Beta-caseins in infant milk formula and use for reducing regurgitation episodes in infants

Abstract

The frequency of regurgitation attacks in infants is reduced by providing an infant milk formula prepared from milk comprising beta-casein, wherein the beta-casein comprises less than 30% by weight of beta-casein having histidine at position 67 of the beta-casein amino acid sequence.

Description

Technical Field

[0002] This invention relates to the milk protein β-casein and to reducing the frequency or duration of infant reflux. In particular, this invention relates to infant formula prepared with milk having β-casein properties that are virtually free of or contain no β-casein A1 or related β-casein variants. The applicant has found that ingestion of infant formula prepared with milk containing β-casein variants that readily produce BCM-7 upon digestion results in an increase in infant reflux compared to infant formula prepared with milk containing β-casein variants that are less readily produced upon digestion. Background of the Invention

[0004] The benefits of breastfeeding for infants for at least the first six months of life, and preferably for an additional six to twelve months, are well-established. Human breast milk is known to protect infants from infections and reduce the incidence of health problems, including diabetes, obesity, and asthma. It is generally accepted that the intact gut microbiota of breastfed infants provides anti-infective properties and is an important stimulant for the development of the immune system after birth. Breast milk is recognized as the best source of nutrition for newborns. However, it is also well known that many mothers are unable to breastfeed their infants, thus making infant formula (also known as baby formula or milk formula) the preferred, or in some cases, only, option.

[0005] Mature breast milk contains 3-5% fat, 0.8-0.9% protein, 6.9-7.2% carbohydrates (calculated as lactose), and 0.2% minerals. The main human milk proteins are whey and casein. Whey protein concentration begins to decline in early lactation and continues to decrease. These changes result in a whey / casein ratio of approximately 90:10 in early lactation, 60:40 in mature milk, and 50:50 in late lactation. The main proteins in human milk are β-casein, α-lactalbumin, lactoferrin, immunoglobulin IgA, lysozyme, and serum albumin. The essential amino acid pattern in human milk is very similar to the optimal amino acid pattern found for human infants.

[0006] Infant formula is designed based on human breast milk from approximately 1-3 months postpartum. The most common protein components of infant formula include casein and whey from cow's milk, along with added purified whey. They also contain blends of vegetable oils as a fat source, lactose as a carbohydrate source, a vitamin-mineral blend, and other ingredients, depending on the manufacturer. Additionally, some infant formulas use soy as a protein source instead of cow's milk, and some use proteins hydrolyzed into their component amino acids for infants allergic to other proteins. Besides human breast milk, infant formula is the only other milk product considered nutritionally acceptable for infants under one year old (unlike cow's milk, goat's milk, or subsequent formulas with different compositions).

[0007] Cow's milk typically contains about 30 grams of protein per liter. Casein makes up the largest component of this protein (80%), and β-casein accounts for about 37% of casein. Over the past three decades, substantial evidence has been growing regarding the involvement of casein, particularly β-casein, in a number of health disorders.

[0008] The β-casein family comprises several variants, commonly referred to as A1, A2, A3, B, C, D, E, F, G, H1, H2, and I. Other variants may also exist that have not yet been identified. β-casein A1 and β-casein A2 are the major β-caseins in milk consumed by most populations. The applicants have previously established a correlation between the consumption of β-casein A1 in milk and dairy products and the occurrence of certain health conditions, including type 1 diabetes (WO 1996 / 014577), coronary heart disease (WO 1996 / 036239), and neurological disorders (WO 2002 / 019832). In addition, the applicant has demonstrated the correlation between β-casein A1 and intestinal inflammation (WO 2014 / 193248), lactose intolerance (WO 2015 / 005804), high blood glucose levels (WO 2015 / 026245), antioxidant capacity (WO2016 / 190750), cognitive function (WO 2017 / 171563), and gut microbiota (WO 2018 / 063008).

[0009] The difference between β-casein A1 and β-casein A2 lies in a single amino acid. Histidine is located at position 67 of the 209-amino acid sequence in β-casein A1, while proline is located at the same position in β-casein A2. However, this single amino acid difference is crucial for the enzymatic digestion of β-casein in the intestine. The presence of histidine at position 67 allows for the easy formation of a seven-amino acid protein fragment called β-casein-7 (BCM-7) during enzymatic digestion. Therefore, BCM-7 is a digestion product of β-casein A1. In the case of β-casein A2, position 67 is occupied by proline, which hinders protein cleavage at that position. Therefore, BCM-7 is not readily formed during the digestion of β-casein A2.

[0010] Other β-casein variants, such as variants B, C, F, G, H1, and H2, also have histidine at position 67, while variants other than A2, such as variants A3, D, E, and I, also have proline at position 67.

[0011] BCM-7 is an opioid peptide that can bind to and activate opioid receptors throughout the body. BCM-7 can cross the gastrointestinal wall and enter the circulation, enabling it to influence systemic and cellular activities via opioid receptors.

[0012] The applicant has now discovered a correlation between infant formula containing β-casein variants with histidine at position 67 of the β-casein amino acid sequence and a higher frequency of infant regurgitation compared to infant formula containing variants with proline at position 67 of the β-casein amino acid sequence. This effect is thought to be caused by BCM-7 produced during the digestion of β-casein A1 and related variants in the intestine. Therefore, any β-casein variant containing histidine at position 67 and capable of readily producing BCM-7 during digestion is undesirable in infant diets.

[0013] The purpose of this invention is to provide a method for reducing the frequency of infantile reflux episodes, or at least to provide a useful alternative to existing methods. Invention Overview

[0015] In a first aspect of the invention, a method is provided to reduce the frequency of infant reflux by providing an infant with an infant formula for its ingestion, wherein the milk used to prepare the infant formula contains β-casein, which contains less than 30% by weight of β-casein having a histidine residue at position 67 of the β-casein amino acid sequence.

[0016] In some embodiments of this aspect of the invention, the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

[0017] In certain embodiments of this aspect of the invention, the β-casein comprises any one or more of less than 25% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of β-casein variants A1, B, C, F, G, H1, and H2, or contains no β-casein variants A1, B, C, F, G, H1, and H2.

[0018] In some embodiments of this aspect of the invention, β-casein comprises less than 30% by weight of β-casein A1.

[0019] In some embodiments of this aspect of the invention, β-casein comprises less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0020] In some embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0021] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0022] In some embodiments of this aspect of the invention, β-casein comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% by weight of β-casein A2.

[0023] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0024] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0025] In a second aspect of the invention, a method is provided for reducing the frequency of infant reflux by feeding an infant formula, wherein the milk used in preparing the infant formula contains β-casein, which contains less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0026] In some embodiments of this aspect of the invention, the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

[0027] In certain embodiments of this aspect of the invention, the β-casein comprises any one or more of less than 25% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of β-casein variants A1, B, C, F, G, H1, and H2, or contains no β-casein variants A1, B, C, F, G, H1, and H2.

[0028] In some embodiments of this aspect of the invention, β-casein comprises less than 30% by weight of β-casein A1.

[0029] In some embodiments of this aspect of the invention, β-casein comprises less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0030] In some embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0031] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0032] In some embodiments of this aspect of the invention, β-casein comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% by weight of β-casein A2.

[0033] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0034] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0035] Another aspect of the invention provides a method for reducing the frequency of infant reflux episodes, wherein the infant's diet comprises an infant formula containing β-casein, which contains at least 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence, the method being carried out by replacing the aforementioned infant formula with an alternative infant formula prepared from milk containing β-casein having less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0036] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises less than 30% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2.

[0037] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 25% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 10% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 5% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 1% by weight, or does not contain any one of β-casein variants A1, B, C, F, G, H1, and H2.

[0038] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula contains less than 30% by weight of β-casein A1.

[0039] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula contains less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0040] In some embodiments of this aspect of the invention, the β-casein alternative to infant formula comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0041] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0042] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% of β-casein A2.

[0043] In a preferred embodiment of this aspect of the invention, the alternative infant formula also contains fat, lactose, minerals, and vitamins.

[0044] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0045] Another aspect of the invention provides a method for reducing the frequency of reflux in infants consuming a diet containing a first infant formula, wherein the first infant formula is replaced in the infant's diet with a second infant formula.

[0046] The first infant formula contains β-casein, which comprises at least 30% by weight of β-casein having a histidine residue at position 67 of the β-casein amino acid sequence; and

[0047] The second infant formula is prepared from milk containing β-casein, wherein the β-casein comprises less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0048] In some embodiments of this aspect of the invention, the β-casein of the second infant formula is prepared from milk containing β-casein, said β-casein comprising any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 30% by weight.

[0049] In some embodiments of this aspect of the invention, the second infant formula is prepared from milk containing β-casein, said β-casein comprising less than 25% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, or without any one of β-casein variants A1, B, C, F, G, H1, and H2.

[0050] In some embodiments of this aspect of the invention, the second infant formula is prepared from milk containing β-casein, said β-casein comprising less than 30% by weight of β-casein A1.

[0051] In some embodiments of this aspect of the invention, the second infant formula is prepared from milk containing β-casein, said β-casein comprising less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or without β-casein A1.

[0052] In some embodiments of this aspect of the invention, the second infant formula is prepared from milk containing β-casein, said β-casein comprising at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0053] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0054] In some embodiments of this aspect of the invention, the second infant formula is prepared from milk containing β-casein, said β-casein comprising at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% of β-casein A2.

[0055] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0056] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0057] Another aspect of the invention provides an infant formula for reducing the frequency of infant reflux, the infant formula being prepared from milk containing β-casein, said β-casein comprising less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0058] In some embodiments of this aspect of the invention, the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

[0059] In certain embodiments of this aspect of the invention, the β-casein comprises any one or more of less than 25% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of β-casein variants A1, B, C, F, G, H1, and H2, or contains no β-casein variants A1, B, C, F, G, H1, and H2.

[0060] In some embodiments of this aspect of the invention, β-casein comprises less than 30% by weight of β-casein A1.

[0061] In some embodiments of this aspect of the invention, β-casein comprises less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0062] In some embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0063] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0064] In some embodiments of this aspect of the invention, β-casein comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% by weight of β-casein A2.

[0065] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0066] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0067] Another aspect of the invention provides the use of milk in the preparation of an infant formula for reducing the frequency of reflux in infants, wherein the milk comprises β-casein, which comprises less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0068] In some embodiments of this aspect of the invention, the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

[0069] In certain embodiments of this aspect of the invention, the β-casein comprises any one or more of less than 25% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of β-casein variants A1, B, C, F, G, H1, and H2, or contains no β-casein variants A1, B, C, F, G, H1, and H2.

[0070] In some embodiments of this aspect of the invention, β-casein comprises less than 30% by weight of β-casein A1.

[0071] In some embodiments of this aspect of the invention, β-casein comprises less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0072] In some embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0073] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0074] In some embodiments of this aspect of the invention, β-casein comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% by weight of β-casein A2.

[0075] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0076] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0077] In another aspect of the invention, a method is provided to reduce the frequency of infant reflux by providing an infant with an infant formula for its ingestion, wherein the infant formula contains β-casein, comprising less than 30% by weight of β-casein having a histidine residue at position 67 of the β-casein amino acid sequence.

[0078] In some embodiments of this aspect of the invention, the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

[0079] In certain embodiments of this aspect of the invention, the β-casein comprises any one or more of less than 25% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of β-casein variants A1, B, C, F, G, H1, and H2, or contains no β-casein variants A1, B, C, F, G, H1, and H2.

[0080] In some embodiments of this aspect of the invention, β-casein comprises less than 30% by weight of β-casein A1.

[0081] In some embodiments of this aspect of the invention, β-casein comprises less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0082] In some embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0083] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E and I.

[0084] In some embodiments of this aspect of the invention, β-casein comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% by weight of β-casein A2.

[0085] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0086] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0087] In another aspect of the invention, a method for reducing the frequency of reflux episodes in infants is provided, the infant's diet comprising an infant formula containing β-casein, which comprises at least 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence, the method being carried out by replacing the aforementioned infant formula with an alternative infant formula containing β-casein having less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0088] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises less than 30% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2.

[0089] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 25% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 10% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 5% by weight, any one or more of β-casein variants A1, B, C, F, G, H1, and H2 at less than 1% by weight, or does not contain any one of β-casein variants A1, B, C, F, G, H1, and H2.

[0090] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula contains less than 30% by weight of β-casein A1.

[0091] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula contains less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0092] In some embodiments of this aspect of the invention, the β-casein alternative to infant formula comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0093] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0094] In some embodiments of this aspect of the invention, the β-casein that replaces infant formula comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% of β-casein A2.

[0095] In a preferred embodiment of this aspect of the invention, the alternative infant formula also contains fat, lactose, minerals, and vitamins.

[0096] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old.

[0097] In another aspect of the invention, a method is provided for reducing the frequency of reflux in infants consuming a diet containing a first infant formula, wherein the first infant formula is replaced in the infant's diet with a second infant formula.

[0098] The first infant formula contains β-casein, which comprises at least 30% by weight of β-casein having a histidine residue at position 67 of the β-casein amino acid sequence; and

[0099] The second infant formula contains β-casein, which contains less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

[0100] In some embodiments of this aspect of the invention, the second infant formula contains less than 30% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2.

[0101] In some embodiments of this aspect of the invention, the second infant formula comprises β-casein, comprising less than 25% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 10% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 5% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, less than 1% by weight of any one or more of β-casein variants A1, B, C, F, G, H1, and H2, or without any one of β-casein variants A1, B, C, F, G, H1, and H2.

[0102] In some embodiments of this aspect of the invention, the second infant formula contains β-casein, which contains less than 30% by weight of β-casein A1.

[0103] In some embodiments of this aspect of the invention, the second infant formula contains β-casein, which contains less than 25% by weight of β-casein A1, less than 10% by weight of β-casein A1, less than 5% by weight of β-casein A1, less than 1% by weight of β-casein A1, or contains no β-casein A1.

[0104] In some embodiments of this aspect of the invention, the second infant formula comprises β-casein, which contains at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

[0105] In certain embodiments of this aspect of the invention, the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 75% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 90% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 95% by weight of any one or more of β-casein variants A2, A3, D, E, and I; at least 99% by weight of any one or more of β-casein variants A2, A3, D, E, and I; or 100% by weight of any one or more of β-casein variants A2, A3, D, E, and I.

[0106] In some embodiments of this aspect of the invention, the second infant formula comprises β-casein, which comprises at least 70% by weight of β-casein A2, at least 75% by weight of β-casein A2, at least 90% by weight of β-casein A2, at least 95% by weight of β-casein A2, at least 99% by weight of β-casein A2, or 100% of β-casein A2.

[0107] In a preferred embodiment of this aspect of the invention, the infant formula also contains fat, lactose, minerals, and vitamins.

[0108] In some embodiments of this aspect of the invention, the infant is less than 6 months or less than 3 months old. Brief description of the attached diagram

[0110] Figure 1 A graph showing the average daily occurrence of vomiting / regurgitation (reflux).

[0111] Figure 2 A graph showing the overall score of the Infant Gastrointestinal Symptom Questionnaire (IGSQ).

[0112] Detailed description

[0113] The term "infant" refers to a child under 12 months of age.

[0114] The term "infant formula" refers to a composition intended for use by infants as a substitute for or in addition to human milk, and is defined under the United States Federal Food, Drug, and Cosmetic Act as "a food that is claimed or represented solely for the purpose of a special diet for infants, due to its suitability as a complete or partial substitute for human milk." Infant formula is typically in powder form and is reconstituted with water before being fed to an infant.

[0115] The term “β-Cauroprin-7” or “BCM-7” refers to the Tyr-Pro-Phe-Pro-Gly-Pro-Ile protein fragment, which is a heptapeptide that is readily produced by enzymes that digest bovine β-casein variants (which have histidine at position 67 of the amino acid sequence) but are not produced or may be produced at low levels by enzymes that digest β-casein variants (which have proline at position 67 of the amino acid sequence).

[0116] The term "reflux" refers to the act of bringing swallowed food back into the mouth.

[0117] The term "reflux attack" refers to a single occurrence or event of reflux.

[0118] The term “frequency” in relation to reflux episodes refers to the number of reflux episodes that occur over a certain period of time.

[0119] The term "variant" used in conjunction with proteins refers to a member of a group of highly similar proteins that are encoded by a single gene or gene family and are the result of genetic differences.

[0120] Any reference to the percentage by weight of β-casein variants in infant formula refers to the percentage by weight of that variant in the total β-casein in the infant formula. For example, the statement "Infant formula contains β-casein, which comprises 100% β-casein A2 by weight" means that 100% of the total β-casein content in the infant formula is β-casein A2.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any determination, method, apparatus, and material similar to or equivalent to those described herein may be used in the implementation or testing of this invention, a variety of determinations, methods, apparatus, and materials are currently described.

[0122] References to numerical ranges disclosed herein (e.g., 1-10) are intended to incorporate all related numerical values ​​within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any reasonable range of values ​​within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are merely examples of specific intent, and all possible combinations of numerical values ​​between the listed minimum and maximum values ​​should be considered as expressly stated in a similar manner in this application.

[0123] As used in this specification, the terms “comprising,” “including,” and similar terms should not be construed as exclusive or exhaustive. In other words, they mean “including but not limited to.”

[0124] Those skilled in the art will understand that the invention described herein is readily adaptable to changes and modifications beyond the specific descriptions. It should be understood that the invention includes all such changes and modifications. The invention also includes all steps or features individually or collectively referred to or indicated in this specification, as well as any and all combinations or any two or more of said steps or features.

[0125] The scope of this invention is not limited to the specific embodiments described in this specification, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this invention.

[0126] Any reference to prior art documents in this specification shall not be construed as an admission that such prior art is well known, nor shall it constitute part of general knowledge in the art.

[0127] This invention relates to infant formula containing the protein β-casein for use in infants and its use in reducing the frequency of reflux. The β-casein comprises less than 30% by weight of β-casein variants having a histidine residue at position 67 of the β-casein amino acid sequence, including variants A1, B, C, F, G, H1, and H2.

[0128] The applicant found that infant formula made from milk containing a β-casein variant that readily produces BCM-7 upon ingestion was associated with a higher frequency of infant reflux compared to infant formula made from milk containing a β-casein variant that does not readily produce BCM-7 upon ingestion. The only structural difference between the two types of β-casein variants is that one type has a histidine residue at position 67, while the other has a proline residue at position 67. This difference allows β-casein A1 (and related variants) to be readily digested in the intestine to produce the opioid peptide BCM-7, while the presence of proline at position 67 spatially binds β-casein A2 (and related variants), making the production of BCM-7 from β-casein A2 extremely difficult. Release of BCM-7 from β-casein A1 (and related variants) in the infant gut is thought to contribute to increased infant reflux. Therefore, it is desirable to reduce or eliminate infant formula containing β-casein A1 or any other β-casein variants with histidine at position 67, primarily by selecting milk from infant diets for the preparation of infant formula.

[0129] β-casein produced in bovine milk is determined using genetic methods. For example, cows homozygous for the A1 allele (A1A1) can produce only the A1 β-casein variant in their milk. Cows homozygous for the A2 allele (A2A2) can produce only the A2 β-casein variant in their milk. Furthermore, cows heterozygous for both the β-casein A1 and A2 genes (A1A2) can produce both A1 and A2 β-casein variants in their milk.

[0130] It is well known that the β-casein gene in cows can be genotyped. Cows genotyped and identified as homozygous for β-casein with proline at position 67 include, but are not limited to, A2A2 and A2I cows. Cows identified as homozygous for β-casein with histidine at position 67 include, but are not limited to, A1A1 and A1B cows. Cows identified as heterozygous for β-casein at position 67 include, but are not limited to, A1A2, A1I, and A2B cows. Cows identified as homozygous for β-casein with proline at position 67 can be separated from other populations, thereby creating a population capable of producing only β-casein with proline at position 67 in their milk. Therefore, infant formula prepared from this milk provides an infant formula free of β-casein A1 or any other variant with histidine at position 67. Therefore, if the main source of β-casein in infant formula includes the A2 variant and not other variants, the infant's dietary intake of the A1 variant from that infant formula is almost eliminated, and the adverse effects of BCM-7 on infant crying are expected to be reduced or even eliminated.

[0131] Therefore, the present invention is based on reducing or eliminating β-casein A1 and other β-casein variants having histidine at position 67 of the amino acid sequence in infant formula, and promoting β-casein A2 and other β-casein variants having proline at position 67 of the amino acid sequence. This is achieved by ensuring that the β-casein in the milk used to prepare infant formula is predominantly or entirely a β-casein variant having proline at position 67 of the amino acid sequence, preferably β-casein A2.

[0132] The A1 and A2 β-casein variants are the dominant variants in most dairy product groups. Therefore, the invention will be further described with reference to β-casein A1 and β-casein A2.

[0133] Ideally, the β-casein in milk used to prepare infant formula contains 100% β-casein A2. However, any infant formula that can reduce the frequency of infant reflux contains β-casein A2 (or other β-casein variants with proline at position 67 of the amino acid sequence), for example, in any amount from 70% to 100% by weight, including but not limited to 75%, 80%, 90%, 95%, 98%, and 99% by weight.

[0134] It is understood that because β-casein variants with histidine at position 67 of the amino acid sequence have an adverse effect on the frequency of reflux in infants, there is no specific lower limit to the amount of β-casein variants in such infant formulas where this effect can be observed. For example, an infant formula containing 49% by weight of β-casein A1 and 51% by weight of β-casein A2 is advantageous over an infant formula containing 50% by weight of β-casein A1 and 50% by weight of β-casein A2. The applicant considers that an infant formula prepared from milk containing less than 30% by weight of any one of β-casein variants A1, B, C, F, G, H1, and H2 is sufficient to reflect the invention; however, it is preferred that no amount of β-casein variants A1, B, C, F, G, H1, and H2 is contained.

[0135] Therefore, it can be understood that the β-casein composition of milk used to prepare infant formula contains less than 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of any one or more of the β-casein variants A1, B, C, F, G, H1, and H2, or contains no amount of any of these β-casein variants.

[0136] Support for this invention can be found in Example 1, which describes a single-point, double-blind, randomized, controlled, parallel pilot study. This study investigated the relative effects of infant formula prepared from milk containing only proline at position 67 of the β-casein amino acid sequence (referred to as A2 β-casein-based infant formula), infant formula prepared from milk containing variants containing proline at position 67 of the β-casein amino acid sequence and variants containing histidine at position 67 of the β-casein amino acid sequence (referred to as A1 / A2 β-casein-based infant formula), and breast milk on infant gastrointestinal health, nutrition, and growth. The inventors presume that infant formula prepared from milk containing only variants containing proline at position 67 of the β-casein amino acid sequence has similar effects to breast milk. It was found that infant formula prepared from milk containing both types of β-casein variants contained a variant with histidine at position 67 and a variant with proline at position 67 in a ratio of approximately 40:60.

[0137] As early as the first week of this study, the inventors found that breastfed infants and those fed A2 β-casein-based infant formula were less irritable, with a significantly reduced frequency of reflux compared to infants fed A1 / A2 β-casein-based infant formula. This difference persisted until the end of the 8-week study period.

[0138] Paired comparisons of intergroup differences in the frequency of reflux episodes showed early significant differences between breastfed infants and infants fed A1 / A2 β-casein-based formula, and between infants fed A1 / A2 β-casein-based formula and infants fed A2 β-casein-based formula. However, no difference was observed between breastfed infants and infants fed A2 β-casein-based formula, consistent with the presumption.

[0139] This study provides, for the first time, clear scientific evidence of a correlation between infants consuming β-casein variants containing histidine at position 67 (particularly β-casein A1) and reflux frequency. The applicant's findings clearly indicate that infants should avoid consuming β-casein that readily produces BCM-7. In fact, the benefits of this invention can be achieved by sourcing milk containing β-casein contents primarily of β-casein A2 and producing infant formula derived from that milk, and by providing this infant formula with the aim of reducing the frequency of reflux in infants.

[0140] Support for the invention can also be found in Example 2. The purpose of this study was to compare and determine biomarkers of infant comfort during the observation period for mixed-fed A2 IMF versus A1 / A2 IMF. The term "A2 IMF" refers to an IMF containing 100% by weight of a β-casein variant with proline at position 67 of the β-casein amino acid sequence, as determined by UPLC and mass spectrometry. The β-casein content was determined to contain 17% by weight of casein I, with the remaining 87% by weight being almost entirely β-casein A2. The term "A1 / A2 IMF" refers to an IMF containing 30% by weight of a β-casein variant with histidine at position 67 of the β-casein amino acid sequence, as determined by UPLC and mass spectrometry; and 70% by weight of a β-casein variant with proline at position 67 of the β-casein amino acid sequence, as determined by UPLC and mass spectrometry.

[0141] The Infant Gastrointestinal Symptom Questionnaire (IGSQ) was used to assess GI-related signs and symptoms in infants as observed by parents / caregivers, including the frequency and duration of reflux episodes (vomiting / spitting up). A total of 280 infants from Shanghai participated in this study, including 140 A2 IMF-mixed-fed infants and 140 A1 / A2 IMF-mixed-fed infants. The mixed-fed infants were fed a mixture of IMF and human milk. Each infant consumed more than 400 mL of IMF per day.

[0142] Individual item scores, scores across all five domains, and total IGSQ scores were similar between baseline groups. At week 2, regarding vomiting / regurgitation, the A2 IMF group had significantly lower mean (SD) daily scores (5.72 [1.45] vs. 6.36 [1.40]) and total IGSQ scores (19.15 [3.74] vs. 21.21 [3.30], all p < 0.05) than the control group. These scores were also significantly lower in the A2 IMF group than in the A1 / A2 IMF control group at week 4.

[0143] Therefore, the data clearly show that after 4 weeks, infants in group A2 had fewer episodes of spitting up / vomiting compared to the control group. This was reflected in the parent / caregiver satisfaction survey, which showed improved relief of gastrointestinal symptoms in infants in group A2, including reduced vomiting after feeding, improved digestion, and improved stool characteristics.

[0144] The relative proportions of β-casein variants containing proline or histidine at position 67 of the amino acid sequence in a cow's milk can be tested to determine whether the milk is suitable for preparing the infant formula of the present invention. Alternatively, the cow can be genetically tested to determine its ability to produce milk containing β-casein A1 (or other β-casein variants containing histidine at position 67 of the amino acid sequence), or to test its ability to produce milk containing β-casein A2 (or other β-casein variants containing proline at position 67 of the amino acid sequence), or a combination of both. These techniques are well known.

[0145] The present invention provides a relatively easy-to-manage solution that ensures that the cow's milk used to prepare infant formula contains β-casein, which is mainly β-casein A2, or any of the variants A3, D, E and I, preferably 100% β-casein A2.

[0146] The infant formula of this invention can be prepared using any known preparation method. The powdered infant formula can be prepared using any standard method, typically employing a dry-mixing method or a wet-mixing / spray-drying method. In a dry-mixing method, the ingredients are combined in dehydrated powder form to achieve a homogeneous blend of the macro- and micronutrients essential for a complete infant formula product.

[0147] In one example of the method, the blended product is passed through a sieve to remove excessively large particles and foreign matter. The sieved product is then transferred to bags, tote bags, or fiberboard-lined drums for storage. In some cases, the powder can be transferred directly to a powder packaging line. On the packaging line, the powder is transferred to a filling hopper, which feeds the powder into the bottling line.

[0148] In one example of a wet blending / spray drying method, the ingredients are mixed together, homogenized, pasteurized, and spray-dried to prepare a powdered product. After pasteurization, the liquid is typically homogenized, and then any heat-sensitive micronutrients (e.g., vitamins, amino acids, and fatty acids) are added. The liquid can be concentrated via an evaporator or pumped directly to a spray dryer. Spray drying can cause the product to aggregate to increase particle size and improve its solubility. Alternatively, the milk can be dried by roller drying, where the milk is applied as a film to the heated roller surface. Freeze drying can also be used. The drying method and heat treatment during milk processing alter the properties of the milk powder, such as its solubility in cold water, flavor, and bulk density. The finished powder is then passed through a sieve and then transferred to bags, pouches, or silos for storage, or directly to a powder packaging line.

[0149] Infant formula is usually in powder form, but it can also be in liquid concentrate or ready-to-eat form. Water is mixed with the powder or liquid concentrate to prepare infant formula for consumption by infants.

[0150] The invention is further described with reference to the following embodiments. It should be understood that the claimed invention is not intended to be limited in any way by these embodiments.

[0151] Example

[0152] Example 1: Infant formula testing and reflux measurement I

[0153] Research Design

[0154] This study was a single-point, double-blind, randomized, controlled, parallel-lead study involving 32 infants aged 40–60 days who were breastfed prior to the study, had normal electrocardiograms (ECGs) and blood pressure at rest, and were not taking any medications, supplements, nutrients, or other dairy products, including lactic acid bacteria milk. The infant formulas used in this study were: (1) those prepared from cow's milk containing approximately 40:60 of histidine-containing β-casein and proline-containing β-casein at position 67 (Group A1 / A2), or (2) those prepared from cow's milk containing 100% proline-containing β-casein at position 67 (Group A2), both confirmed by ultra-high performance liquid chromatography and mass spectrometry in each case.

[0155] Statistical analysis of the study data was performed using SAS statistical software package version 9.3 (SAS Institute Inc., USA). All statistical tests for the hypotheses were two-tailed and performed at a significance level of 0.05. Means and standard deviations (SDs) for continuous variables and frequencies (%) for categorical variables at baseline and after intervention are provided. Baseline individual characteristics of all enrolled individuals, including age, sex, height, weight, head circumference, and chest circumference, were summarized using means and standard deviations (SDs) for the frequencies and percentages of continuous and categorical measures. Differences between study groups were assessed using analysis of variance (ANOVA) for continuous variables. Further paired between-group comparisons were then performed based on the results from ANOVA for outcomes showing significant differences between groups. Multiple comparisons were corrected for using Bonfferoni. Paired t-tests were used to assess differences within each study group.

[0156] result

[0157] The statistical summary of baseline individual characteristics of the study groups is shown in Table 1. Data are the means (standard deviation) of continuous variables or the frequencies (%) of categorical variables. Results from one-way ANOVA showed no significant differences among the three study groups, indicating that randomization was effective.

[0158] Table 1. Baseline Characteristic Evaluation - Mean (SD) or Frequency (%), ANOVA

[0159]

[0160] The baseline and weekly average daily milk reflux frequencies for each study group are summarized in Table 2. Both groups were similar at baseline for all daily living habits outcomes. A significant difference was observed between the BF (breastfeeding) and A1 groups in terms of reflux frequency during the first week after product intervention, but not between the BF and A2 groups.

[0161] Table 2. Summary of weekly averages of daily life habits results based on study group mean (SD) and p-value (ANOVA)

[0162]

[0163] *: p < 0.05; **: p < 0.01; ***: p < 0.001; Unsigned: p ≥ 0.05 / 3

[0164] Paired comparisons (Table 3) showed that there were significant differences between the A1 / A2 group and the breastfed (BF) group starting from week 1, and significant differences between the A1 / A2 group and the A2 group starting from week 2. However, there were no significant differences between the A2 group and the breastfed group throughout the study period.

[0165] Table 3. Paired comparison of weekly averages of daily life habit outcomes

[0166]

[0167]

[0168] *: p < 0.005 / 3; **: p < 0.01; ***: p < 0.001; Unsigned: p ≥ 0.05 / 3, using Bonferroni correction for multiple comparisons.

[0169] Example 2: Infant Formula Trial and Reflux Measurement II

[0170] Research Design

[0171] This was a randomized, open-label, prospective study designed to evaluate the nutritional impact of A2 infant formula on the tolerability of healthy full-term infants. A total of 280 healthy full-term mixed-fed infants (daily formula intake exceeding 400 mL) were recruited from Shanghai, including 140 A2 IMF mixed-fed infants and 140 A1 / A2 IMF mixed-fed infants as a control group. Mixed-fed infants were fed a mixture of IMF and human milk, with a daily intake of more than 400 mL of IMF. The term "A2IMF" refers to an IMF whose β-casein inclusions contain 100% by weight of a β-casein variant with proline at position 67 of the β-casein amino acid sequence, as determined by UPLC and mass spectrometry. The β-casein inclusions were determined to contain 17% by weight of β-casein I, with the remaining 87% by weight almost entirely β-casein A2. The term "A1 / A2 IMF" refers to an IMF whose β-casein contents consist of approximately 30% by weight of β-casein variants containing histidine at position 67 of the β-casein amino acid sequence (as determined by UPLC and mass spectrometry) and approximately 70% by weight of β-casein variants containing proline at position 67 of the β-casein amino acid sequence (also determined by UPLC and mass spectrometry).

[0172] The study period was 56 days prior to the introduction of complementary foods. There were four visits (days 0, 14, 28, and 56). At each visit, a vomiting / regurgitation (reflux) questionnaire and a gastrointestinal tolerance questionnaire were completed. At the first visit, parents / guardians received an “individual diary” containing a record of the daily frequency of vomiting / regurgitation. All parents / guardians received an “individual diary” and were instructed to bring it to each study visit for review. The frequency and volume of formula intake were recorded. Parents / guardians also recorded any other food / liquid intake (including infant cereal, non-study formula, water, baby food, etc.). The Infant Gastrointestinal Symptom Questionnaire (IGSQ) was used to assess GI-related signs and symptoms observed by parents / guardians in different domains, including vomiting / regurgitation as well as defecation, crying, flatulence, and irritability. For each study group, the daily average scores for the domains over the past week at each visit were tabulated. The composite IGSQ score (the sum of scores across all domains) was calculated for each study group and comparisons were made between study groups using ANCOVA, adjusting for baseline composite IGSQ scores, age, sex, and average daily formula intake during the week preceding each visit.

[0173] Statistical analysis

[0174] Descriptive statistics for the study variables were provided by the research team. Mean and standard deviation were summarized for normally distributed continuous variables; median and interquartile ranges were provided for non-normal variables; and frequencies and percentages were provided for categorical variables. A significance level of 0.05 was used for two-tailed statistical tests. Distributional hypotheses of the results were assessed prior to testing, and transformed or nonparametric versions of the tests were used if deemed necessary. A 95% confidence level was used for confidence intervals. All analyses were performed using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA).

[0175] result

[0176] Of the 280 eligible participants enrolled at baseline and who began taking the study formulation, 29 withdrew early in the study (14 in group A2 and 15 in the A1 / A2 control group), resulting in an overall withdrawal rate of 10.36%. The number of individuals who completed each study visit is listed in Table 4 by group.

[0177] Table 4. Number of individuals who completed each visit

[0178]

[0179] One-way ANOVA for normally distributed continuous variables, Kronoswiss test for non-normally distributed continuous variables, and chi-square / Fisher exact test for categorical variables were used to summarize and compare differences in demographic and other baseline characteristics between study groups, as shown in Table 5.

[0180] Table 5. Demographic and baseline characteristics

[0181]

[0182] Infant Gastrointestinal Symptom Questionnaire (IGSQ)

[0183] The two study groups were nearly identical at baseline in all individual item scores, vomiting / regurgitation scores, and total IGSQ scores. At weeks 2 and 4, the A2 group had significantly lower mean daily vomiting / regurgitation scores (p < 0.0001 and p = 0.0002). No significant differences were observed between the groups in other scores at weeks 1 and 8. See also Figure 1 and Figure 2 .

[0184] Table 6. Average daily vomiting / spitting up and IGSQ score

[0185]

[0186]

[0187] CI: Confidence interval.

[0188] Unless otherwise stated, data provided are mean ± standard deviation. Differences between groups were assessed using analysis of covariance, adjusted for age, sex, and mean daily formula intake over the past week. Post-intervention analyses were also adjusted for baseline scores. *: p < 0.05

[0189] a. Baseline data were based on 24-hour recall of symptoms from the previous day. Intergroup differences were assessed using analysis of covariance, adjusted for age and sex.

[0190] a. The data provided are the median (first quartile, third quartile) and range. The Kronoswiss test was used to assess differences between groups.

[0191] Although the invention has been described by way of examples, it should be understood that changes and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents to specific features exist, such equivalents may be incorporated, as specifically mentioned in this specification.

Claims

1. A method for reducing the frequency of infant reflux by providing infant formula to an infant for ingestion, wherein the milk used to prepare the infant formula contains β-casein, which contains less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

2. The method claimed in claim 1, wherein the β-casein comprises any one or more of β-casein variants A1, B, C, F, G, H1, and H2, which are less than 30% by weight.

3. The method claimed in claim 1 or claim 2, wherein the β-casein comprises less than 25% by weight of β-casein A1.

4. The method claimed in any one of claims 1-3, wherein the β-casein comprises less than 10% by weight of β-casein A1.

5. The method claimed in any one of claims 1-4, wherein the β-casein comprises less than 5% by weight of β-casein A1.

6. The method claimed in any one of claims 1-5, wherein the β-casein comprises less than 1% by weight of β-casein A1.

7. The method claimed in any one of claims 1-6, wherein the β-casein does not contain β-casein A1.

8. The method claimed in any one of claims 1-7, wherein the β-casein comprises at least 70% by weight of β-casein having a proline residue at position 67 of the β-casein amino acid sequence.

9. The method claimed in any one of claims 1-8, wherein the β-casein comprises at least 70% by weight of any one or more of β-casein variants A2, A3, D, E and I.

10. The method claimed in any one of claims 1-9, wherein the β-casein comprises at least 75% by weight of β-casein A2.

11. The method claimed in any one of claims 1-10, wherein the β-casein comprises at least 90% by weight of β-casein A2.

12. The method claimed in any one of claims 1-11, wherein the β-casein comprises at least 95% by weight β-casein A2.

13. The method claimed in any one of claims 1-12, wherein the β-casein comprises at least 99% by weight β-casein A2.

14. The method claimed in any one of claims 1-13, wherein the β-casein comprises 100% β-casein A2.

15. The method claimed in any one of claims 1-14, wherein the infant is less than 6 months or less than 3 months old.

16. A method for reducing the frequency of infant reflux episodes, performed by feeding an infant with infant formula, wherein the milk used to prepare the infant formula contains β-casein, which contains less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

17. A method for reducing the frequency of reflux in infants having a diet containing infant formula, wherein the infant formula contains β-casein, comprising at least 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence, the method being carried out by replacing the aforementioned infant formula with an alternative infant formula prepared from milk containing β-casein, wherein the β-casein comprises less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

18. A method for reducing the frequency of reflux episodes in an infant having a diet containing a first infant formula, wherein the first infant formula is replaced in the infant's diet with a second infant formula; The first infant formula contains β-casein, which comprises at least 30% by weight of β-casein having a histidine residue at position 67 of the β-casein amino acid sequence; and The second infant formula is prepared from milk containing β-casein, wherein the β-casein comprises less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

19. An infant formula for reducing the frequency of reflux episodes in infants, the infant formula being prepared from milk containing β-casein, said β-casein comprising less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

20. Use of cow's milk in the preparation of an infant formula for reducing the frequency of reflux in infants, wherein the milk contains β-casein, comprising less than 30% by weight of β-casein having histidine at position 67 of the β-casein amino acid sequence.

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