Beta-casein a2 and antioxidant capacity

Abstract

Improving an animal's antioxidant capacity by increasing glutathione levels in its blood or tissues involves providing animals with a composition containing β-casein for consumption, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion. Uses include as an antioxidant for the treatment or prevention of symptoms of cancer, inflammation, Gashica (protein deficiency), seizures, autism, Down syndrome, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, sickle cell anemia, liver disease, cystic fibrosis, HIV, AIDS, infections, heart attacks, stroke, and diabetes, thereby preventing or mitigating the effects of aging, promoting tissue recovery after physical exercise, and promoting fertility.

Description

[0001] This application is a divisional application of Chinese patent application 201680032032.7, the original application being filed on May 20, 2016, entitled "β-casein A2 and antioxidant capacity". Technical Field

[0002] This invention relates to the milk protein A2 β-casein and to enhancing the antioxidant capacity of animals by increasing glutathione levels in their bodies. Specifically, this invention relates to dairy products and dairy-derived foods. The applicant has discovered that consuming dairy products containing the A2 variant of the protein β-casein and / or avoiding dairy products containing the A1 variant of β-casein helps increase glutathione levels in the body. Regulation of glutathione levels is beneficial in addressing many health problems associated with low levels of antioxidants and increased oxidative stress. Background of the Invention

[0003] Glutathione (GSH) is an antioxidant involved in several important biochemical pathways. GSH is a thiol peptide formed from three amino acids: glutamic acid, cysteine, and glycine. The thiol group (-SH) of the cysteine ​​residue in GSH provides a key site for various conjugation and reduction reactions between GSH and other biomolecules. The oxidized dimer form of GSH (GSSG) can be converted to GSH by reduction with glutathione reductase. Cysteine ​​utilization is the rate-limiting factor in GSH synthesis.

[0004] The term redox state is commonly used to describe the balance of GSH and GSSG (and other types) in biological systems such as cells or organs. Abnormal redox states occur under various harmful conditions, such as hypoxia, shock, and sepsis. Redox mechanisms also control many cellular processes. The primary role of GSH is to prevent damage to vital cellular components caused by reactive oxygen species (ROS). ROS are oxygen-containing chemically reactive molecules. Examples include hydroxyl groups (-OH), superoxide dismutase (O2), and hydroxyl radicals (-OH). - Hydrogen peroxide (H2O2) and peroxynitrite (ONOO) - During environmental or physiological stress, ROS levels can increase significantly. This can lead to significant damage to cellular structure and is commonly referred to as oxidative stress.

[0005] Cellular redox states can change when ROS production or antioxidant utilization alters. GSH plays a crucial role in the detoxification and elimination of ROS. Decreased cellular GSH levels can lead to ROS accumulation and oxidative stress. Regulation of GSH production is essential for cellular survival in oxidative environments.

[0006] The conditions of oxidative stress are determined by an imbalance between the level of ROS or the production of antioxidants and the body's ability to detoxify ROS. It is necessary to maintain ROS levels within physiologically safe ranges and avoid levels that cause pathological tissue damage. Excessive ROS levels lead to oxidative stress, which, if not adequately remedied by tissue repair mechanisms, can result in cell damage or death. Oxidative stress plays a crucial role in the pathogenesis of many diseases, including cancer, inflammation, Gastrocardia (primarily due to protein deficiency), seizures, autism, Down syndrome, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, sickle cell anemia, liver disease, cystic fibrosis, HIV / AIDS, infections, heart attacks, stroke, and diabetes. Therefore, GSH plays an important role in reducing or preventing these diseases and related symptoms. Furthermore, GSH has been reported to minimize age-related oxidative stress to aid tissue repair after physiological stress, such as that caused by physical exercise and various forms of physical activity, and is beneficial for healthy fertility.

[0007] There are many examples of antioxidant dietary supplements on the market. Some are marketed as glutathione supplements. Others claim to increase GSH levels. Whey protein is known to increase GSH levels, and since milk contains whey protein, milk can also increase GSH levels. However, the applicant has discovered that β-casein, also found in milk and the milk of other mammals, and particularly certain types of β-casein, is particularly effective in maximizing GSH levels in the blood and tissues relative to other types of β-casein.

[0008] Dairy products, primarily milk, consumed by populations worldwide, are a major source of protein in the human diet. Milk typically contains approximately 30-35 grams of protein per liter. Casein constitutes the largest component of this protein (80%), with β-casein making up about 37% of casein. Over the past two decades, evidence involving casein, particularly β-casein, has been growing in numerous health disorders. β-casein can be classified as A1 or A2 β-casein, depending on whether they contain a proline or histidine amino acid at position 67 of the β-casein amino acid sequence. This difference affects the ability of β-casein to produce specific heptapeptide fragments during digestion by an enzyme called BCM-7. A1 and A2 β-caseins are the predominant β-caseins in milk consumed by most populations.

[0009] The applicants have previously established associations between the consumption of A1β-casein in dairy products and certain health conditions, including type 1 diabetes (WO 1996 / 014577), coronary heart disease (WO 1996 / 036239), and neurological disorders (WO 2002 / 019832). Furthermore, the applicants have demonstrated associations between A1β-casein and enteritis (WO 2014 / 193248), lactose intolerance symptoms (WO 2015 / 005804), and hyperglycemia (WO 2015 / 026245).

[0010] The applicant has now discovered conclusive scientific evidence demonstrating a direct link between A2β-casein consumption and elevated GSH levels in the blood and tissues. Therefore, the applicant has found a novel approach to treat the aforementioned conditions or manage their symptoms.

[0011] Therefore, the object of the present invention is to provide a method for improving the antioxidant capacity of animals, or at least to provide a useful alternative to existing methods. Invention Overview

[0012] The first aspect of the invention provides a method for improving the antioxidant capacity of animals by providing animals with a composition comprising β-casein, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

[0013] The one or more β-caseins are preferably selected from type A2 β-caseins.

[0014] In some embodiments of the invention, the composition is ingested to increase the level of glutathione in the blood or tissues of an animal.

[0015] Furthermore, in some embodiments, ingestion of the composition avoids or reduces the risk of diseases or conditions associated with oxidative stress. Diseases or conditions associated with oxidative stress may include cancer, inflammation, Gashica (protein deficiency), seizures, autism, Down syndrome, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, sickle cell anemia, liver disease, cystic fibrosis, HIV, AIDS, infections, heart attacks, stroke, and diabetes.

[0016] In other embodiments of the invention, the intake of the composition avoids or reduces the effects of aging, promotes tissue recovery after physical exercise, or promotes fertility.

[0017] In a preferred embodiment of the invention, the animal is a human. Alternatively, the animal can be any other animal sensitive to oxidative stress, including, for example, a dog or a cat.

[0018] In a second aspect of the invention, there is provided a composition for improving the antioxidant capacity of an animal by providing the animal with a composition comprising β-casein, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

[0019] Another aspect of the invention provides the use of the composition for improving the antioxidant capacity of animals by providing the animals with a composition comprising β-casein, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

[0020] In another aspect of the invention, the use of dairy products in the preparation of compositions for improving the antioxidant capacity of animals is provided, wherein the dairy products contain β-casein, and wherein the β-casein contains at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

[0021] In another aspect of the invention, the use of β-casein in the preparation of compositions comprising β-casein for improving the antioxidant capacity of animals is provided, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion. β-casein is preferably a component of dairy products. The dairy products are preferably milk.

[0022] In another aspect of the invention, the use of compositions comprising β-casein as antioxidants is provided, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

[0023] The amount of one or more β-caseins that cannot produce β-casein-7 during enzymatic digestion can be any amount in the range of 75% to 100% of the weight of the β-casein, such as at least 90%, at least 95%, at least 98%, at least 99%, or even 100%.

[0024] In some embodiments of the invention, the composition is a dairy product or dairy product. The dairy product may be milk powder or liquid milk. The dairy product may be cream, yogurt, quark, cheese, butter, ice cream, or any other product derived from milk or containing casein or casein derivatives, including infant formula, adult nutrition products, protein supplements, or pet food.

[0025] In some embodiments of the invention, milk is obtained by genotypic or phenotypic testing of the cows and by milking only those cows that have been identified as producing only A2 β-casein in their milk. The cows can be herded prior to milking, consisting only of those cows identified as producing only A2 β-casein in their milk. Brief description of the attached diagram

[0026] Figure 1 The study showed cysteine ​​levels in the ileum and liver of rabbits fed diets containing A1β-casein and A2β-casein.

[0027] Figure 2 The levels of GSH in the ileum and liver of rabbits fed diets containing A1β-casein and A2β-casein were shown.

[0028] Figure 3 The study showed the cysteine ​​levels in the frontal cortex and hippocampus of rabbits fed diets containing A1β-casein and A2β-casein.

[0029] Figure 4 The levels of GSH in the frontal cortex and hippocampus of rabbits fed diets containing A1β-casein and A2β-casein were shown.

[0030] Figure 5 The median GSH concentration in people before and after consuming dairy products containing only A2β-casein or both A1β-casein and A2β-casein is shown. Invention Details

[0031] This invention relates to compositions comprising the protein β-casein and their use in improving the antioxidant capacity of animals, particularly humans. Importantly, the β-casein is the A2 variant of β-casein. The β-casein in the composition is 100% A2 β-casein or constitutes at least 75% by weight of all β-casein variants present in the composition. The importance of the A2 variant's predominance in the composition is attributed to the fact that the applicant has demonstrated a direct correlation between the consumption of milk containing only the A2 β-casein variant and elevated levels of GSH and cysteine ​​(a GSH precursor) in rabbits and humans. Higher levels of GSH and cysteine ​​were found when the dietary β-casein was A2 β-casein instead of A1 β-casein.

[0032] This is of great significance for the prevention, treatment, or management of diseases or disorders associated with physiologically high levels of ROS. The antioxidant GSH helps regulate ROS levels. Therefore, maximizing GSH levels in the blood and tissues is beneficial for avoiding or alleviating symptoms of various diseases, including cancer, inflammation, Gashica (protein deficiency), seizures, autism, Down syndrome, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, sickle cell anemia, liver disease, cystic fibrosis, HIV, AIDS, infections, heart attacks, stroke, and diabetes, to aid in tissue repair after physiological stress, slow or minimize the effects of aging, and improve fertility.

[0033] Since the primary (if not only) source of β-casein in the diets of most people is dairy products or dairy-derived products, and since most consumed dairy products contain only a mixture of A1 and A2 variants of β-casein (as explained below), the consumption of dairy products (or products made from such dairy products) with high A2 variant content necessarily implies low consumption of the A1 variant. Therefore, the present invention is based on reducing or eliminating A1 β-casein in the diet and promoting A2 β-casein, and this is achieved by ensuring that the β-casein in β-casein-containing food compositions, particularly dairy products and dairy products, is primarily A2 β-casein, preferably only A2 β-casein.

[0034] Ideally, the β-casein in the composition is 100% A2β-casein. Therefore, complete elimination of A1β-casein maximizes the maintenance of high GSH levels in the blood and tissues, thereby avoiding adverse symptoms and outcomes associated with redox imbalance and excessive ROS levels. However, the β-casein in the composition does not necessarily have to be 100% A2β-casein. Beneficial effects of high GSH levels can be observed in any composition where β-casein is predominantly A2β-casein, for example, in any amount between 75% and 100% by weight, including but not limited to 80%, 90%, 95%, 98%, and 99% by weight.

[0035] The compositions of the present invention are typically dairy products, but can also be any dairy product, such as cream, yogurt, quark, cheese, butter, ice cream, or any product containing casein or casein derivatives such as sodium caseinate. Examples of such products include infant formula, adult nutrition products, protein supplements, and pet food. The composition can also be a non-dairy product containing β-casein obtained from dairy products. The composition can be β-casein itself, or can be prepared from β-casein, which can be in solid form such as powder, granules, or solid cake form.

[0036] The dairy product may be fresh milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenized milk, condensed milk, lightly condensed milk, pasteurized milk or non-pasteurized milk or any other form of milk.

[0037] Although dairy products can be obtained from any mammal (including humans, goats, pigs, and buffalo), in a preferred embodiment of the invention, the dairy product is cow's milk.

[0038] The compositions of the present invention are intended primarily for human consumption, but it should be understood that the health benefits are also relevant to some other animals such as cats, dogs and other livestock.

[0039] β-casein is generally classified into A1 and A2 types. A1 and A2 β-casein are the main β-caseins found in dairy products consumed by most populations. A1 and A2 β-casein differ by only one amino acid. Histidine is located at position 67 of the 209-amino acid sequence in A1 β-casein, while proline is located at the same position in A2 β-casein. 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 production of a 7-amino acid protein fragment called β-tyrosine-7 (BCM-7) during enzymatic digestion. Therefore, BCM-7 is a digestion product of A1 β-casein. In the case of A2 β-casein, position 67 is occupied by proline, which hinders the cleavage of the amino acid bond at that position. Therefore, BCM-7 is not a digestion product of A2 β-casein.

[0040] Other β-casein variants, such as Bβ-casein and Cβ-casein, also have histidine at position 67, while other variants, such as A3, D, E, and I, have proline at position 67. However, these variants have been found in very low levels, or not at all, in milk from European cows. Therefore, in the context of this invention, the term A1 β-casein refers to any β-casein having histidine at position 67 and thus possessing the ability to produce BCM-7 upon enzymatic digestion, and the term A2 β-casein refers to any β-casein having proline at position 67 and thus not possessing the ability to produce BCM-7 upon enzymatic digestion.

[0041] The relative proportions of A1 and A2 β-casein in cow's milk can be tested. Alternatively, cows can be genetically tested to determine their ability to produce milk containing A1 or A2 β-casein, or a combination of both. These methods and techniques are well-known.

[0042] Intracellular GSH concentrations average 1–2 mM in most cells, varying from approximately 10 mM in hepatocytes to 0.2 mM in neurons. Hepatocytes provide a wide range of GSH found in plasma. Due to the relatively low GSH content in neurons, supplementing GSH by reducing GSSG is a particularly important process in neurons. GSH can be transported out of cells and blood using carrier-dependent facilitation mechanisms. Some dietary and intestinal GSH can also enter portal plasma. The liver is the primary source of plasma GSH, which is synthesized from cysteine. The brain, kidneys, lungs, and intestines are the main consumers of hepatic GSH. Interorgan metabolism of GSH facilitates the non-toxic transport of cysteine ​​and cysteine ​​between tissues and also helps maintain intracellular GSH concentrations and optimal redox states. Subphysiological levels of GSH can lead to the accumulation of ROS and thus oxidative stress. Conversely, increased GSH synthesis increases antioxidant capacity and promotes metabolic activity.

[0043] Cellular oxidative stress typically results from one of three factors: 1) increased oxidant production, 2) reduced antioxidant protection, and 3) inability to repair oxidative damage. Oxidative damage can occur in DNA, proteins, and lipids. GSH plays a central role in neutralizing almost all ROS responses through both direct and indirect pathways. The main cellular damage caused by ROS is the oxidation of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA.

[0044] Support for the invention can be found in the experiments described in the embodiments.

[0045] Example 1 is a study of rabbits fed a diet containing skim milk powder. The skim milk powder used was derived from milk containing only the A1 variant (A1) of β-casein or milk containing only the A2 variant (A2) of β-casein. The results are as follows: Figures 1 to 4 As shown in the image. Figure 1 and 2 Rabbits fed an A2 diet showed increased uptake of cysteine ​​and GSH in the ileum and liver. Since the liver is the primary storage organ for GSH homeostasis, increased hepatic GSH indicates an overall increase in GSH levels in the body. Elevated GSH levels were also observed in the frontal cortex and hippocampus of rabbits fed an A2 diet compared to rabbits fed an A1 diet. Low GSH levels in the frontal cortex have been associated with disorders such as autism, ADHD, Down syndrome, and schizophrenia. Low levels in the hippocampus may affect memory recovery. Low cysteine ​​levels in the hippocampus have been found in patients with autism and Alzheimer's disease. These findings suggest that rabbits fed an A2 diet have relatively higher antioxidant capacity across various body organs, particularly in the brain, compared to rabbits fed an A1 diet.

[0046] Example 2 describes a double-blind, randomized, controlled, 2×2 crossover study in which healthy participants consumed 2×250 ml of regular milk daily, containing both A1 and A2 β-casein variants, or milk containing only the A2 β-casein variant. Plasma glutathione concentrations were measured. It was found that consumption of milk containing only A2 β-casein resulted in a greater increase in plasma glutathione concentration compared to consumption of milk containing both β-casein variants.

[0047] Previous studies have shown that whey proteins that do not produce BCM-7 during proteolysis promote cysteine ​​uptake and GSH synthesis, while opioid peptides derived from β-casein and wheat inhibit cysteine ​​uptake, reduce GSH concentration, and decrease antioxidant potential (i.e., reduce the GSH / GSSG ratio). The results of Example 2 are consistent with these prior studies and indicate that A1 β-casein limits the amount of cysteine ​​absorbed from milk, and thus limits the ability to synthesize GSH. Elimination of A1 β-casein from a milk diet resulted in a more significant increase in GSH synthesis, possibly by eliminating the inhibitory effect of BCM-7 on cysteine ​​uptake. Therefore, daily consumption of conventional commercial milk is associated with increased GSH levels, likely due to the increased cysteine ​​supply from whey proteins. However, compared to milk containing both A1 and A2 β-caseins, consumption of the A2 variant containing only β-casein showed significantly higher levels of antioxidants. These results suggest that eliminating A1 β-casein from milk can lead to a more significant increase in GSH levels, thereby enhancing antioxidant capacity.

[0048] The above experiments show a clear correlation between A2β-casein consumption and high levels of GSH (and its precursor cysteine) in blood, liver, and brain tissue, compared to A1β-casein consumption. Since GSH is the body's primary defense against ROS, and ROS is known to be closely associated with various diseases, the therapeutic and preventative benefits of replacing dietary A1β-casein with A2β-casein are clear.

[0049] The present invention provides a relatively easy solution by avoiding dairy products or dairy products containing A1β-casein and ensuring that dairy products and dairy products in the diet contain β-casein that is primarily A2β-casein, preferably 100% A2β-casein.

[0050] Any reference to prior art documents in this specification shall not be construed as an admission that such prior art is widely known or forms part of general common knowledge in the art.

[0051] As used in this specification, the words “comprising,” “including,” and similar terms should not be construed as having an exclusive or exhaustive meaning. In other words, they are intended to mean “including but not limited to.”

[0052] 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 examples.

[0053] Example

[0054] Example 1: -Casein A1 and - Effects of casein A2 consumption on cysteine ​​and GSH levels in rabbits

[0055] Ten male rabbits (NZW, age and / or weight matched) were randomly divided into two groups and fed a rabbit diet with skim milk powder (SMP) as the primary protein source for 12 weeks. The total protein content was 16.8%, with 60% of the protein derived from SMP, meaning 10% of the diet consisted of milk protein. The SMP used was derived from milk containing only the A1 variant of β-casein (A1) or milk containing only the A2 variant of β-casein (A2). The palatability of the SMP diet was tested to rule out any issues involving rabbits refusing to eat the diet. At the end of the 12-week period, the rabbits were euthanized. Tissue samples were obtained and stored at -80°C until further use. The tissues were lysed using 1X lysis buffer and sonicated on ice for 15 seconds. Protein content was determined using 100 μL of the sonicated material. The remaining lysate was added to a microcentrifuge tube along with an equal volume of 0.4 N perchloric acid and incubated on ice for 5 minutes. The sample was centrifuged at 13,000 RPM, and the supernatant was transferred to a new microcentrifuge tube. 100 μL of the sample was added to a conical micro-automatic sampling vial and kept in the autosampler cooling tray at 4°C. 10 μL of this sample was injected into the HPLC system. Separation of redox and methylation pathway metabolites was performed using an Agilent Eclipse XDB-C8 analytical column (3 x 150 mm; 3.5 μm) and an Agilent Eclipse XDB-C8 guard column (4.6 x 12.5 mm; 5 μm). Two mobile phases were used. Mobile phase A consisted of 0% acetonitrile, 25 mM sodium phosphate, and 1.4 mM 1-octylsulfonic acid, adjusted to pH 2.65 with phosphoric acid. Mobile phase B consisted of 50% acetonitrile. The initial flow rate was set to 0.6 mL / min, using a discontinuous gradient: 0–9 min 0% B, 9–19 min 50% B, and 19–30 min 50% B. The column was then equilibrated with 5% B for 12 minutes before the next run. The temperature was maintained at 27°C. The electrochemical detector was an ESA CoulArray with a BDD analytical cell (Model 5040), and the operating potential was set to 1500 mV. Sample concentrations were determined based on the peak areas of metabolites using the standard calibration curve and the HPLC software provided by ESA. The sample concentrations were calibrated relative to protein content. In some cases, the sample was diluted with the mobile phase as needed, or up to 50 μL of sample was injected to ensure that the thiol levels were within the range of the standard curve.

[0056] Target tissues were collected from the gastrointestinal tract, liver, and two different parts of the brain (hippocampus and frontal cortex), and homocysteine ​​and GSH levels were analyzed. Results are as follows: Figure 1-4 As shown in the image.

[0057] Example 2: -Casein A1 and - Effect of casein A2 on GSH concentration in human plasma samples

[0058] Eligible Chinese men and women included those aged 25–68 years who consumed milk irregularly and self-reported intolerance to commercial milk, self-reported mild to moderate digestive discomfort after milk consumption, and had normal ECG and blood pressure during quiet breathing. A total of 21 men and 24 women were enrolled, with a mean ± standard deviation (SD) age of 46.6 ± 14.0 years. Based on the results of a urinary galactose test, 23 individuals were confirmed to be lactose deficient.

[0059] Participants consumed commercially available regular milk containing both A1 and A2 variants (A1 / A2) of β-casein in Phase 1 or commercially available milk containing only the A2 variant (A2) of β-casein (A1 / A2) in Phase 2. A2; Sequence 1), or vice versa (A2) A1 / A2; Sequence 2). The A1-A2 ratio in milk containing two β-casein variants was determined to be 42:58 by UPLC-DAD and tandom mas spectrometry. Each study phase lasted 2 weeks, with a 2-week clearance period before entering the first phase and between the first and second phases. Participants were instructed to consume 250 ml of milk after two meals daily. They were prohibited from consuming other dairy products, but were allowed to consume non-dairy products during the study period. This study was conducted in accordance with the Declaration of Helsinki revised in Seoul in 2008 and approved by the ethics committee of the Shanghai Nutrition Society (Approval No.: SNSIRB#2014

[002] ). This study has been registered at ClinicalTrials.gov (identifier: NCT02406469).

[0060] Blood samples were collected at baseline and at the end of each study phase for determination of laboratory variables, including GSH. Plasma was stored at -80°C until the required assay was performed. Plasma samples were thawed on ice, and 5 μL of 0.4N perchloric acid solution was added to 200 μL of plasma to precipitate any remaining proteins.

[0061] Total GSH levels were determined at 412 nm using a recycling reaction of GSH with dithionitrobenzoate in the presence of excess GSH reductase. Results are expressed as nmol (in min) of 5-thio-2-nitrobenzoate formed. -1 mg -1 (Protein). In independent assays, GSH concentration was measured twice for each sample.

[0062] Based on the Cole-Stokes test, GSH concentrations showed a significant deviation. Therefore, the Wilcoxon two-sample test was used to analyze the data, with phases and crossovers treated as fixed effects, and a [missing information - likely a specific method or approach] was employed. P <0.05 indicates a statistically significant stage or cross-effect.

[0063] The median GSH concentration datasets measured at the beginning and end of each study phase in both sequences are shown below. Figure 5 As shown in the table. The results of the Wilcoxon two-sample test are shown in Table 1.

[0064] Table 1. Wilcoxon two-sample test for plasma glutathione concentration

[0065] Compared with consumption of milk containing both β-casein variants, consumption of milk containing only the A2 β-casein variant involved a significant increase in plasma GSH concentration from baseline to the end of the study phase. This increase occurred in both sequences, regardless of which dairy product was consumed first. The GSH concentration of milk containing A2 β-casein changed by a mean ± SEM of 4.01 ± 0.61 nmol / mL from baseline, compared with 1.99 ± 0.50 nmol / mL for milk containing both A1 and A2 β-casein. The trend of change in GSH levels from baseline was greater in phase 1 (sequence A2 → A1 / A2) than in phase 2 (sequence A1 / A2 → A2) (4.07 vs. 2.70 nmol / mL).

[0066] Although the invention has been described by way of examples, it should be understood that variations and modifications can be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents of a particular feature exist, such equivalents are incorporated as specifically mentioned in this specification.

Claims

1. A method of improving the antioxidant capacity of an animal by providing it with a composition comprising β-casein, wherein the β-casein comprises at least 75% by weight of one or more β-caseins that are incapable of producing β-casein-7 upon enzymatic digestion.

2. The method of claim 1, wherein the one or more β-caseins are selected from A2 type β-caseins.

3. The method of claim 1 or claim 2, wherein ingestion of the composition increases glutathione levels in the blood or tissues of an animal.

4. The method of any one of claims 1-3, wherein ingestion of the composition avoids or reduces the risk of diseases or disorders associated with oxidative stress.

5. The method of claim 4, wherein the disease or disorder associated with oxidative stress is selected from cancer, inflammation, Gashica (protein deficiency), seizures, autism, Down syndrome, chronic fatigue syndrome, Alzheimer's disease, Parkinson's disease, sickle cell anemia, liver disease, cystic fibrosis, HIV, AIDS, infection, heart attack, stroke, and diabetes.

6. The method according to any one of claims 1-3, wherein ingestion of the composition avoids or mitigates the effects of aging, promotes tissue recovery after physical exercise, or promotes fertility.

7. The method as described in any one of claims 1-6, wherein the animal is a human, a dog, or a cat.

8. The method of any one of claims 1-7, wherein the β-casein comprises at least 90% by weight A2β-casein.

9. The method of any one of claims 1-8, wherein the β-casein comprises 100% by weight A2β-casein.

10. The method according to any one of claims 1-9, wherein the composition is a dairy product or dairy product.

11. The method of claim 10, wherein the dairy product is fresh milk, milk powder, liquid milk reconstituted from powder, skim milk, homogenized milk, condensed milk, diluted condensed milk, pasteurized milk, or non-pasteurized milk.

12. The method of claim 10, wherein the dairy product is cream, yogurt, quark, cheese, butter, ice cream, infant formula, adult nutrition products, protein supplements, or pet food.

13. The method of claim 10 or claim 11, wherein milk is obtained by genotypic or phenotypic testing of cows and milking only cows that have been determined to produce only A2 type β-casein in their milk.

14. The method of claim 13, wherein a herd of cows is formed comprising only cows that are determined to produce only type A2 β-casein A2 in their milk and then milked from one or more cows in the herd.

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