be emphasized that MS also includes HRMS (ie high resolution mass spectrometry).

[0071] The term "compounds with defined m / z values" means that when measuring a given sample, the MS is set to measure only compounds with m / z values ​​explicitly defined by the user.

[0072] The term "A1 / A1 cow" refers to a cow having the homozygous genotype A1A1. Milk obtained from an A1 / A1 cow is referred to as "A1 milk."

[0073] The term "A2 / A2 cow" refers to a cow having the homozygous genotype A2A2. Milk obtained from A2 / A2 cows is referred to as "A2 milk."

[0074] The term "A1 / A2 cow" refers to a cow with the heterozygous genotype A1A2. Milk obtained from an A1 / A2 cow is referred to as "A1 / A2 milk."

[0075] The term "A1 whey" refers to whey obtained essentially from A1 cow's milk.

[0076] The term "A2 whey" refers to whey obtained substantially from A2 cow's milk.

[0077] The term "A1 / A2 whey" refers to whey made from A1 / A2 milk, or a mixture of A1 whey and A2 whey, or whey made from A1 milk and A2 milk, A1 milk and A1 / A2 milk, A2 milk and A1 / A2 milk, or a mixture of A1 milk, A2 milk and A1 / A2 milk.

[0078] The term "A2 β-casein" refers to the A2 variant (secreted form of the protein) of bovine β-casein having the amino acid sequence according to SEQ ID NO: 1. In the context of the present invention, other variants including proline at position 67 may be included in A2 β-casein.

[0079] The term "A1 β-casein" refers to the A1 variant (secreted form of the protein) of bovine β-casein having the amino acid sequence according to SEQ ID NO: 2. SEQ ID NO: 1 and SEQ ID NO: 2 differ only in that A1 β-casein includes a histidine at position 67, while A2 β-casein includes a proline at position 67. In the context of the present invention, other variants including a histidine at position 67 may be included in A1 β-casein.

[0080] The term "intact β-casein" refers to a protein that has not been cleaved except for removal of the signal sequence, such as the protein disclosed by SEQ ID NO: 1 and SEQ ID NO: 2.

[0081] The term "β-casomorphin 7," also described as "BCM-7," refers to a peptide having the amino acid sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile.

[0082] The term "WPC" means "whey protein concentrate". In this context, WPC includes traditional WPC according to the USP definition as well as lactose-reduced whey and mineral-reduced whey, ie protein levels may be as low as 10% w / w.

[0083] The term "WPI" means "whey protein isolate" and is a whey protein concentrate having a whey protein content of not less than 90% by weight on a dry basis.

[0084] The term "whey protein fraction" refers to a composition comprising whey protein (eg WPC and / or WPI).

[0085] The term "standard SMP" refers to "standard skim milk powder" and is derived from milk obtained from a mixed dairy herd and therefore contains multiple variants of β-casein, including A1 β-casein and A2 β-casein.

[0086] The term "A2 SMP" refers to "A2 skimmed milk powder" and contains only A2 beta-casein and no A1 beta-casein.

[0087] The term "proteoforms" refers to the highly related protein molecules resulting from all combinatorial sources of variation that give rise to the products produced by a single gene. This includes products that differ due to genetic variation, or due to differences in spliced ​​RNA transcripts and post-translational modifications.

[0088] the term" Peptone" is the same as that used by Swaisgood in 1982 Same as peptone, i.e. the term " "Peptone" refers to those proteins / peptides that remain in solution after heating milk at 95°C for 20 minutes and then acidifying the milk to pH 4.7 with 12% trichloroacetic acid.

[0089] The term "β-casein derived "Peptone" refers to a protein derived solely from β-casein Peptones, such as PP-5, PP-8 Fast, and PP-8 Slow.

[0090] the term" "Peptone 5," "PP5," or "PP-5" refers to residues 1-105 and 1-107 derived from beta-casein.

[0091] the term" "Peptone 8 fast," "PP8f," or "PP8 fast" refers to residues 1-28 derived from β-casein. PP8 fast may also be referred to as "bcas4P 1-28."

[0092] the term" "Peptone 8 slow," "PP8s," or "PP8 slow" refers to residues 29-105 and 29-107 derived from β-casein. PP8 slow may also be referred to as "bcas1P 29-105" and "bcas1P 29-107."

[0093] The term "infant" refers to a child under the age of 12 months. In one embodiment of the present invention, the term "infant" can be expanded to include children of any age up to 18 months, or to include children of any age up to 24 months.

[0094] As used herein, the term "infant formula" refers to a food that is intended for specific nutritional use for infants during the first few months after birth (such as from 0 to 12 months, 0 to 10 months, 0 to 8 months, 0 to 6 months or 0 to 4 months), and that itself meets the multiple nutritional needs of such a population (in compliance with Article 2(c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formula and stage 2 infant formula). It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and Special Infants (including foods for special medical purposes). The expression "infant formula" covers both "stage 1 infant formula (starter infant formula)" and "stage 2 infant formula (follow-up formula)" or "follow-on formula for older infants".

[0095] "Stage 2 infant formula" or "follow-on formula" refers to a formula given from the 6th month onwards. Infant formula constitutes the main liquid element of the gradually diversified diet of this group of people.

[0096] In the context of the present invention, the term "powder" refers to a dry, bulk solid composed of a plurality of very fine particles that flow freely when shaken or tilted. The powder may contain water in an amount not exceeding 10%, such as not exceeding 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1% or 0.5%.

[0097] The term "nutritional composition" refers to a composition that provides nutrients to an individual. The nutritional composition is typically taken 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 (e.g., powder) or liquid form.

[0098] In a particular embodiment, the composition of the invention is a hypoallergenic nutritional composition.The expression "hypoallergenic nutritional composition" refers to a nutritional composition that is less likely to cause an allergic reaction.

[0099] In a specific embodiment, the nutritional composition of the present invention is a "synthetic nutritional composition." The expression "synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological methods, the chemical properties of which may be identical to the mixture naturally occurring in mammalian milk (i.e., the synthetic nutritional composition is not human milk).

[0100] "Probiotics" refers to microbial cell preparations or microbial cell components that have a beneficial effect on the health or well-being of the host. The definition of probiotics was given by Salminen S et al. in 1999.

[0101] "Prebiotics" are selectively fermented ingredients that produce specific changes in the composition and / or activity of the gastrointestinal microbiota that confer a health benefit on the host. Roberfroid MB discussed prebiotics in a 2007 article;

[0102] Detection and / or quantification of β-casein-derived Peptone and / or β-casein

[0103] According to a first aspect, the present invention relates to a method for determining and / or quantifying β-casein derived A method for producing peptone and / or β-casein, said method comprising the steps of:

[0104] (i) providing a dairy-based product to be analyzed;

[0105] (ii) subjecting the product to analysis using liquid chromatography-mass spectrometry;

[0106] (iii) determining and / or quantifying the β-casein derived compounds in the product by detecting compounds with defined m / z values ​​or calculating monoisotopic masses by deconvoluting one or more mass spectra. Peptone and / or β-casein.

[0107] The dairy based product may be selected from a range of finished products such as infant formula, maternal nutrition, adult nutrition or dairy products such as milk, milk powder, liquid whey, whey powder, caseinate, WPC or WPI.

[0108] In one embodiment, the milk-based product to be analyzed is an infant formula or a whey protein fraction.

[0109] 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. Preferably, the powder is dissolved by dispersing it in water. However, the powder may also be dissolved in other liquids, such as a reducing agent, a buffer (such as ammonium bicarbonate, Tris, trisodium citrate, HEPES, TEAB (triethylammonium bicarbonate)) or a denaturing buffer as defined below, and combinations thereof.

[0110] In one embodiment, the powder is dissolved in a reducing agent and a buffer solution, and then optionally a denaturing buffer is added. In another embodiment, the powder is dissolved in a reducing agent and a denaturing buffer, and then optionally a buffer solution is added. In yet another embodiment, the powder is dissolved in a buffer solution and a denaturing buffer, and then optionally a reducing agent is added.

[0111] In one embodiment, the powder is dispersed in a liquid to a concentration of at most 10% w / v protein, such as at most 9.5% w / v protein in the liquid, such as at most 9% w / v protein in the liquid, such as at most 8.5% w / v protein in the liquid, such as at most 8% w / v protein in the liquid, such as at most 7.5% w / v protein in the liquid, such as at most 7% w / v protein in the liquid, such as at most 6.5% w / v protein in the liquid, such as at most 6% w / v protein in the liquid, such as at most 5.5% w / v protein in the liquid. / v protein in the liquid, such as up to 5% w / v protein in the liquid, such as up to 4.5% w / v protein in the liquid, such as up to 4% w / v protein in the liquid, such as up to 3.5% w / v protein in the liquid, such as up to 3% w / v protein in the liquid, such as up to 2.5% w / v protein in the liquid, such as up to 2% w / v protein in the liquid, such as up to 1.5% w / v protein in the liquid, such as up to 1% w / v protein in the liquid, such as up to 0.5% w / v protein in the liquid.

[0112] After the sample is dissolved, or in order to dissolve the sample, the sample is denatured and reduced using a denaturing and reducing buffer (such as urea, thiourea, guanidine hydrochloride, DTT, β-mercaptoethanol, and TCEP).

[0113] In one embodiment, the sample is dissolved in water and further diluted in a mixture of trisodium citrate, guanidine hydrochloride, and DTT.

[0114] In another embodiment, the sample is dissolved in Tris and urea and further diluted in ammonium bicarbonate and DTT.

[0115] Preferably, the sample is also clarified before analysis. This is done by centrifugation. However, this can also be done by filtration.

[0116] In one embodiment, the product to be analyzed is analyzed using intact protein analysis, wherein the protein is intact when the product is analyzed using liquid chromatography followed by mass spectrometry (LC-MS), such as liquid chromatography high resolution mass spectrometry (LC-HRMS). Different protein forms of β-casein and genetic variants of full-length β-casein (including A1 and A2 variants) can be distinguished using intact protein analysis. Thus, measurement of intact proteins from a product can yield an intact protein fingerprint of the product. Additionally, intact protein analysis methods can be used to distinguish Different genetic variants of peptones.

[0117] In another embodiment, the product is analyzed by peptide analysis, which includes a step of enzymatic digestion of the product prior to analysis. Thus, the product is enzymatically digested, for example by trypsin or GluC (endoproteinase GluC (Staphylococcus aureus protease V8)) before analysis by LC-MS.

[0118] Enzymatic digestion can be used to determine whether A1 β-casein is present in a product, or whether β-casein derived from A1 and / or A2 β-casein is present. However, since the method development involves protein digestion, it is not possible to distinguish peptone and full-length β-casein, but only able to distinguish between the A1 and A2 genetic variants. This is done by detecting a sequence covering amino acid position 67, which differs between the A1 and A2 variants. Therefore, this will not provide a complete protein fingerprint, but will detect the presence of the A1 variant and possibly the BCM-7 precursor.

[0119] In another embodiment, the enzymatic digestion is performed by trypsin digestion or GluC digestion.

[0120] In one embodiment, the liquid chromatography is high performance liquid chromatography. In another embodiment, the liquid chromatography is ultra performance liquid chromatography. In yet another embodiment, the liquid chromatography is nano liquid chromatography.

[0121] Standard setups for performing liquid chromatography known to those skilled in the art can be used in the measurements of the present invention.

[0122] In a preferred embodiment for intact protein analysis, the sample can be separated using a 0.5 mL / min gradient on a C4 column. Preferably, a buffer containing trifluoroacetic acid is used. However, the gradient can be varied from 0.1 mL / min to 1 mL / min, such as from 0.1 mL / min to 0.5 mL / min. Alternatively, the following buffers can be used for LC separations, such as water / methanol or water / acetonitrile, which may include formic acid, difluoroacetic acid, and / or trifluoroacetic acid.

[0123] In a preferred embodiment for peptide analysis, the sample can be separated using a 75 μl / min gradient on a C18 column. Preferably, a buffer containing formic acid is used. However, the gradient can be varied from 0.1 μl / min to 100 μl / min, such as from 0.4 μl / min to 85 μl / min. Alternatively, the following buffers can be used for LC separations, such as water / methanol or water / acetonitrile, which may include formic acid, difluoroacetic acid, and / or trifluoroacetic acid.

[0124] MS chromatograms can be recorded on a machine such as the Thermo Orbitrap Elite or the Thermo Q-Exactive HF.

[0125] In one embodiment, MS chromatograms were recorded on a Thermo Orbitrap Elite (heater temperature: 60°C, sheath gas: 20, auxiliary gas: 5, sweep gas: 0, spray voltage: 3.8 kV, capillary temperature: 320°C, S-Lens RF level: 60%, mass range: 400 m / z to 2'000 m / z, resolution: 240'000, AGC target: 1e6).

[0126] In another embodiment, MS chromatograms were recorded on a Thermo Q-Exactive HF (heater temperature: 100°C, sheath gas: 53, auxiliary gas: 14, sweep gas: 3, spray voltage: 3.5 kV, capillary temperature: 320°C, S-Lens RF level: 70%, mass range: 400 m / z to 2'000 m / z, resolution: 240'000, AGC target: 1e6, maximum IT: 200 ms).

[0127] In one embodiment, the mass spectrometry is high resolution mass spectrometry.

[0128] In one embodiment, HRMS acquisition is preferably performed at a resolution higher than 100'000, such as between 120'000 and 240'000.

[0129] According to one embodiment, the β-casein derived compounds are determined and / or quantified by detecting compounds having m / z values ​​as defined above. Peptone. This allows the presence and content of specific peptide sequences to be detected. This is possible because enzymatic digestion produces specific peptides due to specific cleavage.

[0130] In one embodiment, the peptide method uses a non-targeted (data dependent acquisition - DDA) approach. In another embodiment, the peptide method uses a non-targeted (data independent acquisition - DIA) approach.

[0131] In yet another embodiment, the peptide method uses a PRM (parallel reaction monitoring) or MRM (multiple reaction monitoring) approach.Preferably, the precursor is selected and fragmented on the first quadrupole and the MS2 data recorded.

[0132] In one embodiment, the precursor can be selected based on the peptide list set forth as SEQ ID NOs: 3-10.

[0133] According to another embodiment, the mass spectral data obtained by measuring each sample using LC-MS is deconvoluted to calculate the monoisotopic mass. Thus, in one embodiment, the monoisotopic mass is calculated by deconvoluting one or more mass spectra to determine and / or quantify the β-casein derived molecule in the product. Peptone.

[0134] Deconvolution can be performed by commercially available software such as Thermo BioPharma Finder.

[0135] In one embodiment, a sliding window algorithm is used for deconvolution. In another embodiment, a fixed window algorithm is used for deconvolution.

[0136] In a preferred embodiment, deconvolution is performed by using Thermo BioPharma Finder 1.0 software using the Xtract algorithm (S / N threshold: 3, relative abundance threshold: 1%, match factor: 80%, retention threshold: 25%, overlap, charge state: 5 to 50, minimum intensity: 1, expected intensity error: 3, m / z: 600 to 2000, minimum number of detected charge states: 3) and a sliding window (time: 5 min to 20 min, target average mass spectrum width: 0.1 min, target average mass spectrum offset: 50%, merging tolerance: 1.5 Da, maximum retention time interval: 0.5 min, minimum number of detection intervals: 3, XIC).

[0137] The deconvoluted monoisotopic masses were compared with the main milk protein components αS1-CN, αS2-CN, β-CN, κ-CN, γ-CN, α-lactalbumin, β-lactoglobulin, CGMP and β-casein. The protein database of peptones was compared to the peptones. Combinatorial additions of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation, and pyroglutamate) were tested to identify the majority of signals.

[0138] Quantification of β-casein-derived One approach is to compare the signal intensity obtained by measuring peptone and / or β-casein with that derived from measuring known amounts of different The standard curves of peptone and / or β-casein are compared. From this, the amount of The absolute amount of peptone and / or β-casein was determined and the amount was quantified.

[0139] Alternatively, the focus The signal intensity of peptones can be expressed in relative terms, such as as a percentage of the total amount of any one protein in the sample or as a percentage of the β-casein content.

[0140] The compound to be identified and / or quantified is a β-casein derived In one embodiment, Peptone is derived from A1β-casein In another embodiment, the β-casein derived The peptone is PP8 fast, PP8 slow and / or PP-5. In yet another embodiment, the A1 β-casein derived The peptones were PP8 Fast, PP8 Slow and / or PP-5.

[0141] In one embodiment, the β-casein to be detected and / or quantified is A1 β-casein.

[0142] Derived from reduced beta-casein Whey protein fractions with peptone content (selected whey protein fractions)

[0143] In a second aspect, the present invention relates to a method for producing a β-casein-derived A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps:

[0144] (i) providing a whey protein fraction;

[0145] (ii) determining and quantifying the β-casein derived protein in the whey protein fraction as described herein Peptone; and

[0146] (iii) selecting a whey protein fraction having at most 10% by weight of β-casein derived proteins based on the total protein in the whey protein fraction The whey protein fraction is purified by the peptone to form a selected whey protein fraction.

[0147] Due to standard production methods, the available whey protein fractions to be used in finished products (such as nutritional compositions including infant formula) typically include A1 whey and / or A1A2 whey. However, this has not previously been considered an issue (e.g. for the production of A2 infant formula) because A1 β-casein will precipitate during whey preparation and will not become part of the whey protein fraction. However, as shown in the Examples, A1 β-casein derived from A1 β-casein can be used to produce a whey protein fraction that is not part of the whey protein fraction. Peptone is detected in whey protein fractions such as WPC.

[0148] In one embodiment the whey protein fraction is based on A1 whey and / or A1A2 whey.

[0149] Due to the advantages of the above methods for determining and quantifying β-casein variants and protein forms, β-casein derived proteins can be easily detected and quantified. The whey protein fractions can therefore be separated into fractions containing different amounts of peptone. Thus, it may be chosen to contain up to 10% by weight of the total amount of protein in the whey protein fraction. Protein fraction of peptone.

[0150] In one embodiment, the β-casein derived protein content of the selected whey protein fraction is based on the total protein in that whey protein fraction. Peptone content such as up to 9.5% by weight of beta-casein derived Peptone, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, further more preferably at most 6.5% by weight, such as at most 6% by weight, still more preferably at most 5.5% by weight, such as at most 5% by weight, most preferably at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, %, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, further more preferably at most 0.75% by weight, such as at most 0.50% by weight, still more preferably at most 0.25% by weight, such as at most 0.10% by weight, most preferably at most 0.05% by weight, such as at most 0.01% by weight.

[0151] Preferably, the total amount of protein in the whey protein fraction is measured using the Kjeldahl method (ISO 8968-1:2014).

[0152] In one embodiment, The peptone is PP8 fast, PP8 slow and / or PP-5. In another embodiment, The peptones were PP8 slow and / or PP-5.

[0153] In one embodiment, the PP8 slow and / or PP-5 content of the selected whey protein fraction is at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, still more preferably at most 5.5% by weight, such as at most 5% by weight, and most preferably at most 7.5% by weight. Preferably, the content of the compound of the present invention is at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, still more preferably at most 0.25% by weight, such as at most 0.10% by weight, most preferably at most 0.05% by weight, such as at most 0.01% by weight.

[0154] In yet another embodiment, The peptone was PP-5.

[0155] With reduced Whey protein fraction with reduced peptone content (whey protein fraction)

[0156] In a third aspect, the present invention relates to a method for producing a protein having reduced β-casein derived A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps:

[0157] (i) providing a whey protein fraction;

[0158] (ii) based on the total protein in the whey protein fraction, the β-casein-derived The peptone content is reduced to a concentration of at most 10% by weight, thereby forming a whey protein fraction with a reduced content.

[0159] In another aspect, the present invention relates to a method for producing a β-casein-derived A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps:

[0160] (i) Provide whey protein fraction with initial content of peptone;

[0161] (ii) derivatizing the β-casein in the whey protein fraction The peptone content is reduced to a concentration of at least 5× less than the initial content of the whey protein fraction, such as at least 8× less, such as at least 10× less, such as at least 15× less than the initial content of the whey protein fraction, thereby obtaining a whey protein fraction with a reduced content.

[0162] In one embodiment the whey protein fraction is based on A1 whey and / or A1A2 whey.

[0163] By way of example, this at least 5× less is to be understood as meaning that if the initial content was 5 μg / mg, a reduction of 5× (five-fold) would mean that the whey protein fraction with a reduced content contains at most 1 μg / mg, and similarly, if the signal intensity of the initial content was 500 000 000, the signal intensity of the whey protein fraction with a reduced content would be at most 100 000 000.

[0164] The at least 5× less, 8× less, 10× less, or 15× less reduction can be measured by the signal intensity obtained by the determination and quantification methods described herein.

[0165] In one embodiment, the determination and quantification of Method for testing whey protein fractions for the content of peptone to determine whether the whey protein fraction needs to be reduced or not before use Peptone content.

[0166] Based on the total protein in the whey protein fraction, the β-casein derived protein in the whey protein fraction was The peptone content is reduced to at most 10% by weight.

[0167] Preferably, the total amount of protein in the whey protein fraction is measured using the Kjeldahl analysis method (ISO 8968-1:2014).

[0168] In one embodiment, the reduction of However, it can also be reduced by ion exchange chromatography, affinity chromatography or membrane separation methods. Peptone content.

[0169] In another embodiment, the reduced whey protein content of the whey protein fraction can be determined and quantified by the method described above. Final content of peptone.

[0170] In one embodiment, the whey protein fraction derived from whey protein with reduced β-casein content is The peptone content is at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, still more preferably at most 5.5% by weight, such as at most 5% by weight, most preferably at most 4.5% by weight, such as at most 4% by weight, The amount of the present invention is at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, still more preferably at most 0.25% by weight, such as at most 0.10% by weight, most preferably at most 0.05% by weight, such as at most 0.01% by weight.

[0171] In one embodiment, The peptones were PP8 Fast, PP8 Slow and / or PP-5.

[0172] In another embodiment, The peptones were PP8 slow and / or PP-5.

[0173] In one embodiment, the whey protein fraction with a reduced content of PP8 and / or PP-5 has a content of at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, still more preferably at most 5.5% by weight, such as at most 5% by weight, and most preferably at most 7.5% by weight, based on the total protein in the whey protein fraction. The content of the present invention is preferably at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, still more preferably at most 0.25% by weight, such as at most 0.10% by weight, most preferably at most 0.05% by weight, such as at most 0.01% by weight.

[0174] In yet another embodiment, The peptone was PP-5.

[0175] Nutritional compositions (such as those with reduced Peptone content of infant formula)

[0176] In a fourth aspect, the present invention relates to a method for producing a protein having reduced β-casein derived A method for preparing a nutritional composition having a high peptone content, the method comprising the steps of:

[0177] (i) providing a selected whey protein fraction or a whey protein fraction having a reduced content as described herein;

[0178] (ii) preparing a whey protein fraction having a reduced content of whey protein from said selected whey protein fraction, said whey protein fraction having a reduced content of whey protein or a mixture thereof; The nutritional composition has a peptone content.

[0179] In a fifth aspect, the present invention relates to a method for producing a protein having reduced β-casein derived A whey protein fraction having a peptone content, which whey protein fraction is obtainable by the method described herein.

[0180] In a sixth aspect, the present invention relates to a nutritional composition comprising said whey protein fraction having a reduced content of β-casein derived proteins as described herein. Peptone may be obtained by the methods described herein.

[0181] In another embodiment, the beta-casein derived protein of a nutritional composition (such as an infant formula) is based on the total protein in the nutritional composition. The peptone content is at most 9% by weight.

[0182] Preferably, the total amount of protein in the whey protein fraction is measured using the Kjeldahl method (ISO 8968-1:2014).

[0183] In yet another embodiment, the beta-casein derived protein of the nutritional composition is based on the total protein in the whey protein fraction. The peptone content is at most 8.5% by weight, more preferably at most 8% by weight, such as at most 7.5% by weight, even more preferably at most 7% by weight, such as at most 6.5% by weight, still more preferably at most 6% by weight, such as at most 5.5% by weight, most preferably at most 5% by weight, such as at most 4.5% by weight, such as at most 4% by weight, at most 3.5% by weight, more preferably at most 3% by weight, such as at most 2.5% by weight, even more preferably at most 2% by weight, such as at most 1.5% by weight, still more preferably at most 1% by weight, such as at most 0.75% by weight, most preferably at most 0.50% by weight, such as at most 0.25% by weight, such as at most 0.1% by weight, such as at most 0.05% by weight, such as at most 0.01% by weight.

[0184] The nutritional composition of the present invention may be intended for use with any mammal, such as, for example, humans and pets (such as cats and dogs).In a preferred embodiment, the mammal is a human.

[0185] Examples of nutritional compositions are infant formula, maternal nutrition, adult nutrition or dairy products.

[0186] In one embodiment, the nutritional composition is an infant formula.

[0187] The general composition of a nutritional composition for use according to the invention, such as an infant formula, may optionally contain substances that may have a beneficial effect, such as probiotics, fiber, lactoferrin, nucleotides, nucleosides and / or other substances that are present in conventional amounts in nutritional compositions to be fed to infants.

[0188] The probiotic bacteria may be selected from Lactobacilli, such as Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus reuteri, and Bifidobacteria, such as Bifidobacterium lactis, Bifidobacterium breve and Bifidobacterium longum.

[0189] Nutritional compositions, such as infant formula, may also optionally contain prebiotics, such as non-digestible carbohydrates that promote the growth of beneficial intestinal bacteria.

[0190] In a preferred embodiment, the nutritional composition, such as an infant formula, comprises a prebiotic selected from the group consisting of fructooligosaccharides (FOS), levo-oligofructose, inulin, levo-inulin, lactulose, milk oligosaccharides (CMOS) or galacto-oligosaccharides (GOS).

[0191] The nutritional composition may also contain all vitamins and minerals considered necessary for a daily diet, which are present in the composition in nutritionally significant amounts.

[0192] Therefore, a preferred embodiment relates to a nutritional composition, such as an infant formula, which further comprises vitamins.

[0193] Minimum requirements for certain vitamins and minerals have been established. Examples of minerals, vitamins, and other nutrients that may be 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 typically added in the form of salts.

[0194] If necessary, nutritional compositions such as infant formula may contain emulsifiers and stabilizers, such as soy, lecithin, mono- and di-citrin, etc. This is particularly true if the composition is provided in liquid form.

[0195] A preferred embodiment relates to a nutritional composition, such as an infant formula, wherein the at least one carbohydrate source is selected from lactose, corn syrup solids, fructose, glucose, maltodextrin, dry glucose syrup, sucrose, trehalose, galactose, maltose, honey powder, starch, oligosaccharides, Left inulin or Left oligofructose.

[0196] Another preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising anhydrous milk fat.

[0197] Yet another preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising LC-PUFAs, such as DHA, EPA, DPA and / or ARA.

[0198] Nutritional compositions, such as infant formula, may also include flavoring agents, such as, but not limited to, vanillin.

[0199] A particularly preferred embodiment relates to a nutritional composition, such as an infant formula, further comprising fructo-oligosaccharides, such as LAFT inulin and / or LAFT oligofructose.

[0200] The nutritional composition, such as an infant formula, may be a liquid formula or a powder to be reconstituted before use. A preferred embodiment is a powdered infant formula.

[0201] In a preferred embodiment, the nutritional composition is a liquid composition prepared by reconstitution of a powder.

[0202] The present invention also provides a nutritional composition according to the invention for use as a complementary feeding for infants in association with human breast milk, such as an infant formula.

[0203] According to a specific embodiment, the infant is between 0 and 12 months old, preferably between 0 and 6 months old.

[0204] With reduced Therapeutic and non-therapeutic uses of nutritional compositions or infant formulas containing peptone .

[0205] In a seventh aspect of the present invention, the nutritional composition as described herein may be used as a medicament.

[0206] In an eighth aspect of the present invention, the infant formula as described herein relates to use for treating, preventing and / or ameliorating colic in infants.

[0207] In a ninth aspect of the present invention, the infant formula as described herein is directed to use for treating, preventing and / or improving lactose intolerance in infants.

[0208] A tenth aspect of the present invention relates to the use of an infant formula as described herein for improving stool consistency.

[0209] BCM-7 is known to affect opioid receptors and is associated with stool consistency and abdominal pain. Therefore, it does not contain or contains only very limited amounts of BCM-7 precursors (i.e., A1 β-casein derived Infant formulas containing peptones PP-5 and PP8) will result in less formation of BCM-7 during consumption of the infant formula, thereby having less adverse effects on stool consistency and abdominal pain.

[0210] Furthermore, it is believed that administering an infant formula that contains less BCM-7 precursor molecules, and therefore results in less formation of BCM-7, will result in less impact on affected opioid receptors, thereby preventing lactose intolerance in the infant later in life. There is growing evidence that events early in life may contribute to the disease, even if it may only manifest later in life.

[0211] In this context, treatment is understood as the situation where the disease is established, rather than prevention which occurs before the disease is established. Amelioration is understood as the situation where the disease is established but the disease is not treated in a way that makes it go away but only improves the health status of the individual suffering from the disease.

[0212] In another aspect, the present invention relates to a method for treating, preventing and / or ameliorating colic in infants.

[0213] In yet another aspect, the present invention relates to a method for treating, preventing and / or ameliorating lactose intolerance in infants.

[0214] It should be noted that embodiments and features described in the context of one aspect of the invention may also apply to the other aspects of the invention.

[0215] All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.

[0216] The present invention will now be described in further detail in the following non-limiting examples.

[0217] Example

[0218] Materials and methods

[0219] Chemical reagents and samples

[0220] Guanidine hydrochloride (RDD001), trisodium citrate dihydrate (S1804), DL-dithiothreitol (43819), and β-casein standard (C6905) were purchased from Sigma-Aldrich (St. Louis, MI, USA), TFA (1.08178.0050) was purchased from VWR International (Radnor, PA, USA), and LC-MS grade water (1.15333.1000) and acetonitrile (1.00029.1000) were purchased from Merck (Darmstadt, Germany).

[0221] milk samples

[0222] Raw milk from cows genotyped as A1 / A1 was collected from Teagasc Moorepark dairy farm of Fermoy (Co. Cork, Ireland), skimmed, and pasteurized using a Microthermics (UHT / HTSTElectric Model 25HV Hybrid) apparatus from Liquid Technologies (Wexford, Ireland) heated to 85°C for 23 seconds and then subjected to a 2-stage homogenization (GEA Niro Soavi SpA; Type: NS2006H (non-sterile)) at a total homogenization pressure of 2500 psi. The sample was spray dried to generate a skim milk powder sample referred to as A1|A1 SMP. Commercially available A1 / A2 and A2 / A2 SMPs were purchased from Europe and the United States, buffalo SMP was purchased from India, and pasteurized human breast milk from a pooled human donor (991-01-P) was purchased from Lee Biosolutions, Maryland Heights, MO USA.

[0223] Different batches from the same or different manufacturers are numbered A, B, C, etc.

[0224] Finished products and whey protein concentrates

[0225] The infant formula powder that commercially available is applicable to baby (0 month to 12 months) and children (1 year old and above) is purchased from different manufacturers, for different age groups.In experiment, different samples are expressed as IF1-IF22 (referring to 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.

[0226] Table 1

[0227] ID type stage protein% Casein*% β-Casein**% IF1 A2 1 10.2 35 1.19 IF2 A2 2 15.3 60 3.06 IF3 A2 3 13.9 60 2.78 IF4 A2 4 17.0 60 3.40 IF5 A2 1 10.4 30 1.04 IF6 A2 2 15.0 50 2.50 IF7 A2 3 15.0 60 3.00 IF8 A1 / A2 1 10.6 40 1.41 IF9 A1 / A2 2 14.9 <![CDATA[40 § ]]> 1.99 IF10 A1 / A2 3 15.3 <![CDATA[70 § ]]> 3.57 IF11 A1 / A2 3 22.2 78 5.77 IF12 A1 / A2 1 11.0 40 1.47 IF13 A1 / A2 1 10.2 35 1.25 IF14 A1 / A2 2 15.3 60 3.02 IF15 A1 / A2 3 15.0 60 3.07 IF16 A1 / A2 4 17.0 60 3.48 IF17 A2 1 9.9 30 1.01 IF18 A2 2 10.4 30 1.03 IF19 A2 3 12.8 50 1.94 IF20 A2 1 10.1 35 1.32 IF21 A2 2 11.7 60 2.41 IF22 A2 3 15.2 60 3.12

[0228] *As indicated on label (based on assumptions except §), **Calculated by multiplying protein content by casein content and dividing by 3 (as described in Swaiswell, 1995).

[0229] Three different commercially available WPC batches were tested (denoted A, B and C). WPC35 is a whey protein concentrate with 35% w / w protein.

[0230] Two different commercially available batches of WPC28 were tested (denoted A and B).WPC28 is a demineralised WPC with 28% w / w protein.

[0231] WPC80 is a commercially available alpha-lactalbumin enriched whey protein concentrate having 80% w / w protein.

[0232] Sample preparation

[0233] The powder was dispersed in water using a volumetric flask to form a 3.5% (w / v) protein solution, stirred at room temperature for at least 30 minutes, denatured with 4 volumes of denaturation buffer (7.5 M guanidine hydrochloride, 6.25 mM trisodium citrate, 23 mM DTT), incubated at room temperature for 30 minutes, and clarified by centrifugation at 16'000g for 10 minutes.

[0234] Trypsin digestion of intact proteins

[0235] The sample powder was dispersed into a 3.5% (w / v) protein solution of 50 mM Tris and 6 M urea by mixing on an orbital shaker for one hour.

[0236] 200 μL sample was diluted with 1000 μL ammonium bicarbonate (100 mM) and vortexed. 10 μL 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, quickly spun down, and 10 μL iodoacetamide (100 mM) was added. The sample was then incubated at room temperature and in the dark for 30 minutes, and then 6 μL trypsin (0.2 μg / μL) was added and incubated at 37° C. overnight. Trypsin digestion was stopped by adding 6 μL formic acid (10%). The digested sample was centrifuged at 14'000 g for 10 minutes, and the liquid phase was then transferred to a vial for injection and determined using LC-MS.

[0237] LC-MS analysis

[0238] Clarified samples were analyzed using LC-MS based on existing methods (Bonfatti et al., 2008; Frederiksen et al., 2011).

[0239] For intact protein analysis, the following 0.5 mL / min gradient was used on a C4 column (Acquity UPLC ProteinBEH C4, 1.7 μm, 2.1 mm × 150 mm) on the separation sample (Table 2):

[0240] Table 2

[0241] Time (minutes) 0 4 7 22 23 24 25 30 Buffer B% 15 35 42.5 52.5 80 80 15 15

[0242] Buffer A: 0.1% trifluoroacetic acid (TFA) aqueous solution; Buffer B: 0.1% TFA solution, acetonitrile: water = 90:10

[0243] MS signals were recorded in Full MS mode from 4 min to 20 min on a Thermo Q-Exactive HF (heater temperature: 100 °C, sheath gas: 53, auxiliary gas: 14, sweep gas: 3, spray voltage: 3.5 kV, capillary temperature: 320 °C, S-Lens RF level: 70%, mass range: 400 m / z to 2'000 m / z, resolution: 240'000, AGC target: 1e6, maximum IT: 200 ms).

[0244] For peptide analysis, i.e., trypsin digestion or GluC digestion, the following 75 μL / min gradient was used on a C18 column (Acquity UPLC BEH C18, 1.7 μm, 1.0 mm × 150 mm) for peptide separation (Table 3):

[0245] Table 3

[0246] Time (minutes) 0 30 31 33 35 45 Buffer B% 2 60 100 100 2 2

[0247] MS signals were recorded from 3.5 min to 35 min in PRM mode on a Thermo Q-Exactive HF (heater temperature: 30 °C, sheath gas: 8, auxiliary gas: 0, sweep 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, maximum IT: 100 ms, isolation window: 1.5 m / z, isolation offset: 0.5 m / z).

[0248] The inclusion list is shown in Table 4:

[0249] Table 4

[0250]

[0251] Data deconvolution

[0252] Raw data were deconvoluted using the Xtract algorithm of Thermo BioPharma Finder 1.0 software (S / N threshold: 3, relative abundance threshold: 1%, match factor: 80%, retention threshold: 25%, overlap, charge state: 5 to 50, minimum intensity: 1, expected intensity error: 3, m / z: 600 to 2000, minimum number of charge states detected: 3) and a sliding window (time: 5 min to 20 min, target average mass spectrum width: 0.1 min, target average mass spectrum offset: 50%, merging tolerance: 1.5 Da, maximum retention time interval: 0.5 min, minimum number of detection intervals: 3, XIC). Monoisotopic mass, total signal intensity, and apex RT were exported as csv files and used in the following steps.

[0253] Detection limit experiments were also performed by deconvolving the raw data files using a fixed window centered on the β-casein peak. The following parameters were used: S / N threshold: 1, relative abundance threshold: 1%, match factor: 25%, retention threshold: 25%, overlap, charge states: 12 to 30, minimum intensity: 1, expected intensity error: 3, m / z: 800 to 2000, minimum number of detected charge states: 3, time: 11 to 13.5 minutes, and relative intensity threshold: 1%. Monoisotopic mass and total signal intensity were used in the following steps.

[0254] Properties of protein forms

[0255] The deconvoluted monoisotopic masses were compared with the protein mass of the main milk protein components αS1-CN, αS2-CN, β-CN, κ-CN, γ-CN, α-lactalbumin, β-lactoglobulin, CGMP and The protein database of peptone was compared. The combined addition of standard protein modifications (phosphorylation, oxidation, lactosylation, glycosylation and pyroglutamate) was tested to identify most signals. Misjudged signals were corrected by manual verification. The total signal intensity of the relevant proteins or protein forms (i.e., total β-casein, β-casein lactosylation state, β-casein genetic variants, etc.) was extracted. For clarity, only β-casein genetic variants A1, A2 and B are detailed in the figure. β-casein I / H2 co-eluted with β-casein and merged with β-casein A2. Both variants have proline at position 67 and belong to the A2 type.

[0256] Glycation of solid proteins

[0257] The solid-state saccharification experiment is based on Fenaille, 2003 (Fenaille et al., 2003). Briefly, 45g of milk powder was incubated in a saturated potassium bicarbonate package for 8 to 10 days to reach 5.4% humidity (initial % was 3.8%). Nine 2.1g aliquots of the humidified powder were incubated in a 25mL glass tube in an oven at 60°C for 45 minutes to 24 hours. The glass tubes were moved to ice for 5 minutes to stop saccharification. Then, 20g of water was added to make a 3.5% (w / v) protein solution, which was mixed on a roller mixer at room temperature for 1 hour and 30 minutes. The solution was stored at 4°C overnight until the end of the experiment and was placed at room temperature for 2 hours before analysis. The samples were prepared as described above, except that the incubation step was carried out on a 60°C thermomixer (650rpm) for 10 minutes. MS chromatograms were recorded on a Thermo Orbitrap Elite (heater temperature: 60°C, sheath gas: 20, auxiliary gas: 5, sweep gas: 0, spray voltage: 3.8 kV, capillary temperature: 320°C, S-Lens RF level: 60%, mass range: 400 m / z to 2'000 m / z, resolution: 240'000, AGC target: 1e6).

[0258] Quantification of β-casein

[0259] Multiple injections of skim milk powder (SMP) samples were performed in each analytical series to normalize the signal across experiments.

[0260] An external calibration curve was established using a β-casein standard adjusted for protein purity (see text). The β-casein signal intensity was normalized using the average β-casein signal of the SMP samples (corrected for glycation, see above). The calibration curve was normalized to zero (y = 0.0524x, where x is the amount of β-casein injected in μg and y is a dimensionless value).

[0261] For each sample, the β-casein intensity value was first corrected for glycation and normalized using the average β-casein signal of the SMP sample (corrected for glycation). The injected amount of β-casein was then calculated using an external calibration curve. This calculation can be performed for total β-casein, for a given variant (e.g., A1 β-casein), or for a panel of variants (results for β-casein A2 also include the related I / H2 variant).

[0262] Method performance was evaluated for seven infant formula samples (stages 1 to 4, prepared using type A2 skim milk powder) using three replicate injections on five different days by two operators.

[0263] Example 1—LC-MS Method, Data Output, Deconvolution, and Visualization

[0264] To analyze intact proteins, infant formula and skim milk powder samples were dispersed in water to form a 3.5% (v / w) protein solution, denatured in 6 M guanidine, reduced with DTT, and clarified by centrifugation. Intact proteins were separated by UPLC on a C4 column using a water / ACN gradient with 0.1% TFA and analyzed by high-resolution mass spectrometry ( Figure 3 A). Chromatograms were sequentially deconvoluted using a sliding window and the Xtract algorithm to obtain the monoisotopic mass of the protein ( Figure 3 B). Finally, a bubble plot is used to visualize the protein forms, where the X-axis is their retention time, the Y-axis is the monoisotopic size, and the bubble area is the signal intensity ( Figure 3 C).

[0265] Example 2 - Global Fingerprint

[0266] Figure 4 A shows the method output for raw bovine milk and highlights the detection of the major milk proteins (αS1-, αS2-, β-, κ-, and γ-casein, α-lac, and β-lg). Zoom in on the β-casein region ( Figure 4 B) allows for detailed analysis of various β-casein protein forms including genetic variants and protein modifications (lactosylation adducts caused by processing were detected in all skim milk powder samples). The mass accuracy of the raw milk samples is shown in Table 5. Since a 1.5 Da merging tolerance was applied between the sliding windows to avoid splitting the signal into two masses 1 Da apart, the 1 Da difference may be caused by misinterpreted monoisotopic signals that were transmitted during the deconvolution process. This method can be used to determine the spectra of various raw materials and finished products ( Figure 5 Interestingly, the method readily detected caseinoglycomacropeptide (CGMP), the C-terminal portion of kappa-casein released by chymosin during cheesemaking, a peptide that is notoriously difficult to detect by gel staining or UV imaging.

[0267] Table 5: Quality Accuracy of Bovine Raw Milk Samples

[0268]

[0269] Example 3—Detection of β-casein A1 in β-casein A2 infant formula

[0270] Seven β-casein A2 infant formulas were spiked with A1|A1 SMP to contain 5% β-casein A1 (5 g β-casein A1 per 100 g total β-casein). At this concentration, β-casein A1 signals were detected in all cases ( Figure 6 ).

[0271] Example 4—Protein Quantification and Glycation

[0272] Protein quantification requires not only suitable standards to establish calibration curves, but also that different protein forms exhibit similar signal responses. This is essential for applying the calibration curve to unknown samples, given that standards and samples may have different protein form distributions. Alternatively, the method should provide a method to correct for protein form distribution.

[0273] To investigate whether protein lactosylation affects MS signal intensity, glycation was induced in skim milk powder samples by heating as outlined in Materials and Methods ( Figure 7 A). Increase in glycated β-casein ( Figure 7 B) accompanied by a decrease in total β-casein signal ( Figure 7 C). The total β-casein signal was plotted as a percentage of the unglycosylated β-casein signal, and a linear relationship was obtained ( Figure 7 D), the linear relationship is converted into a multiple signal reduction in the form of y = 1 / (ax + b) ( Figure 7 E), where a and b correspond to 0.878 and 0.122, respectively. Therefore, for β-casein, the percentage of unglycated β-casein (%UG β ) was used to calculate the glycation correction factor (GCF).

[0274]

[0275] The measured β-casein signal was multiplied by the GCF to obtain the corrected β-casein signal (βcas corr ), if all β-casein is unglycosylated, then βcas corr corresponds to the expected signal (Formula 2).

[0276] βcas corr =βcas meas ×GCF (Formula 2)

[0277] Example 5—Quantification of β-casein

[0278] A sliding window algorithm was used to evaluate quantification. An external calibration curve was established using commercially purified β-casein. This standard also contained αS1-casein, κ-casein, and various unidentified peptides and protein fragments, most of which matched cleavage products of β-casein ( Figure 8A). In the seven most concentrated calibration samples, the total signal attributable to intact β-casein accounted for 85.6 ± 0.4%, and this purity factor was used to adjust the concentration of the standard β-casein. For each concentration, the total β-casein signal was normalized using the average signal (corrected for glycation) of the injected SMP samples in the same series (and all subsequent quantification experiments that resulted in variations in MS performance), and the normalized values ​​were plotted against the amount of β-casein loaded on the column. Calibration curve ( Figure 8 B) shows good linearity with respect to the random distribution of the residues (R 2 =0.996). The amount of β-casein injected onto the column (in μg) can be calculated from this calibration curve using Equation 3:

[0279]

[0280] Among them, βcas corr,norm is the corrected β-casein signal in the sample (Equation 2) divided by the average of the corrected signals of the nonfat dry milk samples injected in the same series (Equation 4).

[0281]

[0282] The quantification of β-casein variants in the dairy matrix was further evaluated by mixing skim milk powder containing only the A1 variant with skim milk powder containing only the A2 type ( Figure 8 C). The response to β-casein A2 showed good linearity (R 2 =0.996), which indicates that the matrix effect is negligible. The measured and expected values ​​of the A1 / A2 ratio show a strong linear relationship (% A2 calc =1.05%A2 exp , R 2 =0.996). This means that the response factors for the two variants are very similar and that the calibration curve is suitable for quantifying each genetic variant. Quantification of β-casein in the two SMP samples yielded an average of 28.3 g of β-casein per 100 g of protein, which is consistent with the 27% reported in the Handbook of Composition of Cow's Milk (Swaiswell, 1995) (Table 6).

[0283] Table 6: Quantification of β-casein in A1|A1 and A2|A2 SMP

[0284]

[0285] Example 6—Method Performance (Quantification of β-Casein)

[0286] The performance of the method was evaluated on seven infant formulas containing only β-casein A2. Five different samples were prepared for each sample and analyzed by three replicate injections. The amount of β-casein measured was roughly consistent with the theoretical value (83%-136%) calculated by multiplying the protein content by the casein content and then by 33%, which represents the generally accepted proportion of β-casein in total casein (Swaiswell, 1995) ( Figure 9 A).

[0287] This approach was further tested using another range of commercially available infant formulas (either based on A2 or made with standard SMP) and the results showed broad agreement with the theoretical values ​​( Figure 9 B, whey % assumptions based on shaded values). The discrepancy between measured and theoretical β-casein content was greatest for samples with minimal β-casein content. This may be due to the less abundant protein forms being too close to the noise to be effectively measured, particularly in samples with multiple genetic variants such as IF8 and IF13.

[0288] Example 7 - Detection of A1 Specificity in Whey Protein Concentrate by Intact Protein Analysis Peptone

[0289] Six different batches of whey protein concentrate were analyzed by LC-HRMS using the intact protein analysis method as described in the Methods and Materials section.

[0290] Raw materials analyzed:

[0291] Three batches of WPC35

[0292] Two batches of WPC28

[0293] ·A batch of WPC80 (α-lac enriched)

[0294] Figure 10 Individual analyses of six different whey protein concentrates are shown.

[0295] Since no signal was detected within the circle with a solid line border (ie, the circle in the upper right corner), none of the WPCs tested showed any trace of β-casein.

[0296] In contrast, all WPCs examined showed a signal corresponding to Peptone signal.

[0297] In addition, the analysis showed that all ingredients contained CGMP (Casein Glycomacropeptide), a casein-derived peptide identified in the lower left corner of each graph in the chart.

[0298] Figure 11 yes Figure 10 The enlarged view of the inner area of ​​the circle with a dotted line edge is shown. This enlarged view shows that the corresponding The signal is of the expected quality for peptone. Black indicates β-casein A2 derived Peptone, gray indicates β-casein A1 derived peptone, and white indicates unassigned compounds;

[0299] Some of these peptones are: PP5 A1 1-105, PP5 A1 1-107, PP5 A21-105, PP5A2 1-107, bcas1P A1 29-105 (i.e., PP8s A1 29-105), bcas1P A129-107 (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).

[0300] No gamma-casein was detected in the WPCs examined.

[0301] WPC80 does not contain PP8 (29-105 / 7). This may be due to the ultrafiltration step in the preparation of WPC80, which may remove PP8.

[0302] exist Figure 10 and Figure 11 in Figure 10 Several signals (shown in white) both inside and outside the circle with a dashed edge are associated with the peptide to be identified.

[0303] Therefore, the A1 (and A2) Peptones are present in all whey raw materials tested. Therefore, they will be present in the finished product and are most likely responsible for the high A1 signal picked up in all finished products tested.

[0304] Example 8—Detection of A1 Specificity in Finished Product by Intact Protein Analysis Peptone

[0305] Analyze different finished products of different brands of different infant formula to test specificity Peptone. All infant formulas are considered A2 finished products.

[0306] The finished product was dissolved and analyzed by LC-HRMS as described above in the Materials and Methods section.

[0307] Figure 12 Shows that in all tested batches Peptone, even between finished products Peptones have different profiles.

[0308] A1β-casein derived Peptone was found in all infant formulas tested, even if they were based on cow's milk containing A2 β-casein.

[0309] Example 9—Quantification of A1 and A2 specificity by intact protein analysis Peptone

[0310] The relative quantification of PP5 A1 and PP5 A2 in dairy matrices was further evaluated by mixing skim milk powder containing only the A1 variant with skim milk powder containing only the A2 type ( Figure 13 Responses to PP5 A2 and PP5 A1 showed good dose responses with quadratic regression (R 2 =0.999 and R 2 =0.995), which indicates that the matrix effect is negligible. This means that the response factors of the two variants are very close and that the calibration curve is also suitable for quantifying each genetic variant.

[0311] Finally, it was demonstrated that specificity can be quantified using intact protein analysis even in matrix Peptone.

[0312] Example 10—Detection of A1 specificity in finished product by trypsin digestion Peptone

[0313] The different finished products are standard skimmed milk powder (SMP), skimmed milk powder containing only the A2 version of beta-casein, or different infant formulas of different brands and for different age groups. All infant formulas are considered A2 finished products.

[0314] The finished product was dispersed into a 3.5% (w / v) protein solution of 50mM Tris and 6M urea and then mixed on an orbital shaker for one hour. 200 μL of sample was diluted with 1000 μL of ammonium bicarbonate (100mM) and vortexed. 10 μL of DTT (45mM) was added to the 100 μL diluted solution. The sample was incubated at 60°C for 30 minutes, then cooled to room temperature and quickly spun to collect evaporating droplets. 10 μL of iodoacetamide (100mM) in an ammonium bicarbonate solution (100mM) was added; the sample was incubated at room temperature and in the dark for 30 minutes. 6 μL of trypsin (0.2 μg / μL) was added and the sample was incubated overnight at 37°C. 6 μL of formic acid (10%) was added to stop the digestion and the solution was centrifuged at 14'000 g for 10 minutes; the liquid phase was transferred to vials for injection and analyzed by LC-MS as described above in the Materials and Methods section.

[0315] The assay focuses on the detection of A1S peptides, which have Figure 14 The A1S peptide is shown in Figure 2. It is clear from the position of the A1S peptide that it can be distinguished from the A2S peptide as it covers the region of β-casein including amino acid 67.

[0316] Figure 15 It is shown that A1S was not detected in the A2 SMP samples. A1S was detected in one A2 SMP sample, which was later shown to contain β-casein A1. However, the peptide was significantly expressed in the standard SMP. Surprisingly, a strong signal for the peptide A1S was detected in all infant formulas tested. Figure 16 ) and A1T( Figure 17 ), and similar results were obtained.

[0317] It can be predicted that peptide A2S( Figure 18 )、A2N( Figure 19 )、A2T( Figure 20 )、Tot1( Figure 21 ) and Tot2( Figure 22 ) showed strong signals in all tested areas.

[0318] Example 11 - Detection of A1 Specificity in Whey Protein Concentrate by Trypsin Digestion Peptone

[0319] Different batches of whey protein concentrate were analyzed alongside skim milk powder, where both the standard and skim milk powder contained only the A2 version of beta-casein.

[0320] Raw materials (RM) analyzed:

[0321] Three batches of WPC35

[0322] Two batches of WPC28

[0323] ·A batch of WPC80 (α-lac enriched)

[0324] Two different commercially available lactose raw materials

[0325] The products were solubilized and exposed to trypsin digestion as described above in the Materials and Methods section and then analyzed by LC-MS.

[0326] The analysis focused on detecting Tot1 and Tot2 peptides (i.e., peptides common to β-casein of A1 and A2), A1S peptide (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 peptides are located in Figure 14 Shown in.

[0327] Figure 23 and Figure 24 Results obtained by separately determining the amounts of the Tot1 and Tot2 peptides are shown. These are common peptides (for both A1 and A2), but are also present only in intact β-casein and γ-casein. The signal for the common β-casein peptide is significantly reduced in all whey fractions and absent in the lactose fraction, but present in SMP and A2 SMP, as expected.

[0328] This indicates that there is very little intact β-casein or γ-casein (~1%) in RM compared to SMP, even when equivalent protein contents are used, as expected.

[0329] Figure 25 The results obtained by determining the amount of A1S peptide are shown. It is shown that the signal for A1S peptide is the strongest for SMP, but is absent for A2 SMP, as expected. Furthermore, the lactose sample shows no signal. For the whey protein sample, a surprising amount, approximately one-quarter of that observed for SMP, was measured.

[0330] Similar results were obtained when the samples were tested for the presence of two other A1-specific peptides, A1N and A1T ( Figure 26 and Figure 27 ).

[0331] Figure 28The results obtained for determining the amount of A2S are shown. It is shown that the signal for the A2S (A2-specific) peptide is the strongest for A2 SMP, but the signal for SMP is weaker, as expected. The lactose sample shows no signal, as expected. For the whey protein sample, a surprising amount, about a quarter of that observed for SMP, was measured.

[0332] Similar results were obtained when the samples were tested for the presence of two other A2-specific peptides, A2N and A2T ( Figure 29 and Figure 30 ).

[0333] Therefore, from the above results, it can be concluded that in all analyzed whey RMs, the signal intensity of peptides common to β-casein was severely reduced (~100×) compared to SMP. In addition, the A1- and A2-specific peptides were only reduced by ~4 to 5× compared to SMP. Therefore, there was a selective enrichment of A1- and A2-specific peptides.

[0334] Thus, using the trypsin digestion method and identifying the different peptides, it appears that the whey fraction still contains ∼20% of β-casein fragments (compared to SMP), which may be at least partially the known Peptones, such as PP5 and PP8s.

[0335] Example 12 - Whey protein fraction Reduction of peptone

[0336] In order to obtain a whey protein fraction with a reduced content, the whey protein fraction determined in Example 9 was Three batches of WPC35 were subjected to gel filtration in the presence of peptone.

[0337] Three batches of WPC35 were further purified by gel filtration on a Sephadex G-75 column (550 mm x 22 mm) composed and equilibrated in volatile NH4HCO3 (pH 8.0-8.5) buffer (0.1 M).

[0338] 0.5g to 0.7g of each of these batches were dissolved in 5mL to 7mL of buffer (a few mL of 1M NaOH was added to neutralize small amounts of residual TCA and aid dissolution) and applied to the column. The flow rate was adjusted to 0.5mL / min and 5mL fractions were collected.

[0339] Fractions were analyzed by the detection and quantification methods described herein (i.e., LC-HRMS) according to the Materials and Methods section. The slow fractions of peptones PP5 and PP8 were combined to form a fraction with reduced whey protein content.

[0340] References

[0341] Bonfatti, V. et al., J Chromatogr A, 2008; 1195(1-2): 101-106.

[0342] de Jong et al., J Chromatogr A, 1993; 652(1): 207-213.

[0343] EFSA Scientific Report, Scientific Report of EFSA prepared by a DATEXWorking Group on the potential health impact of b-casomorphins and related peptides. 2009; 231; 1-107.

[0344] Fenaille, F. et al., Rapid Commun Mass Spectrom, 2003; 17(13): 1483-1492.

[0345] Fenaille, F. et al., International Dairy Journal, 2006; 16(7): 728-739.

[0346] Feng, P. et al., J AOACInt, 2017; 100(2): 510-521.

[0347] Frederiksen, PD et al., J Dairy Sci, 2011; 94(10): 4787-4799.

[0348] Ho, S. et al., Eur. J. Clin. Nutr. 2014, 68, 994–1000.

[0349] Karamoko, G. et al., Biotechnologie, Agronomie, Société et Environnement, 2013;17(2):373-382.

[0350] Poulsen, NA et al., Acta Agriculturae Scandinavica, Section A—AnimalScience, 2016;66(4):190-198.

[0351] Roberfroid MB.J Nutr.2007;137:830S.

[0352] Salminen S et al., Trend Food Sci. Technol., 1999; 10 107-110.

[0353] Swaisgood, HE Handbook of Milk Composition. RG Jensen, ed. Academic Press, San Diego 1995; 464-468.

[0354] Swaisgood. Developments in Dairy Chemistry. Fox (Ed), Proteins, Volume 1, 1982; 63-110.

[0355] Vallejo-Cordoba, BJ Capillary Electrophor, 1997;4(5):219-224.

[0356] Visser, S. et al., J Chromatogr A, 1995; 711(1): 141-150.

[0357] project

[0358] 1. For the determination and / or quantification of β-casein-derived Peptone and / or β-casein

[0359] The method comprises the following steps:

[0360] (i) providing a dairy-based product to be analyzed;

[0361] (ii) subjecting the product to analysis using liquid chromatography-mass spectrometry;

[0362] (iii) determining and / or quantifying the β-casein derived compounds in the product by detecting compounds with defined m / z values ​​or calculating monoisotopic masses by deconvoluting one or more mass spectra. Peptone and / or β-casein.

[0363] 2. The method according to item 1, wherein said Peptone is derived from A1β-casein Peptone.

[0364] 3. The method according to item 2, wherein the β-casein-derived Peptones are PP8 fast, PP8 slow and / or PP-5, such as A1 β-casein derived The peptones were PP8 Fast, PP8 Slow and / or PP-5.

[0365] 4. The method according to any of the preceding items, wherein the product is an infant formula or a whey protein fraction.

[0366] 5. The method according to any of the preceding items, wherein the product is a powder which is dissolved before analyzing the product.

[0367] 6. The method according to any of the preceding items, wherein the product is analyzed by intact protein analysis.

[0368] 7. The method according to item 6, wherein the mass spectrometry analysis is high-resolution mass spectrometry analysis.

[0369] 8. The method according to any one of items 6 to 7, wherein the β-casein derived molecule in the product is determined and / or quantified by calculating the monoisotopic mass by deconvolution of one or more mass spectra. Peptone.

[0370] 9. The method of claim 8, wherein the deconvolution is performed by a sliding window algorithm.

[0371] 10. The method according to any one of items 1 to 5, wherein the product is analyzed by peptide analysis, the peptide analysis comprising a step of enzymatic digestion of the product before analyzing the product.

[0372] 11. The method according to item 10, wherein the enzymatic digestion is performed by trypsin digestion or GluC digestion.

[0373] 12. The method according to any one of items 10 to 11, wherein the β-casein derived protein is determined and / or quantified by detecting compounds with defined m / z values. Peptone.

[0374] 13. For producing a product derived from β-casein with reduced Whey protein grade with peptone content

[0375] The method comprises the following steps:

[0376] (i) providing a whey protein fraction;

[0377] (ii) determining and quantifying the β-casein-derived β-casein in the whey protein fraction according to any one of items 1 to 12 Peptone; and

[0378] (iii) selecting a whey protein fraction having at most 10% by weight of β-casein derived proteins based on the total protein in the whey protein fraction The whey protein fraction is purified by the peptone to form a selected whey protein fraction.

[0379] 14. For producing a product derived from β-casein with reduced Whey protein grade with peptone content

[0380] The method comprises the following steps:

[0381] (i) providing a whey protein fraction;

[0382] (ii) based on the total protein in the whey protein fraction, the β-casein-derived The peptone content is reduced to a concentration of at most 10% by weight, thereby forming a whey protein fraction with a reduced content.

[0383] 15. The method according to item 14 further comprising determining and quantifying the β-casein derived protein in the whey protein fraction according to any one of claims 1 to 12. Steps before and / or after step (ii) of peptone.

[0384] 16. The method according to any one of items 14 to 15, wherein the Peptone content.

[0385] 17. The method according to any one of items 13 to 16, wherein the β-casein derived from the selected or reduced content whey protein fraction is based on the total protein in the whey protein fraction. The peptone content is at most 9.5% by weight, such as at most 9% by weight, preferably at most 8.5% by weight, such as at most 8% by weight, more preferably at most 7.5% by weight, such as at most 7% by weight, even more preferably at most 6.5% by weight, such as at most 6% by weight, still more preferably at most 5.5% by weight, such as at most 5% by weight, most preferably at most 4.5% by weight, such as at most 4% by weight, The amount of the present invention is at most 3.5% by weight, such as at most 3% by weight, preferably at most 2.5% by weight, such as at most 2% by weight, more preferably at most 1.5% by weight, such as at most 1% by weight, even more preferably at most 0.75% by weight, such as at most 0.50% by weight, still more preferably at most 0.25% by weight, such as at most 0.10% by weight, most preferably at most 0.05% by weight, such as at most 0.01% by weight.

[0386] 18. The method according to any one of items 13 to 17, wherein the The peptones were PP8 Fast, PP8 Slow and / or PP-5.

[0387] 19. For producing a product derived from β-casein with reduced A method for preparing a nutritional composition having a high peptone content, the method comprising the steps of:

[0388] (i) providing a selected whey protein fraction or a whey protein fraction having a reduced content according to any one of items 13 to 18;

[0389] (ii) preparing a whey protein fraction having a reduced content of whey protein from said selected whey protein fraction, said whey protein fraction having a reduced content of whey protein or a mixture thereof; The nutritional composition has a peptone content.

[0390] 20. A whey protein fraction having reduced β-casein derived Peptone content, the whey protein fraction can be obtained by the method according to any one of items 14 to 18.

[0391] 21. A nutritional composition comprising the protein derived from β-casein having reduced Peptone content or the whey protein fraction obtainable by the method according to item 19.

[0392] 22. The nutritional composition according to item 21, wherein the nutritional composition is an infant formula.

[0393] 23. The nutritional composition according to any one of items 21 to 22, wherein the The peptone content is at most 9% by weight.

[0394] 24. Nutritional composition according to any one of items 21 to 23 for use as a medicament.

[0395] 25. The infant formula according to any one of items 22 to 23, which is used for treating, preventing and / or improving colic in infants.

[0396] 26. The infant formula according to any one of items 22 to 23, which is used for treating, preventing and / or improving lactose intolerance in infants.

[0397] 27. Use of the infant formula according to any one of items 22 to 23 for improving stool consistency.

[0398] Sequence Listing

[0399] SEQ ID NO: 1 (amino acid sequence of A2 β-casein) :

[0400] RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVYPFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKHKEMP FPKYPVEPFT ESQSLTLTDVENLHLPLPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGPVRGPFPIIV

[0401] SEQ ID NO: 2 (amino acid sequence of A1 β-casein) :

[0402] RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVYPFPGPIHNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKHKEMP FPKYPVEPFT ESQSLTLTDVENLHLPLPLL QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF LLYQEPVLGPVRGPFPIIV

[0403] SEQ ID NO: 3 (amino acid sequence of A1N): AQTQSLVYPF PGPIHN

[0404] SEQ ID NO: 4 (amino acid sequence of A2N): AQTQSLVYPF PGPIPN

[0405] SEQ ID NO: 5 (amino acid sequence of Tot2): AVPYPQR

[0406] SEQ ID NO: 6 (amino acid sequence of A1S): IHPFAQTQSL VYPFPGPIHN

[0407] SEQ ID NO: 7 (amino acid sequence of A1T):

[0408] IHPFAQTQSL VYPFPGPIHN SLPQNIPPLT QTPVVVPPFL QPEVMGVSK

[0409] SEQ ID NO: 8 (amino acid sequence of A2S): IHPFAQTQSL VYPFPGPIPN

[0410] SEQ ID NO: 9 (amino acid sequence of A2T):

[0411] IHPFAQTQSL VYPFPGPIPN SLPQNIPPLT QTPVVVPPFL QPEVMGVSK

[0412] SEQ ID NO: 10 (amino acid sequence of Tot1): VLPVPQK

Claims

1. For the determination and / or quantification of β-casein-derived A method for producing peptone and / or β-casein, said method comprising the steps of: (i) providing a dairy-based product to be analyzed; (ii) subjecting the product to analysis using liquid chromatography-mass spectrometry; (iii) determining and / or quantifying the β-casein derived compounds in the product by detecting compounds with defined m / z values ​​or calculating monoisotopic masses by deconvoluting one or more mass spectra. Peptone and / or β-casein.

2. The method according to claim 1, wherein the β-casein derived Peptones are PP8 fast, PP8 slow and / or PP-5, such as A1 β-casein derived The peptones were PP8 Fast, PP8 Slow and / or PP-5.

3. The method according to any one of the preceding claims, wherein the product is analysed by intact protein analysis.

4. The method according to any one of claims 1 to 2, wherein the product is analyzed by peptide analysis comprising a step of enzymatic digestion of the product prior to analyzing the product.

5. For the production of products with reduced β-casein derivatives A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps: (i) providing a whey protein fraction; (ii) determining and quantifying the β-casein-derived β-casein in the whey protein fraction according to any one of claims 1 to 4 Peptone; as well as (iii) selecting a whey protein fraction having at most 10% by weight of β-casein derived proteins based on the total protein in the whey protein fraction The whey protein fraction is purified by the peptone to form a selected whey protein fraction.

6. For the production of products with reduced β-casein derivatives A method for preparing a whey protein fraction having a peptone content, the method comprising the following steps: (i) providing a whey protein fraction; (ii) based on the total protein in the whey protein fraction, the β-casein-derived The peptone content is reduced to a concentration of at most 10% by weight, thereby forming a whey protein fraction with a reduced content.

7. The method according to claim 6, wherein the Peptone content.

8. For the production of products with reduced β-casein derivatives A method for preparing a nutritional composition having a high peptone content, the method comprising the steps of: (i) providing a selected whey protein fraction or a whey protein fraction having a reduced content as claimed in any one of claims 5 to 7 (ii) preparing a whey protein fraction having a reduced content of whey protein from said selected whey protein fraction, said whey protein fraction having a reduced content of whey protein or a mixture thereof; The nutritional composition has a peptone content.

9. A whey protein fraction having reduced β-casein derived Peptone content, the whey protein fraction can be obtained by the method according to any one of claims 5 to 7.

10. A nutritional composition comprising the protein derived from a protein having reduced β-casein according to claim 9. Peptone content or the whey protein fraction obtainable by the method according to claim 8.

11. The nutritional composition of claim 10, wherein the nutritional composition is an infant formula.

12. The nutritional composition according to any one of claims 10 to 11 for use as a medicament.

13. The infant formula according to any one of claims 11, which is used for treating, preventing and / or improving infant colic, and / or improving the intestinal comfort of the infant.

14. The infant formula according to any one of claims 11, which is used for treating, preventing and / or improving lactose intolerance in infants.

15. Use of the infant formula according to any one of claim 11 for improving stool consistency.

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