Reducing the incidence of adverse effects in breastfed infants

Abstract

The present disclosure relates to methods of reducing the incidence of adverse effects in breastfed infants, including methods of delivering nutrition to an infant so as to reduce the incidence of certain adverse effects, including adverse gastrointestinal (GI) symptoms, immune response, inflammation, crying and / or dermatitis, comprising orally administering to the infant breast milk from a lactating subject whose diet is substantially free of A1 type beta-casein variants.

Description

27369392.1:DCC-25 / 07 / 2025 - 1 - REDUCING THE INCIDENCE OF ADVERSE EFFECTS IN BREASTFED INFANTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from International Patent Application No. PCT / CN2024 / 107433 filed 25 July 2024 and Australian Provisional Patent Application No. 2024902438 filed 6 August 2024, the entire contents of each of which are incorporated herein by cross-reference. TECHNICAL FIELD

[0002] The present disclosure relates generally to methods of reducing the incidence of adverse effects in breastfed infants. In particular, the present disclosure relates to methods of delivering nutrition to an infant subject so as to reduce the incidence of certain adverse effects, including adverse gastrointestinal (GI) symptoms, immune response, inflammation, crying and / or dermatitis, comprising orally administering to the infant breast milk from a lactating subject whose diet is substantially free of A1 type beta-casein variants. BACKGROUND

[0003] Milk-based nutrition, such as breast milk and infant formula, is typically the exclusive source of nutrition in infants for the first 4 to 6 months after birth, and is often used to supplement their diet for several months or even years thereafter.

[0004] The primary source of milk-based nutrition in infants is breast milk or infant formula derived from bovine milk. The benefits to infants of breast feeding for at least the first six months of life, and preferably for another 6 to 12 months, are well-established. For example, human breast milk is known to protect infants from infections and to reduce the rates of health problems occurring including diabetes, obesity and asthma (see, e.g., WO 1996 / 014577, WO 1996 / 036239 and WO 2002 / 019832). It is widely accepted that the entire intestinal flora of breast-fed infants provides anti-infective properties and is an important stimulating factor for the postnatal development of the immune system.

[0005] However, regardless of whether infants receive nutrition from breast milk or infant formula, this milk-based diet often leads to adverse effects, including adverse27369392.1:DCC-25 / 07 / 2025 - 2 - gastrointestinal (GI) symptoms, immune response, inflammation, crying and / or dermatitis. For example, after consuming milk-based nutrition, infants may experience constipation, flatulence, regurgitation (spit up), crying, and the like. Certain milk-based nutrition has also been associated with dermatitis in infants. Such adverse GI symptoms and dermatitis may cause discomfort, mood disturbances and sleep disturbances in infants, which can also adversely affect the mood and sleep of the infant’s caregivers.

[0006] While a number of infant formulas have been developed to reduce the incidence of such adverse effects in infants, breast milk is widely regarded as the best source of nutrition for a new baby. Therefore, there is an ongoing need for improved or alternative methods for reducing the incidence of adverse effects, such as adverse GI symptoms, immune response, inflammation, crying and / or dermatitis in infants who receive nutrition via breast milk. SUMMARY

[0007] In one aspect, the present disclosure provides a method of reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence.

[0008] In another aspect, the present disclosure provides a method of delivering nutrition to an infant subject so as to reduce the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence.

[0009] In another aspect, the present disclosure provides a method of reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence.27369392.1:DCC-25 / 07 / 2025 - 3 -

[0010] In another aspect, the present disclosure provides a method of delivering nutrition to an infant subject so as to prevent or reduce the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence.

[0011] In another aspect, the present disclosure provides a method of reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

[0012] In another aspect, the present disclosure provides a method of delivering nutrition to an infant subject so as to reduce the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

[0013] In another aspect, the present disclosure provides a method of reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

[0014] In another aspect, the present disclosure provides a method of delivering nutrition to an infant subject so as to prevent or reduce the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in the infant subject, the method comprising orally27369392.1:DCC-25 / 07 / 2025 - 4 - administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

[0015] In another aspect, the present disclosure provides use of milk comprising beta- casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, wherein the milk or milk-derived product is to be consumed by a lactating subject whose breast milk is to be orally administered to the infant subject.

[0016] In another aspect, the present disclosure provides use of milk comprising beta- casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, wherein the milk or milk-derived product is to be consumed by a lactating subject whose breast milk is to be orally administered to the infant subject.

[0017] In another aspect, the present disclosure provides milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived product for reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, wherein the infant subject is orally administered breast milk from a lactating subject whose diet includes the milk or milk-derived products.

[0018] In another aspect, the present disclosure provides a milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived product for reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, wherein the infant subject is orally27369392.1:DCC-25 / 07 / 2025 - 5 - administered breast milk from a lactating subject whose diet includes the milk or milk- derived products. GENERAL DEFINITIONS

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0020] Unless otherwise specified, the indefinite articles “a”, “an” and “the” as used herein, include plural aspects. Thus, for example, reference to “an agent” includes a single agent, as well as two or more agents; reference to “the composition” or “formulation” includes a single composition or formulation, as well as two or more compositions or formulations; and so forth.

[0021] As used herein, the term “about”, as applied to one or more reference values, refers to a value that is similar or approximate to a stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In a particular embodiment, the term “about” means ±10% of the recited value.

[0022] Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0023] The term “consisting of” means “consisting only of”, that is, including and limited to the integer or step or group of integers or steps, and excluding any other integer or step or group of integers or steps.

[0024] The term “consisting essentially of” means the inclusion of the stated integer or step or group of integers or steps, but other integer or step or group of integers or steps that do not materially alter or contribute to the working of the invention may also be included.27369392.1:DCC-25 / 07 / 2025 - 6 -

[0025] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge.

[0026] Other more specific definitions relevant to understanding the present invention may be found throughout the description. DETAILED DESCRIPTION

[0027] The present disclosure generally provides methods of reducing the incidence of certain adverse effects in infants who receive nutrition via breast milk. In particular, the present inventors have found that infants who consume breast milk from a lactating subject (e.g., the infant’s mother) whose diet is low in (or substantially free of) beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence (i.e., A1 type beta- casein variants) may have a reduced incidence of certain adverse effects, such as adverse gastrointestinal (GI) symptoms, immune response, inflammation, crying and / or dermatitis. In other words, the reduction or elimination of A1 type beta-casein variants from the diet of breastfeeding mothers may result in a reduced incidence of certain adverse effects in breastfed infants.

[0028] A major source of A1 type beta-casein variants in human populations is bovine milk or bovine milk-derived products. Bovine milk typically comprises around 30 grams per litre of protein. Caseins make up the largest component (80%) of that protein, and beta-caseins make up about 37% of the caseins. In the past three decades, the body of evidence implicating casein proteins, especially A1 type beta-casein variants, in a number of health disorders (e.g., type I diabetes, coronary heart disease, neurological disorders, among others) has been growing. The beta-casein family comprises several variants, routinely known as A1, A2, A3, B, C, D, E, F, G, H1, H2 and I. There may also be other variants not yet identified. Beta-casein A1 and beta-casein A2 are the predominant beta-caseins in milk consumed in most human populations. Beta-casein A1 differs from beta-casein A2 by a single amino acid. A histidine amino acid residue is located at position 67 of the 209 amino acid sequence of beta-casein A1, whereas a proline residue is located at the same position of beta-casein A2.27369392.1:DCC-25 / 07 / 2025 - 7 -

[0029] The term “variant” when used in connection with a protein means a member of a set of highly similar proteins that are encoded by a single gene or gene family and are the result of genetic differences. Unless otherwise specified, the terms “beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence” and “A1 type beta- casein variants” are used interchangeably herein and have the same meaning. Such A1 beta- casein variants may include, but are not limited to, beta-casein A1, B, C, F, G, H1 and H2. In some embodiments, reference to an A1 type beta-casein variant means a beta-casein selected from the group consisting of beta-casein A1, B, C, F, G, H1 and H2, and any combination thereof. In a particular embodiment, reference to an A1 type beta-casein variant means beta-casein A1. Similarly, the terms “beta-caseins having a proline residue at position 67 of the beta-casein amino acid sequence” and “A2 type beta-casein variants” are used interchangeably herein and have the same meaning. Such A2 beta-caseins may include, but are not limited to beta-casein A2, A3, D, E and I. In some embodiments, reference to an A2 beta-casein variant means a beta-casein selected from the group consisting of beta-casein A2, A3, D, E and I, and any combination thereof. In a particular embodiment, reference to an A2 beta-casein variant means beta-casein A2.

[0030] The defining difference in structure between A1 and A2 type beta-casein variants is that A1 type beta-casein variants have a histidine residue at position 67 of the beta-casein amino acid sequence and A2 type beta-casein variants have a proline residue at position 67 of the beta-casein amino acid sequence. The presence of a histidine residue at position 67 enables A1 type beta-casein variants to be readily digested in the gut to produce the opioid peptide BCM-7, whereas the presence of a proline residue at position 67 sterically constrains A2 type beta-casein variants, making any production of BCM-7 from A2 type beta-casein variants very difficult. The term “beta-casomorphin-7” or “BCM-7” refers to the protein fragment Tyr-Pro-Phe-Pro-Gly-Pro-Ile, a heptapeptide produced readily on enzymatic digestion of bovine beta-casein variants that have a histidine at position 67 of the amino acid sequence, but not produced or may be produced at low levels on enzymatic digestion of bovine beta-casein variants that have a proline at position 67 of the amino acid sequence. BCM-7 is able to cross the gastrointestinal wall and enter the circulation, enabling it to influence systemic and cellular activities via opioid receptors. BCM-7 also has the potential to cross the blood-brain barrier and activate opioid receptors in the central nervous system.27369392.1:DCC-25 / 07 / 2025 - 8 -

[0031] Conventional wisdom is that macromolecules or protein fragments (including BCM-7) are not transferred from the diet to the breast milk of a lactating subject. Rather, the components of breast milk are believed to be produced by the cells in the mammary glands, which in turn obtain the basic nutrients (amino acids, minerals, co-enzymes, vitamins) from the circulation. Without wishing to be bound by theory or a particular mode of action, the present inventors postulate that BCM-7, or protein fragments containing BCM-7, are taken up by macrophages in the gut and transferred to breast milk via the lymphatic system. Thus, it is postulated that when a sufficient quantity of A1 type beta-casein variants are consumed by a lactating subject, BCM-7 (or fragments containing BCM-7) may be transferred to an infant subject who consumes (i.e., eats, drinks and / or otherwise ingests) the breast milk of the lactating subject, leading to an increased incidence of certain adverse effects in the infant, such as adverse GI symptoms, immune response, inflammation, crying and / or dermatitis.

[0032] The present inventors surprisingly found in a randomized controlled trial to investigate effects of conventional (bovine) milk versus A1 type beta-casein protein free milk (“A1PF milk”) on infant gut health, crying and sleep patterns that healthy full term infants who were breastfed by mothers who consume A1PF milk (being bovine milk that is substantially free of A1 type beta-casein variants) had a reduced incidence of certain adverse effect compared with healthy full term infants who were breastfed by mothers who consume conventional bovine milk. For example, the A1PF milk group had a reduced incidence of adverse GI symptoms, including a decreased volume of regurgitation (spit up), reduced discomfort or fuss during regurgitation, fewer hard stools, reduced frequency of constipation (difficulty passing bowel movements), reduced frequency of flatulence (gas), and / or reduced discomfort or fuss during flatulence. The A1PF milk group also had a reduced incidence of crying, including a reduced duration and / or frequency of crying. Further, the A1PF milk group had a reduced incidence of dermatitis, or less itching and / or scratching associated with dermatitis.

[0033] Thus, the present invention provides methods for reducing the incidence of adverse GI symptoms, immune response, inflammation, crying and / or dermatitis in an infant subject. The present invention also provides methods for delivering nutrition to an infant subject so as to reduce the incidence of adverse GI symptoms, immune response, inflammation, crying and / or dermatitis in the infant subject. As demonstrated in Example 1, the methods of present27369392.1:DCC-25 / 07 / 2025 - 9 - invention may reduce the incidence of adverse GI symptoms, which may include, but is not limited to, decreasing volume of regurgitation, reducing discomfort or fuss during regurgitation, reducing number of hard stools, reducing frequency of constipation, reducing frequency of flatulence, or reducing discomfort or fuss during flatulence, or any combination thereof. In some embodiments, the methods of the present invention may reduce the incidence of dermatitis, examples of which may include, but is not limited to, atopic dermatitis (eczema), contact dermatitis, dyshidrotic eczema and seborrheic dermatitis, or any combinations thereof. In a particular embodiment, the dermatitis is eczema, as demonstrated in Example 1.

[0034] The present inventors also surprisingly found that the concentrations of certain biomarkers were decreased or increased in the A1PF milk group relative to baseline. For example, fecal concentrations of acetic acid and total short chain fatty acids (SCFA) increased from baseline in the A1PF milk group, while fecal concentrations of lactic acid decreased from baseline in the A1PF milk group. Fecal lactic acid is generated by colonic fermentation of carbohydrates, such as lactose, that have not been digested and absorbed in the upper GI tract. Accordingly, a decrease in fecal lactic acid may be indicative of improved carbohydrate or lactose digestion. Further, salivary concentrations of IFN-γ and GSH increased from baseline in the A1PF milk group, while salivary concentrations of TNF-α, IL-1, IL-1β, IL-4, IL-6, peroxidase, IgA, IgG, IgG1, IgE and mucin decreased from baseline in the A1PF milk group. These changes in immune biomarker concentrations may be indicative of a reduced incidence of immune response and / or inflammation, and / or improved gut health in infants who are breastfed by mothers whose diet is substantially free of A1 type beta-casein variants. In particular, decreased salivary concentrations of pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-1β, IL-4, IL-6) may be indicative of a reduction in inflammation throughout the body (systemically), including but not limited to inflammation of the GI tract. Without wishing to be bound by theory or a particular mode of action, the present inventors postulate that the decreased salivary concentrations of immune biomarkers such as pro-inflammatory cytokines (e.g., TNF-α, IL-1, IL-1β, IL-4, IL-6) may be due, at least in part, to the reduction or elimination of A1 type beta-casein variants from the diet of the lactating subjects and / or to the increased consumption of A2 type beta-casein variants. Thus, it is to be understood that the methods (and uses) of the present invention may also27369392.1:DCC-25 / 07 / 2025 - 10 - improve digestion and / or reduce the incidence of immune response and / or inflammation in the infant subject.

[0035] Additionally, the concentration of certain minerals, such as iron and zinc, in the breastmilk of lactating subjects was found to be significantly higher in the A1PF group than the conventional milk group. This further suggests that reducing or eliminating A1 type beta- casein in the diet of lactating subjects may improve the nutritional value of their breast milk, thereby improving the overall health of infant subjects who consume that breast milk.The present applicant has also previously found that levels of beneficial gut microbiota are improved by reducing or eliminating A1 type beta-casein in the diet, as was apparent from higher fecal concentrations of SCFAs, such as acetic acid and butanoic acid (see WO 2018 / 063008, the entire contents of which are incorporated herein by reference). Thus, it is to be understood that the methods (and uses) of the present invention may also improve levels of beneficial gut microbiota and / or promote the growth of bacteria, and may be associated with fewer adverse GI symptoms.

[0036] In some embodiments, the methods and uses of the present invention comprise orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence (i.e., A1 type beta-casein variants). In other embodiments, the methods of the present invention comprise orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising A1 type beta-casein variants in an amount that is less than about 30% by weight of total beta-caseins present in the milk or milk-derived products. For example, the milk or milk-derived products referenced herein may comprise A1 type beta-casein variants in an amount that is less than about 30% by weight, or about 25% by weight, or less than about 20% by weight, or less than about 10% by weight, or less than about 5% by weight, or less than about 1% by weight of total beta-caseins present in the milk or milk-derived products. Conversely, the milk or milk-derived products may comprise beta-casein having a proline residue at position 67 of the beta-casein amino acid sequence in an amount that is at least about 70% by weight, or at least about 75% by weight, or at least about 80% by weight, or at least about 90% by weight, or at least about 95% by weight, or at least about 99% by weight of total beta-caseins present27369392.1:DCC-25 / 07 / 2025 - 11 - in the milk or milk-derived products. Preferably, the milk or milk-derived products consumed by the lactating subject are substantially free of A1 type beta-casein variants.

[0037] The term “substantially free” when used herein with reference to a milk or milk- derived product, means that the amount of A1 type beta-casein variants in the milk or milk- derived product is less than about 1% by weight, or less than about 0.5% by weight, or less than about 0.1% by weight of total beta-caseins present in the diet or product. Preferably, the milk or milk-derived product contains no detectable A1 type beta-casein variants, particularly beta-casein A1, when measured using standard techniques known in the art. The present applicant has also developed a method for determining the amount of A1 and A2 type beta-casein variants present in a milk or milk-derived product (see WO 2020 / 091608, the entire contents of which are incorporated herein by reference).

[0038] Thus, a diet that is substantially free of A1 type beta-casein variants may be achieved by the lactating subject consuming a diet that is free of bovine milk or milk-derived products (i.e., a “dairy free” diet), or by consuming only milk or milk-derived products comprising A1 type beta-casein variants in an amount that is less than about 30% (e.g., less than about 25%, 20%, 10% 5% or 1% by weight) by weight of total beta-caseins present in the milk or milk-derived products. Preferably, the milk or milk-derived products consumed by the lactating subject are predominantly milk or milk-derived products that are substantially free of A1 type beta-casein variants. Such milk or milk-derived products may be any animal or plant-based milk or milk-derived products, including but not limited to bovine (e.g., cows, bison, buffalo), carpine (e.g., goats), ovine (e.g., sheep), soy, oat, nut (e.g., almond), coconut, or rice milk or milk-derived products, and the like. In a particular embodiment, the milk or milk-derived product is a bovine milk or milk-derived product that is substantially free of A1 type beta-casein variants (e.g., A1PF milk or milk-derived products).

[0039] As used herein, the terms “infant” and “infant subject” may include a child under the age of 24 months, e.g., a child under the age of 24 months, 18 months, 12 months, 6 months or 4 months. In an embodiment, the term “infant” means a child aged from 0 months to 12 months. In another embodiment, the term “infant” means a child aged from 0 months to 6 months. It is to be understood that reference herein to an “infant”, “infant subject” or “breastfed infant” means an infant whose primary source of nutrition is breast27369392.1:DCC-25 / 07 / 2025 - 12 - milk. Further, the terms “breastfed” or “breastfeeding” when used in relation to an infant is to be understood to encompass infants who consume breast milk either directly or indirectly from the breast of a lactating subject (e.g., by bottle feeding). In some embodiments, the infant’s only source of nutrition is breast milk. Further, as used herein, the term “lactating subject” includes any subject who produces breast milk. In an embodiment, the lactating subject is the infant subject’s biological mother. In another embodiment, the lactating subject is a breast milk donor (e.g., a wet nurse). In some embodiments, the infant subject and the lactating subject are both mammals. The term “mammal” as used herein includes humans, primates, livestock animals (e.g., horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g., mice, rats, guinea pigs), companion animals (e.g., dogs, cats) and captive wild animals (e.g., kangaroos, deer, foxes). Preferably, the infant subject and the lactating subject are both human.

[0040] The breast milk from the lactating subject may be orally administered to the infant subject by any suitable means. For example, the breast milk may be orally administered to the infant subject by breastfeeding from a breast of the lactating subject, or it may be orally administered to the infant subject by bottle feeding after having been pumped from a breast of the lactating subject. In some embodiments, the breast milk may be stored for a period of time (e.g., about 1 day, 2 day, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 1 month or more) at a suitable temperature (e.g., from about 4 °C to about -20 °C) prior to being orally administered to the infant subject. In some embodiments, the breast milk may be dried (e.g., by freeze-drying) to provide a powder, which may be reconstituted in water prior to being orally administered to the infant subject.

[0041] The methods described herein may result in a reduced incidence of adverse effects, such as adverse GI symptoms, immune response, inflammation, crying and / or dermatitis in infants. Depending on the context, reference to a “reduced incidence” (or a grammatical variant thereof) of an adverse effect may be a reference to the frequency, severity, duration and / or volume of an adverse effect. For example, a reduced incidence of regurgitation may refer to at least one of a reduced frequency, duration or volume of regurgitation. In another example, a reduced incidence of crying may refer to a reduced frequency or duration of crying episodes. In some embodiments, a reduced incidence may encompass prevention of an adverse effect. For example, a reduced incidence of dermatitis may include a reduced27369392.1:DCC-25 / 07 / 2025 - 13 - frequency, severity or duration of the dermatitis, or it may include prevention of dermatitis. The term “prevent” (and grammatical variants thereof) with regard to an adverse effect, such as dermatitis, refers to averting the cause, effects, or progression of the adverse effect prior to the adverse effect fully manifesting itself.

[0042] It is to be understood that any reference herein to a “reduced” incidence of an adverse effect in an infant who is orally administered breast milk from a lactating subject whose diet is substantially free of A1 type beta-casein variants is relative to an infant who is orally administered breast milk from a lactating subject whose diet is not substantially free of A1 type beta-casein variants. Similarly, it is to be understood that any reference herein to a “reduced” incidence of an adverse effect in an infant who is orally administered breast milk from a lactating subject whose diet includes milk or milk-derived products comprising A1 type beta-casein variants in an amount that is less than 30% by weight of total beta- caseins present in the milk or milk-derived products is relative to an infant who is orally administered breast milk from a lactating subject whose diet includes milk or milk-derived products comprising A1 type beta-casein variants in an amount that is at least 30% by weight of total beta-caseins present in the milk or milk-derived products.

[0043] Reducing the incidence of certain adverse effects in an infant subject disclosed herein may be associated with improved mood and / or sleep in the infant subject. For example, as demonstrated in Example 1, a reduced incidence of itching and / or scratching associated with dermatitis was found to be associated with improved mood in infants. It is to be understood that any reference herein to an “improvement” (or a grammatical variant thereof), such as improved mood or sleep in an infant subject, is relative to an infant subject who is orally administered breast milk from a lactating subject whose diet is not substantially free of A1 type beta-casein variants, or to an infant subject who is orally administered breast milk from a lactating subject whose diet includes milk or milk-derived products comprising A1 type beta-casein variants in an amount that is greater than or equal to 30% by weight of total beta-caseins present in the milk or milk-derived products, as the case may be.

[0044] In some embodiments, the reduced incidence of adverse effects as described herein is achieved by the lactating subject consuming a diet that is substantially free of A1 type beta-casein variants for at least 7 days, preferably at least 14 days prior to orally administering the breast milk to the infant subject. In other embodiments, the reduced27369392.1:DCC-25 / 07 / 2025 - 14 - incidence of adverse effects as described herein is achieved by the lactating subject consuming a diet that includes milk or milk-derived products comprising A1 type beta- casein variants in an amount that is less than 30% by weight (e.g., less than about 25%, 20%, 10% 5% or 1% by weight) of total caseins present in the milk or milk-derived products, for at least 7 days, preferably at least 14 days prior to orally administering the breast milk to the infant subject. In a particular embodiment, the lactating subject consumes milk or milk- derived products that are substantially free of beta-casein A1 for at least 7 days, preferably at least 14 days prior to orally administering the breast milk to the infant subject.

[0045] The present invention also provides use of milk comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight (e.g., less than about 25%, 20%, 10% 5% or 1% by weight) of total caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of adverse GI symptoms, immune response, inflammation, crying and / or dermatitis in an infant subject, including those adverse GI symptoms described elsewhere herein (e.g., as demonstrated in Example 1). The milk or milk-derived product so manufactured are to be consumed by a lactating subject whose breast milk is to be orally administered to the infant subject. In some embodiments, the milk used to manufacture the milk or milk-derived product is substantially free of A2 type beta-casein variants. This may be achieved by ensuring that the beta-casein in the milk used to manufacture the milk or milk-derived product is predominantly or exclusively beta-casein variants having proline at position 67 of the amino acid sequence, preferably beta-casein A2.

[0046] The present inventors also found that lactating subjects whose diet is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence may also experience certain benefits, such as a reduced incidence of physical and / or mental fatigue. Thus, the present disclosure also provides methods of reducing the incidence of physical and / or mental fatigue in a lactating subject, the method comprising the lactating subject consuming a diet that is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence, or a diet that includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight (e.g., less than about 25%, 20%, 10% 5% or 1% by weight) of total beta-caseins present in the milk or milk-27369392.1:DCC-25 / 07 / 2025 - 15 - derived products. The present disclosure also provides use of milk comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of physical and / or mental fatigue in a lactating subject.

[0047] The milk or milk-derived products disclosed herein may be any suitable products, including food or beverage products that comprise or consist essentially of the milk or milk- derived products. For example, the milk or milk-derived products are selected from the group consisting of fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk, non-pasteurised milk, cream, yoghurt, quark, cheese, butter and ice cream. In some embodiments, the milk or milk-derived products are selected from the group consisting of fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk and non-pasteurised milk. In a particular embodiment, the milk product is fresh milk. Examples of other suitable milk or milk-derived products will be apparent to those skilled in the art. Such products may be consumed by the lactating subject by eating or drinking a food or beverage product consisting essentially of the milk or milk- derived product, or by eating or drinking a food or beverage product comprising the milk or milk-derived product as a component or ingredient of the food or beverage product.

[0048] The A1 and A2 beta-casein variants are the predominant variants in most dairy populations. The beta-casein in the milk used to manufacture the milk or milk-derived products for use in the present invention may comprise at least 70%, 75%, 80%, 90%, 95%, 98% and 99% or 100% A2 beta-casein variants. Ideally, the beta-casein in the milk comprises 100% beta-casein A2. The production of beta-caseins in the milk of a bovine cow is determined by genetics. For example, a bovine cow homozygous for the A1 allele (A1A1) can produce only the A1 beta-casein variant in its milk. A bovine cow homozygous for the A2 allele (A2A2) can produce only the A2 beta-casein variant in its milk. Further, a bovine cow heterozygous for the beta-casein A1 and A2 genes (A1A2) is able produce both the A1 and A2 beta-casein variants in its milk. It is well-established that bovine cows can be genotyped for the beta-casein gene. Cows that are genotyped and determined to be27369392.1:DCC-25 / 07 / 2025 - 16 - homozygous for beta-casein having proline at position 67 include, but are not limited to, A2A2 and A2I cows. Cows determined to be homozygous for beta-casein having histidine at position 67 include, but are not limited to, A1A1 and A1B cows. Cows determined to be heterozygous beta-casein at position 67 include, but are not limited to, A1A2, A1I and A2B cows. Those cows that are determined to be homozygous for beta-casein having proline at position 67 can be separated from all others to produce a herd capable of producing only beta-caseins having proline at position 67 in their milk. The manufacture of milk or milk- derived products from this milk therefore provides products that are substantially free of A1 type beta-casein variants.

[0049] The milk of cows may be tested for relative proportions of beta-casein variants containing proline or histidine at position 67 of the amino acid sequence to determine whether the milk is suitable for use in preparing milk or milk-derived products for use in the present invention. Alternatively, cows can be genetically tested for their ability to produce milk containing beta-casein A1 (or other beta-casein variants having histidine at position 67 of the amino acid sequence) or tested for their ability to produce milk containing beta-casein A2 (or other beta-casein variants having proline at position 67 of the amino acid sequence) or a combination of both. Suitable techniques are well known in the art.

[0050] Those skilled in the art will be aware that the disclosure provided herein is subject to variations and modifications other than those specifically described. It is to be understood that the disclosure provided herein includes all such variations and modifications. The present disclosure also includes all such steps, features, methods, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0051] Certain embodiments of the disclosure will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.27369392.1:DCC-25 / 07 / 2025 - 17 - EXAMPLES Example 1. Randomized controlled trial to investigate effects of conventional vs A1PF milk on breast milk composition and subsequent infant gut health / crying and sleep patterns in healthy term infants Abbreviations ELISA Enzyme linked Immunosorbent Assay LC-MS Liquid Chromatography-Mass Spectrometry HMO Human Milk Oligosaccharide Content BCM Beta-casomorphin IL-1 Interleukin-1 IL-4 Interleukin-4 IL-6 Interleukin-6 IL-10 Interleukin-10 IL-1β Interleukin-1β SCFA Short Chain Fatty Acids MPO Myeloperoxidase CRP C-reactive Protein hsCRP High-sensitivity C-reactive Protein IgA Immunoglobulin A IgG Immunoglobulin G IgG1 Immunoglobulin G1 IgE Immunoglobulin E TNFα Tumor Necrosis Factor α Eczema severity and life quality index related to dermatitis via the Infants’ IDQoL Dermatitis Quality of Life Index IGSQ Infant Gastrointestinal Symptom Questionnaire GSRS Gastrointestinal Symptom Rating Scale AE / SAE Adverse Event / Serious Adverse Event Conv. Conventional milk27369392.1:DCC-25 / 07 / 2025 - 18 - ICF Informed Consent Form International Council for Harmonisation of Technical Requirements for ICH Pharmaceuticals for Human Use 1. STUDY OBJECTIVES 1.1. Primary Objectives

[0052] Mother: · Mother’s gastrointestinal tolerance via questionnaire; · Mother’s salivary and faecal, markers of inflammation and immune response; and · Breast milk mineral composition analysis.

[0053] Infants of these mothers: · IGSQ; · Infant’s salivary and faecal markers of inflammation and immune response; and · Infant crying frequency, duration and sleep patterns. 1.2. Secondary Objectives

[0054] Mother: · Mother’s overall energy / fatigue.

[0055] Infants of these mothers: · Eczema severity and life quality index related to dermatitis via the Infants’ IDQoL; · Anthropometric parameters including weight, length, head circumference; BMI and their corresponding z-scores calculated using the 2006 WHO Growth Standards; and · Adverse effects.27369392.1:DCC-25 / 07 / 2025 - 19 - 2. STUDY DESIGN 2.1. Overview

[0056] This is a two arm, parallel-designed, randomized controlled, double-blind study to investigate the effects of conventional vs A1PF milk on nursing mothers’ gut health, markers of inflammation and immune response, and subsequent infant gut health / crying, sleep patterns and markers of inflammation and immune response in Healthy Term Infants. Up to three study visits were made by the participant over a 14-day period. 2.2. Study Products

[0057] Study milk for mothers: · Conventional milk 200ml / carton: twice per day · A1PF milk (a2 Milk® produced and supplied by The a2 Milk Company) 200ml / carton: twice per day

[0058] Duration of feeding: Subjects received conventional milk or A1PF milk at baseline and continue in-taking for 14 days. 2.3. Study Participants

[0059] Number of participants to be enrolled: 50 mothers and 50 babies were enrolled at baseline and randomized to two groups, with 25 mothers and 25 babies in each group. 2.3.1. Informed consent

[0060] The informed consent process occurred prior to enrolment according to ICH Guidelines and local regulations. Parents or legal guardians signed the informed consent. Fifty mothers must sign the informed consent for themselves and babies to be eligible for the study.

[0061] All ICFs will be signed and archived in two study hospitals. 2.3.2. Participant Inclusion Criteria · Mothers who are exclusively breastfeeding; · Healthy and term infants (37-42 weeks gestation, 2- or 3-month-old infants);27369392.1:DCC-25 / 07 / 2025 - 20 - · Infants of these mothers who have been exclusively breastfed since birth, and their parent(s) decides to continue exclusively breastfeeding until at least 120 days of age Birth weight between 2500g and 4500g; · Agree to stop taking any dairy products 5 days before the start of the study; · Agree not to drink / consume strong tea, coffee, cola, and other foods that may affect the results of the study during the study intervention; · Agree not to consume any dairy products other than the study product, containing animal milk related products such as cheese, milk tea, cream cakes, ice cream, cheese, etc. during the study intervention; · Willing to sign ICF before the study launched. 2.3.3. Participant Exclusion Criteria · Mothers with infection or have recently taken the flu / COVID vaccine in the last 4 weeks; · Infant with inborn malformation and with hereditary and / or chronic and / or inborn diseases that could interfere with the survey; · Evidence of feeding difficulties or intolerance / allergy to cow’s milk (mixed- fed group only); · Conditions requiring infant feedings other than those specified in the protocol; · Significant systemic disorders (cardiac, respiratory, endocrinological, hematologic, gastrointestinal, or other); or parental refusal to participate; · Infants with an acute infection or gastroenteritis at time of enrolment; · Participation in another clinical trial; · Investigator's uncertainty about the willingness or ability of the parents to comply with the protocol requirements. 2.4. Randomization

[0062] Prior to randomization to study milk, all inclusion and exclusion criteria were verified to ensure the potential participant qualifies for study enrolment. Randomization envelopes remained sealed until subject was determined eligible for enrolment by confirming that the subject met all of the inclusion criteria and none of the exclusion criteria,27369392.1:DCC-25 / 07 / 2025 - 21 - and that permission had been obtained from the mother through a signed informed consent form.

[0063] When a subject was considered eligible for enrolment, the participant was randomly assigned to one of the two study milks based upon the randomization envelopes. 2.5. Participant Withdrawal Criteria

[0064] The Investigator and / or subject may elect to withdraw from the study at any time. Reasons for withdrawing a participant include, but are not limited to: · Study milk never consumed · Failure to thrive · Participant was lost to follow-up after 3 unsuccessful attempts to contact · Subject elected to remove from the study for any reason

[0065] All participants who withdraw from the study prior to the last study day should be followed to determine the participant’s condition approximately days after stopping study milk. The follow-up will consist of a phone call or home visit with the subject or physician or a review of the participant’s medical records and will be documented on the appropriate case report form.

[0066] A participant is considered lost to follow-up after three unsuccessful attempts to contact. All attempts to contact the subjects should be documented in the participant's file. 3. STUDY OUTCOMES 3.1. Physical Exam – Medical History

[0067] Medical staff at the study site performed physical exams of each participant and record any abnormalities found. Demographic and social economy information were collected at baseline. 3.2. Questionnaires

[0068] Mothers’ GSRS (day 0, 1, 7, 14), IGSQ (day 0, 7, 14), Infant Gastrointestinal Tolerance Questionnaire (day 0, 1), infants’ crying frequency and duration (day 0, 1), sleep patterns (day 0, 7, 14), eczema severity and life quality index related to dermatitis via the27369392.1:DCC-25 / 07 / 2025 - 22 - IDQoL (day 0, 7, 14), and mother’s overall energy / fatigue scores (day 0, 7, 14) were collected. 3.3. Anthropometric Measurements

[0069] During the study period, participants were weighed on the study site’s scale and the weight was recorded. Participants’ body length / height without shoes and head circumference were measured in centimeters at the study sites. 3.4. Bio-samples Analysis

[0070] Breast milk and saliva samples were collected at each of the 3 study visits and stored at study sites. All bio-samples were analyzed in the lab appointed by Jinhua Hospital of TCM.27369392.1:DCC-25 / 07 / 2025 - 23 -27369392.1:DCC-25 / 07 / 2025 - 24 - 4. STATISTICAL METHODS 4.1. Sample Size Determination:

[0071] The primary outcome for this study is for breast milk characterization and composition analysis during the study period. A sample size of 20 mothers and 20 infants per group was needed. Taken into consideration of a 20% possible attrition rate, 25 mothers and 25 infants in each group were enrolled at baseline.

[0072] Mean and standard deviation (SD) were provide for continuous variables with normal distribution; median, lower and upper quartiles were provided for non-normal or ordinal variables; counts and percentage were provided for discrete variables. All descriptive analyses were presented by study group.

[0073] Between-group comparisons were performed using analysis of variance (ANOVA) for continuous variables with normal distribution (post intervention analysis also adjusted for baseline measurements), Kruskal-Wallis test for non-normal or ordinal variables, and Fisher’s exact test for discrete variables. Within-group changes from baseline were evaluated using paired t-test for normal distribution continuous variables, Wilcoxon signed rank test for non-normal or ordinal variables. A two-sided 0.05 level of significance was used for statistical testing unless specified otherwise. Prior to testing, distributional assumptions for the outcomes were assessed and transformations or nonparametric versions of the tests were used if deemed necessary.

[0074] All analyses were performed using the SAS 9.4 statistical software. 5. RESULTS 5.1. Baseline Characteristics

[0075] The infants and their parent in the A1PF group and the conventional milk group were comparable in all baseline characteristics (Table 1). Mothers from both groups had no diagnosis or history of diabetes, renal insufficiency, liver disease, heart disease, chronic obstructive pulmonary disease, hypertension, pelvic floor dysfunction, pelvic / abdominal surgery, depression, anxiety, or gastrointestinal disorders.27369392.1:DCC-25 / 07 / 2025 - 25 - Table 1. Baseline Characteristics p-27369392.1:DCC-25 / 07 / 2025 - 26 - p-27369392.1:DCC-25 / 07 / 2025 - 27 - p-Unless otherwise stated, data presented are mean±standard deviation for continuous variables and frequency (%) for categorical variables. Between-group difference was evaluated using analysis of variance for continuous variables and Fisher’s exact test for categorical variables. a Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. 5.2. Laboratory Analysis 5.2.1. Biomarkers of Mothers

[0076] The levels of metals in breast milk were comparable between the two study groups at baseline (Table 2). Calcium concentration in breast milk increased significantly after one day of A1PF milk consumption (p=0.025), however the difference compared to the conventional group was not significant. After 14 days of milk consumption, calcium, iron and zinc concentration increased significantly from baseline in both the A1PF group (p<0.0001, p<0.0001 and p=0.002, respectively) and the conventional milk group (p=0.0002, p<0.0001 and p=0.029, respectively). The A1PF group had significantly higher iron (p=0.0001) and zinc (p=0.026) concentration compared to the conventional milk group; it had slightly higher calcium concentration than the conventional milk group, but the difference between the two groups was not significant. In addition, selenium concentration in the breast milk of the A1PF group also increased significantly from baseline (p=0.015), although test of group difference resulted in lack of significance.27369392.1:DCC-25 / 07 / 2025 - 28 - Table 2. Breast Milk Laboratory Analysis v. 3 2* 0 0 3 01* 3 0 4 1 8 9*Data presented are mean±standard deviation. Between-group difference was evaluated using analysis of variance; post-intervention comparisons were adjusted for baseline measurements; the difference was27369392.1:DCC-25 / 07 / 2025 - 29 - presented as the difference of least-squares means (95% confidence interval). Within-group difference was evaluated using paired t-test. *: p<0.05.

[0077] Mothers in the two study groups had similar levels of all fecal biomarkers measured at baseline (Table 3). No significant change or significant difference between the two groups was observed in these biomarkers after one day of milk consumption.

[0078] After 14 days of A1PF milk consumption, mothers’ fecal concentrations of CRP (p=0.0004), lactic acid (p=0.002) decreased significantly from baseline; and were significantly lower than that of the conventional milk group (p=0.023 and 0.008, respectively). Fecal acetic acid, propionic acid and total SCFA concentration of the A1PF group increased significantly from baseline (all p<0.0001); and were significantly higher than that of the conventional milk group (p=0.018, 0.021 and 0.029, respectively). Fecal butyric acid concentration of the A1PF group also increased significantly from baseline (p=0.001), but the difference compared to the conventional milk group was not significant. Table 3. Fecal Laboratory Analysis for Mothers o nv. 44 80 25 9727369392.1:DCC-25 / 07 / 2025 - 30 - o nv. 04 76 12 45 53 39 26 19 57 02Unless otherwise stated, data presented are mean±standard deviation. Between-group difference was evaluated using analysis of variance; post-intervention comparisons were adjusted for baseline measurements; the difference was presented as the difference of least-squares means (95% confidence interval). Within-group difference was evaluated using paired t-test. *: p<0.05. a Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test.

[0079] Mothers in the two study groups had similar levels of all salivary biomarkers measured at baseline (Table 4). No significant change or significant difference between the two groups was observed in these biomarkers after one day of milk consumption.27369392.1:DCC-25 / 07 / 2025 - 31 -

[0080] After 14 days of A1PF milk consumption, mothers’ salivary concentrations of hsCRP (p=0.007), IL-4 (p=0.007), IL-6 (p=0.008), IgA (p=0.004) and IgE (p=0.014) decreased significantly from baseline. Among these biomarkers, the concentration of IL-4 (p=0.025), IL-6 (p=0.001), IgA (p=0.023) and IgE (p=0.004) were significantly lower than that of the conventional milk group. Mothers in the A1PF group had significantly increased salivary concentration of IFN-γ (p=0.007) and GSH (p=0.001) compared to baseline. Their GSH level was significantly higher than that of the conventional milk group (p=0.049); group difference in IFN-γ concentration was not significant.

[0081] Compared to baseline measurements, salivary lysozyme concentration of the A1PF group decreased slightly, while that of the conventional milk group increased slightly. Significant between-group difference was observed in lysozyme concentration (p=0.005), although within-group changes from baseline was not significant in both groups. Table 4. Salivary Laboratory Analysis for Mothers o v. 12 14 90 9* 00 9027369392.1:DCC-25 / 07 / 2025 - 32 - v. 0 1 0 3 6* 7 7 7 6 8 0 827369392.1:DCC-25 / 07 / 2025 - 33 - o v. 53 98 31 87 37 53 91 18 24 76 16 82 69 6327369392.1:DCC-25 / 07 / 2025 - 34 - o v. 07 54 02 63 82 61 90 95Unless otherwise stated, data presented are mean±standard deviation. Between-group difference was evaluated using analysis of variance; post-intervention comparisons were adjusted for baseline measurements; the difference was presented as the difference of least-squares means (95% confidence interval). Within-group difference was evaluated using paired t-test. *: p<0.05. a Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.2.2. Biomarkers of Infants

[0082] Infants in the two study groups had similar levels of all fecal biomarkers measured at baseline (Table 5). No significant change or significant difference between the two groups was observed in these biomarkers after one day of milk consumption.27369392.1:DCC-25 / 07 / 2025 - 35 -

[0083] After 14 days of milk consumption, fecal concentrations of acetic acid (p<0.0001), propionic acid (p=0.016), butyric acid (p=0.001) and total SCFA (p<0.0001) of the A1PF group increased significantly from baseline. Among these biomarkers, the concentration of acetic acid and total SCFA were significantly higher than that of the conventional milk group (p=0.048 and 0.041, respectively). Fecal lactic acid concentration decreased slightly from baseline among infants in the A1PF group and increased slightly from baseline among those in the conventional milk group; although none of the within-group change from baseline was significant, the A1PF group showed significantly lower lactic acid concentration compared to the conventional milk group (p=0.041). Table 5. Fecal Laboratory Analysis for Infants . 5 3 0 6 7 4 1 927369392.1:DCC-25 / 07 / 2025 - 36 - . 0 6 8 4 6 2Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test.

[0084] Infants in the two study groups had similar levels of all salivary biomarkers measured at baseline (Table 6). No significant change or significant difference between the two groups was observed in these biomarkers after one day of milk consumption.

[0085] After 14 days of A1PF milk consumption, infants’ salivary concentrations of cortisol (p=0.002), TNF-α (p<0.0001), IL-1 (p=0.0002), IL-1β (p=0.045), IL-4 (p=0.0003), IL-6 (p=0.008), peroxidase (p=0.003), IgA (p=0.0003), IgE (p<0.0001), IgG (p<0.0001), IgG1 (p<0.0001) and mucin (p=0.006) decreased significantly from baseline. Among these biomarkers, the concentration of TNF-α (p=0.002), IL-1 (p=0.049), IL-4 (p=0.001), peroxidase (p=0.037), IgA (p=0.0003), IgG (p<0.0001), IgG1 (p<0.0001) and mucin (p=0.013) were significantly lower than that of the conventional milk group.

[0086] Salivary concentration of IFN-γ (p=0.001) and GSH (p<0.0001) of infants in the A1PF group increased significantly from baseline and were significantly higher than that of the conventional milk group (p=0.023 and 0.0001, respectively).27369392.1:DCC-25 / 07 / 2025 - 37 - Table 6. Salivary Laboratory Analysis for Infants o v. 1 1* 6 4 8 6 5 5 3 1 8 727369392.1:DCC-25 / 07 / 2025 - 38 - o v. 8 9 7 0 8 8 0 4 6 8 1 4 327369392.1:DCC-25 / 07 / 2025 - 39 - o v. 8 1 3 1 9 9 6 0 8 2 9 9 427369392.1:DCC-25 / 07 / 2025 - 40 - o v. 6 9Unless otherwise stated, data presented are mean±standard deviation. Between-group difference was evaluated using analysis of variance; post-intervention comparisons were adjusted for baseline measurements; the difference was presented as the difference of least-squares means (95% confidence interval). Within-group difference was evaluated using paired t-test. *: p<0.05. a Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.3. Mothers’ Gastrointestinal Symptom Rating Scale (GSRS)

[0087] Mothers in the two study groups had similar scores in all GSRS individual items and the total score at baseline (Table 7). No significant change or significant difference between the two groups was observed in these scores after one day of milk consumption.

[0088] After 7 days of milk consumption, mothers in the A1PF group had significantly less symptoms of rumbling (p=0.035), bloating (p=0.016), burping (p=0.031), flatus (p=0.031), constipation (p=0.031), diarrhea (p=0.016), loose stools (p=0.031), and urgent need to have a bowel movement (p=0.031) compared to baseline. The conventional milk group had significantly increased diarrhea (p=0.035), loose stools (p=0.016), and urgent need to have a bowel movement (p=0.031) compared to baseline. Between-group differences were significant in the scores of the above symptoms. Total GSRS score of the A1PF group decreased significantly from baseline and was significantly lower than that of the conventional milk group (p<0.0001).

[0089] After 14 days of milk consumption, the above-mentioned symptoms further reduced in the A1PF group. Between-group differences were significantly for rumbling (p=0.017), bloating (p=0.017), burping (p=0.016), flatus (p=0.035), constipation (p=0.017), and total GSRS score (p<0.0001). The score for diarrhea, loose stools, and urgent need to have a bowel movement of the conventional milk group reduced from day 7 and there was27369392.1:DCC-25 / 07 / 2025 - 41 - no significant difference compared to its own baseline scores and the A1PF group. The A1PF group also showed significant lower scores in abdominal pain (p=0.031), heartburn (p=0.031) and sensation of not completely emptying the bowels (p=0.016) compared to baseline and to that of the conventional milk group (p=0.033, 0.008 and 0.017, respectively). Table 7. Gastrointestinal Symptom Rating Scale (GSRS) for Mothers ine .27369392.1:DCC-25 / 07 / 2025 - 42 - ine . * * *27369392.1:DCC-25 / 07 / 2025 - 43 - line . 3 60 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.4. Infant Gastrointestinal Symptom Questionnaire (IGSQ)

[0090] Infants in the two study groups had similar scores in all IGSQ individual items and the total score at baseline (Table 8).

[0091] After 7 days of milk consumption, the amount of milk that usually comes out when baby spat up and the number of times baby seems unconformable / fussy when spitting up reduced significantly in the A1PF group (both p=0.031), and were significantly lower than that in the conventional milk group (p=0.036 and 0.043, respectively). The total IGSQ score of the A1PF group also reduced significantly from baseline (p=0.002) and were significantly lower than that of the conventional milk group (p=0.028).

[0092] After 14 days of milk consumption, the A1PF group showed further decrease in the amount of milk that usually comes out when baby spat up and the number of times baby seems unconformable / fussy when spitting up (p=0.031 and 0.016 compared to baseline, respectively). In addition, the A1PF group showed significantly reduced number of times baby passed hard stool (p=0.031), number of times baby had difficulty passing bowel movements (p=0.031), amount of time baby usually cries in a day (p=0.005), number of times unable to soothe baby to stop crying (p=0.004), number of times baby cried during or right after feeding (p=0.008), number of days baby was fussy (p=0.031), number of times unable to sooth the baby when being fussy (p=0.031), number of times baby passed a lot of gas / was gassy (p=0.031), and number of times gas seems to make baby uncomfortable / fussy (p=0.031). All the above-mentioned scores were significantly lower in the A1PF group compared to the conventional milk group. The total IGSQ score of the A1PF group was significantly lower than its own baseline (p<0.0001) and that of the conventional milk group (p<0.0001).27369392.1:DCC-25 / 07 / 2025 - 44 - Table 8. Infant Gastrointestinal Symptom Questionnaire (IGSQ) Score v. 0 0 5 8 4 0 0 0 6 0 0 0 6 4 9 5 6 0 3 4427369392.1:DCC-25 / 07 / 2025 - 45 - v. 1 5 5 0 6 00 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.5. Infant Gastrointestinal Tolerance Questionnaire

[0093] Infants in the two study groups had similar stool frequency and stool characteristics at baseline (Table 9). Degree of burp reduced significantly in the A1PF group at day 1, although the difference compared to the conventional milk group. No significant change or significant difference between the two groups was observed in other stool and gastrointestinal symptoms after one day of milk consumption. Table 9. Infant Gastrointestinal Tolerance Questionnaire line v. 9 0 927369392.1:DCC-25 / 07 / 2025 - 46 - line v. 9 0 3 0 0 0 0Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.6. Infant Daily Sleep and Crying Duration

[0094] Infants in the two study groups had similar daily sleep and crying durations at baseline (Table 10). No significant change or significant difference between the two groups was observed in these outcomes after one day of milk consumption.27369392.1:DCC-25 / 07 / 2025 - 47 - Table 10. Infant Daily Sleep and Crying Durations . 3 8 0 0 4 5 0 9 527369392.1:DCC-25 / 07 / 2025 - 48 - . 0 0 2 6 00 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.7. Infant Sleep Questionnaire during the Past Week

[0095] Infants in the two study groups had similar sleep patterns during the past week of baseline (Table 11). No significant change or significant difference between the two groups was observed in infant sleep patterns during the study period.27369392.1:DCC-25 / 07 / 2025 - 49 - Table 11. Infant Sleep Questionnaire during the Past Week line . 4 2 2 9 4 0 0 5 0 6 6 1 00 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.8. Chalder Fatigue Questionnaire for Mothers

[0096] Mothers in the two study groups had similar scores in all individual items and total scores of the Chalder Fatigue Questionnaire during the past week of baseline (Table 12).27369392.1:DCC-25 / 07 / 2025 - 50 -

[0097] After 7 days of milk consumption, mothers in the A1PF group showed significant decreases from baseline in the scores for sleepy or drowsy feeling (p=0.008), physical fatigue score (p=0.044) and total fatigue score (p=0.047). The A1PF group had significantly lower score for sleepy or drowsy feeling (p=0.021) compared to the conventional milk group, while between-group difference was not significantly for physical fatigue score and total fatigue score.

[0098] After 14 days of milk consumption, mothers in the A1PF group showed significant decrease from baseline in the scores for problems with tiredness (p=0.016), sleepy or drowsy feeling (p=0.008), difficulties concentrating (p=0.031), as well as in the physical fatigue score (p=0.042), mental fatigue score (p=0.038), and total fatigue score (p=0.006). All of these scores of the A1PF group were significantly lower than that of the conventional milk group (p=0.021, 0.021, 0.015, 0.007, 0.036, and 0.003, respectively). Table 12. Chalder Fatigue Questionnaire for Mothers line v. 0 0 0 0 0 0 0 0 5 0027369392.1:DCC-25 / 07 / 2025 - 51 - line v. 0 0 0 0 6 0 0 5 0 0 0 0 0 6 6 45 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.9. Infants' Dermatitis Quality of Life Index (IDQoL)

[0099] Eczema severity and life quality index were similar among infants of the two study groups at baseline (Table 13). No significant change or significant difference between the two groups was observed after 7 days of milk consumption.27369392.1:DCC-25 / 07 / 2025 - 52 -

[0100] After 14 days of milk consumption, infants in the A1PF group showed significant lower scores for itching and scratching (p=0.016) and mood (p=0.016), which were significantly lower than the scores of the conventional milk group (p=0.017 and 0.034, respectively). Table 13. Infants' Dermatitis Quality of Life Index (IDQoL) line . 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 027369392.1:DCC-25 / 07 / 2025 - 53 - line . 0 0 0 0 03 Data presented are median (1stquartile, 3rdquartile). Between-group difference was evaluated using Kruskal Wallis test. Within-group difference was evaluated using Wilcoxon signed rank test. 5.10. Infant Growth

[0101] Infants from the two study groups were comparable at baseline and day 14 in all anthropometric parameters including weight, length, head circumference, BMI and their corresponding z-scores calculated using the WHO Growth Standards. Table 14. Anthropometric Measurements for Infants ue 7 6 0 5 2 3 6 6 3 1 5 2227369392.1:DCC-25 / 07 / 2025 - 54 - ue 8 6 8 38 Data presented are mean±standard deviation. Between-group difference was evaluated using analysis of variance, adjusted for gender; post-intervention comparisons were also adjusted for baseline measurements. The difference was presented as the difference of least-squares means (95% confidence interval). *: p<0.05. 5.11. Adverse effects

[0102] There were five AEs among mothers during the study period, including cold (3), cough with no other related symptoms (1), and torticollis (1). There was one AE (cold) among infants during the study period. No between-group difference was observed in the overall AE rate among mothers and infants. Table 15. Anthropometrical Measurements for Infants ue 0 0Data presented are frequency (%). Between-group difference was evaluated using Fisher’s exact test.27369392.1:DCC-25 / 07 / 2025 - 55 - 6. CONCLUSIONS

[0103] The present study primarily evaluated the effect of the A1PF milk with comparison to a conventional milk after 14 days of consumption in breast milk characterization and composition, mother’s gastrointestinal tolerance, infants’ gastrointestinal tolerance, as well as infants’ crying frequency, duration and sleep patterns. The study also evaluated mothers’ overall energy / fatigue, infants’ eczema severity and life quality index related to dermatitis, and infant growth. For mothers

[0104] With a continuous consumption for 7 days, the A1PF milk outperformed conventional milk in the following aspects: · Fewer symptoms of rumbling, bloating, burping, flatus, constipation, diarrhea, loose stools, and urgent need to have a bowel movement, and also lower total GSRS score; and · Less sleepy or drowsy feeling.

[0105] With a continuous consumption for 14 days, the A1PF milk outperformed conventional milk in the following aspects: · Higher iron and zinc concentration in the breast milk; · Lower fecal concentrations of CRP, lactic acid, higher fecal acetic acid, propionic acid and total SCFA concentration; · Lower salivary concentrations of IL-4, IL-6, IgA and IgE, as well as higher salivary concentration of GSH and lysozyme; · Fewer symptoms of rumbling, bloating, burping, flatus, constipation, abdominal pain, heartburn, sensation of not completely emptying the bowels, and lower total GSRS score; and · Fewer problems with tiredness, sleepy or drowsy feeling, difficulties concentrating, as well as less overall physical fatigue, mental fatigue, and total fatigue.27369392.1:DCC-25 / 07 / 2025 - 56 -

[0106] In addition, with 14 days of milk consumption: · Breast milk calcium concentration increased with consumption of A1PF milk or conventional milk, with no significant difference between the two groups; and · Fecal butyric acid concentration increased, salivary hsCRP concentrations decreased and salivary IFN-γ concentrations increased from baseline in the A1PF milk group but the difference compared to the conventional milk group was not significant. For infants

[0107] With a continuous consumption for 7 days, the A1PF milk outperformed conventional milk in the following aspects: · Reduced amount of milk that usually comes out when baby spat up; · Reduced number of times baby seems unconformable / fussy when spitting up; and · Fewer overall gastrointestinal symptoms.

[0108] With a continuous consumption for 14 days, the A1PF milk outperformed conventional milk in the following aspects: · Higher fecal concentrations of acetic acid and total SCFA, lower fecal concentration of lactic acid concentration; · Lower salivary concentration of TNF-α, IL-1, IL-4, peroxidase, IgA, IgG, IgG1 and mucin, as well as higher salivary concentration of IFN-γ and GSH; · Further decreased amount of milk that usually comes out when baby spat up and number of times baby seems unconformable / fussy when spitting up; reduced number of times baby passed hard stool, number of times baby had difficulty passing bowel movements, amount of time baby usually cries in a day, number of times unable to soothe baby to stop crying, number of times baby cried during or right after feeding, number of days baby was fussy, number of times unable to sooth the baby when being fussy, number of times baby passed a lot of gas / was gassy, and number of times gas seems to make27369392.1:DCC-25 / 07 / 2025 - 57 - baby uncomfortable / fussy, and reduced overall gastrointestinal symptoms; and · Less itching and scratching and improved mood related to dermatitis.

[0109] In addition, with 14 days of milk consumption: · Fecal propionic acid and butyric acid increased from baseline in the A1PF milk group, with no significant difference compared to that of the conventional milk group; and · Salivary concentrations of cortisol, IL-1β, IL-6 and IgE decreased significantly from baseline in the A1PF milk group, with no significant difference compared to that of the conventional milk group.

Claims

27369392.1:DCC-25 / 07 / 2025 - 58 - CLAIMS 1. A method of reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence.

2. A method of delivering nutrition to an infant subject so as to reduce the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence.

3. A method of reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-caseins having a histidine residue at position 67 of the beta-casein amino acid sequence.

4. A method of delivering nutrition to an infant subject so as to prevent or reduce the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence.

5. A method of reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

6. A method of delivering nutrition to an infant subject so as to reduce the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising27369392.1:DCC-25 / 07 / 2025 - 59 - beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk- derived products.

7. A method of reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk- derived products comprising beta-casein having a histidine residue at position 67 of the beta- casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

8. A method of delivering nutrition to an infant subject so as to prevent or reduce the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in the infant subject, the method comprising orally administering to the infant subject breast milk from a lactating subject whose diet includes milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived products.

9. The method of any one of claims 5 to 8, wherein the milk or milk-derived products comprise beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 25% by weight, or less than 20% by weight, or less than 10% by weight, or less than 5% by weight, or less than 1% by weight of total beta- caseins present in the milk or milk-derived products.

10. The method of any one of claims 5 to 9, wherein the milk or milk-derived products comprise beta-casein having a proline residue at position 67 of the beta-casein amino acid sequence in an amount that is at least about 70% by weight, or at least about 75% by weight, or at least about 80% by weight, or at least about 90% by weight, or at least about 95% by weight, or at least about 99% by weight of total beta-caseins present in the milk or milk- derived products.

11. The method of any one of claims 5 to 10, wherein the diet of the lactating subject is substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence.27369392.1:DCC-25 / 07 / 2025 - 60 - 12. The method of any one of claims 5 to 11, wherein the milk or milk-derived products are selected from the group consisting of fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk, non-pasteurised milk, cream, yoghurt, quark, cheese, butter and ice cream.

13. The method of claim 12, wherein the milk or milk-derived products are selected from the group consisting of fresh milk, raw milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenised milk, condensed milk, evaporated milk, flavoured milk, UHT milk, pasteurised milk and non-pasteurised milk.

14. The method of any one of claims 1, 2, 5 and 6, wherein reducing the incidence of adverse GI symptoms comprises decreasing volume of regurgitation, reducing discomfort or fuss during regurgitation, reducing number of hard stools, reducing frequency of constipation, reducing frequency of flatulence, reducing discomfort or fuss during flatulence, improving digestion, or reducing inflammation in the GI tract, or any combination thereof.

15. The method of any one of claims 3, 4, 7 and 8, wherein the dermatitis is selected from the group consisting of atopic dermatitis (eczema), contact dermatitis, dyshidrotic eczema and seborrheic dermatitis, or any combinations thereof.

16. The method of any one of claims 1 to 15, wherein the beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence comprises any one or more A1 type beta-casein variants A1, B, C, F, G, H1 and H2.

17. The method of any one of claims 1 to 16, wherein the lactating subject has consumed milk or milk-derived products that are substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence for at least 7 days prior to orally administering the breast milk to the infant subject.

18. The method of claim 17, wherein the lactating subject that are substantially free of beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence for at least 14 days prior to orally administering the breast milk to the infant subject.

19. The method of any one of claims 1 to 18, wherein the lactating subject consumes milk or milk-derived products that are substantially free of beta-casein A1.27369392.1:DCC-25 / 07 / 2025 - 61 - 20. The method of any one of claims 1 to 19, wherein the breast milk is orally administered to the infant subject by breastfeeding from a breast of the lactating subject.

21. The method of any one of claims 1 to 19, wherein the breast milk is orally administered to the infant subject by bottle feeding after having been pumped from a breast of the lactating subject.

22. The method of any one of claims 1 to 21, wherein the infant subject is aged from 0 months to 12 months, or from 0 months to 6 months.

23. Use of milk comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta- caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, wherein the milk or milk-derived product is to be consumed by a lactating subject whose breast milk is to be orally administered to the infant subject.

24. Use of milk comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta- caseins present in the milk in the manufacture of a milk or milk-derived product for reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant subject, wherein the milk or milk-derived product is to be consumed by a lactating subject whose breast milk is to be orally administered to the infant subject.

25. Milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived product for reducing the incidence of adverse gastrointestinal (GI) symptoms, immune response, inflammation and / or crying in an infant subject, wherein the infant subject is orally administered breast milk from a lactating subject whose diet includes the milk or milk-derived products.

26. Milk or milk-derived products comprising beta-casein having a histidine residue at position 67 of the beta-casein amino acid sequence in an amount that is less than 30% by weight of total beta-caseins present in the milk or milk-derived product for reducing the incidence of dermatitis, or itching and / or scratching associated with dermatitis, in an infant27369392.1:DCC-25 / 07 / 2025 - 62 - subject, wherein the infant subject is orally administered breast milk from a lactating subject whose diet includes the milk or milk-derived products.

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