US20160113997A1 - Beta-casein a2 and prevention of inflammation of the bowel - Google Patents

Beta-casein a2 and prevention of inflammation of the bowel Download PDF

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US20160113997A1
US20160113997A1 US14/894,711 US201414894711A US2016113997A1 US 20160113997 A1 US20160113997 A1 US 20160113997A1 US 201414894711 A US201414894711 A US 201414894711A US 2016113997 A1 US2016113997 A1 US 2016113997A1
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casein
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milk
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Andrew John Clarke
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A2 Milk Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A23K1/1631—
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to the use of the milk protein beta-casein A2 for preventing or reducing the risk of inflammation of the bowel.
  • the invention relates to milk and milk derived food products and the avoidance of consuming milk and milk products that contain high levels of the protein beta-casein A1 which has been shown to cause both immediate and ongoing inflammation of the bowel.
  • the invention relates to the prevention of bowel inflammation, including general bowel irritation as well as inflammatory bowel diseases and irritable bowel syndrome.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • IBS affects people of all ages, including children. It is estimated that approximately 10 to 25% of the worldwide population suffer from IBS. The impact of IBS can range from mild inconvenience to severe debilitation. People with moderate to severe IBS struggle with symptoms that often impair their physical, emotional, economic, educational and social well-being.
  • Bovine milk mainly bovine milk, consumed in populations throughout the world, is a major source of protein in human diets.
  • Bovine milk typically comprises around 30 grams per litre of protein. Caseins make up the largest component (80%) of that protein.
  • casein proteins especially beta-caseins, in a number of health disorders.
  • beta-caseins can be categorised as beta-casein A1 and beta-casein A2. These two proteins are the predominant beta-caseins in the milk consumed in most human populations. Beta-casein A1 differs from beta-casein A2 by a single amino acid. A histidine amino acid is located at position 67 of the 209 amino acid sequence of beta-casein A1, whereas a proline is located at the same position of beta-casein A2. This single amino acid difference is, however, critically important to the enzymatic digestion of beta-caseins in the gut. The presence of histidine at position 67 allows a protein fragment comprising seven amino acids, known as beta-casomorphin-7 (BCM-7), to be produced on enzymatic digestion.
  • BCM-7 beta-casomorphin-7
  • BCM-7 is a digestion product of beta-casein A1.
  • position 67 is occupied by a proline which hinders cleavage of the amino acid bond at that location.
  • BCM-7 is not a digestion product of beta-casein A2.
  • beta-casein variants such as beta-casein B and C, also have histidine at position 67, and other variants, such as A3, D and E, have proline at position 67. But these variants are found only in very low levels, or not found at all, in milk from cows of European origin.
  • beta-casein A1 refers to any beta-casein having histidine at position 67
  • beta-casein A2 refers to any beta-casein having proline at position 67.
  • BCM-7 is an opioid peptide and can potently activate opioid receptors throughout the body. BCM-7 has the ability to cross the gastrointestinal wall and enter circulation enabling it to influence systemic and cellular activities via opioid receptors.
  • the applicant and others have previously determined a link between the consumption of beta-casein A1 in milk and milk products and the incidence of certain health conditions including type I diabetes (WO 1996/014577), coronary heart disease (WO 1996/036239) and neurological disorders (WO 2002/019832).
  • BCM-7 can also affect digestive function. It has been reported that opioid receptors play a role in controlling gastrointestinal function, including regulating gastrointestinal motility, mucus production and hormone production. (for example, Mihatsch, W. A, et al., Biol. Neonate , 2005, 87(3):160-3). The caseins found in milk are thought to be associated with inhibiting intestinal motility, which can lead to constipation (Gunn T. R. and Stunzer D., NZ Med. J ., 1986, 99(813):843-6) and research on casomorphins and synthetic casomorphin derivatives indicates that BCM-7 contributes to this opioid receptor mediated effect (Charlin V. et al., Rev. Med.
  • beta-casein A1 The applicant has now found conclusive scientific evidence for a direct link between the consumption of beta-casein A1 and inflammation of the bowel relative to the consumption of beta-casein A2. Given the myriad of factors in human diet that can influence bowel health, and that milk and milk products contain a wide array of protein components and other components, the applicant's finding of a clear direct association between beta-casein A1 consumption and inflammatory conditions of the bowel is surprising. Notably, the applicant has found evidence, not only of an acute bowel inflammation response to the ingestion of beta-casein A1, but also of long term ongoing inflammation of the bowel resulting from a short term exposure to beta-casein A1.
  • compositions for preventing or reducing the risk of inflammation of the bowel in an animal wherein the composition contains beta-casein and wherein the beta-casein comprises at least 50% by weight beta-casein A2.
  • compositions for preventing or reducing the risk of inflammation of the bowel in an animal wherein the composition contains beta-casein and wherein the beta-casein comprises at least 50% by weight beta-casein A2.
  • milk in the manufacture of a composition for preventing or reducing the risk of inflammation of the bowel of an animal, wherein the milk contains beta-casein and wherein the beta-casein comprises at least 50% by weight beta-casein A2.
  • a method of preventing or reducing the risk of inflammation of the bowel in an animal comprising the consumption by the animal of a composition containing beta-casein, or providing the composition to the animal for consumption, wherein the beta-casein comprises at least 50% by weight beta-casein A2.
  • the amount of beta-casein A2 may be any amount in the range of 50% to 100% by weight of the beta-casein, for example at least 90%, at least 99%, or even 100%.
  • the composition is milk or a milk product.
  • the milk may be milk powder or liquid milk.
  • the liquid milk may be in the form of fresh milk, liquid milk reconstituted from a powder, skim milk, homogenised milk, condensed milk, evaporated milk, pasteurised milk or non-pasteurised milk, UHT milk or any other form of milk.
  • the milk product may be cream, yoghurt, quark, cheese, butter, ice cream, or any other milk product.
  • the inflammation of the bowel may be, although is not limited to, an inflammatory bowel disease or irritable bowel syndrome.
  • the inflammatory bowel disease may be, although is not limited to, Crohn's disease or ulcerative colitis.
  • the animal is a human.
  • the animal may be a dog, cat, or any other domestic animal where feed is supplemented with milk.
  • FIG. 1 shows colon myeloperoxidase activity in rats fed the diets of Example 1.
  • FIG. 2 shows jejunum myeloperoxidase activity in rats fed the diets of Example 1.
  • FIG. 3 shows levels of serum amyloid A in plasma in rats fed the diets of Example 1.
  • FIG. 4 shows histology scores for intestine sections taken from rats fed the diets of Example 1.
  • FIG. 5 shows the morphine and BCM-7 concentration dependent uptake of cysteine in neuronal cells and GI epithelial cells.
  • FIG. 6 shows the uptake of cysteine in neuronal cells and GI epithelial cells over time.
  • FIG. 7 shows the effect of BCM-7 and morphine on cysteine, GSH/GSSG and SAM/SAH over time.
  • FIG. 8 shows CpG methylation changes in genes implicated in inflammatory response (NFkappaB and IL10) under the influence of BCM-7.
  • FIG. 9 shows CpG methylation changes in genes implicated in inflammatory response (MPO) and genes implicated in immune response (IL1R).
  • the invention relates to a composition containing the protein beta-casein and its use for preventing inflammation of the bowel or at least reducing the risk of developing inflammation of the bowel.
  • the beta-casein is the A2 variant of beta-casein, or makes up at least 50% by weight of the total beta-casein variants present in the composition.
  • the importance of the predominance of the A2 variant in the composition is due to the fact that the applicant has shown that there is a direct link between the A1 variant and biological markers for inflammation of the bowel in humans. Therefore, an improvement in bowel health can be expected if the consumption of the A1 variant is avoided.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • A1 milk as used in this specification is intended to mean milk containing beta-casein where that beta-casein is predominantly in the form of beta-casein A1 (or any other beta-casein variant having histidine at position 67 of its 209 amino acid sequence).
  • A1 milk may have a beta-casein component that is greater than 50%, typically greater than 90% (ideally 100%), beta-casein A1.
  • A2 milk as used in this specification is intended to mean milk containing beta-casein where that beta-casein is predominantly in the form of beta-casein A2 (or any other beta-casein variant having proline at position 67 of its 209 amino acid sequence).
  • A2 milk may have a beta-casein component that is greater than 50%, typically greater than 90% (ideally 100%), beta-casein A2.
  • the primary, if not only, source of beta-caseins in the diet of most human populations is milk or products derived from milk, and since most milk consumed contains a mixture of the A1 and A2 variants of beta-casein only, the consumption of milk (or products made from such milk) having a high content of the A2 variant will necessarily mean that the consumption of the A1 variant is low.
  • the only dietary source of beta-casein contains the A2 variant and no other variant, the dietary intake of the A1 variant is eliminated and the adverse health consequence of bowel inflammation arising from beta-casein A1 consumption can therefore also be expected to be eliminated.
  • the invention of this application is based on the reduction or elimination of beta-casein A1 in the diet of animals (especially humans) and this is achieved by ensuring that the beta-casein in beta-casein containing food compositions, especially milk and milk products, is predominantly or even exclusively beta-casein A2.
  • the beta-casein in the composition is 100% beta-casein A2.
  • the complete elimination of beta-casein A1 therefore maximises the associated health benefit by reducing or eliminating altogether the risk of inflammation of the bowel caused by beta-casein A1 (and BCM-7).
  • the risk may be reduced in any composition where the beta-casein is predominantly beta-casein A2, that is, any amount between 50% by weight and 100%, including but not limited to 60%, 70%, 75%, 80%, 90%, 95%, 98% and 99% by weight.
  • the invention relates to the use of a composition containing beta-casein where the beta-casein comprises at least 50% by weight beta-casein A2, and as explained above beta-casein A1 and beta-casein A2 are essentially the only beta-casein variants present in milk consumed by most populations, the invention also relates to the use of a composition containing beta-casein where the beta-casein comprises less than 50% by weight beta-casein A1.
  • the composition contains less than 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% by weight beta-casein A1, and ideally no beta-casein A1 at all.
  • the composition of the invention is typically milk, but may also be any milk-derived product such as cream, yoghurt, quark, cheese, butter, or ice cream.
  • the composition may also be a non-milk product containing beta-casein that has been obtained from milk.
  • the composition may be beta-casein itself, or may be prepared from beta-casein, which beta-casein may be in solid form such as powder or granules or in the form of a solid cake.
  • the milk may be obtained from any mammal, including humans, goats, pigs and buffalo, in preferred embodiments of the invention the milk is bovine milk.
  • the milk may be in the form of fresh milk, milk powder, liquid milk reconstituted from a powder, skim milk, homogenised milk, condensed milk, evaporated milk, pasteurised milk or non-pasteurised milk, UHT milk, or any other form of milk.
  • composition of the invention is applicable for consumption by humans primarily, but it should be appreciated that the health benefit is also relevant for some other animals such as cats, dogs and other domestic animals.
  • Example 2 relates to the effect of beta-casein A1 and beta-casein A2 diets on myeloperoxidase (MPO) activity in the colon of rats.
  • MPO activity is a marker for inflammation (Krawisz, et al., Gastroenterology , 1984, 87(6):1344-1350 and Dommels, Y. E. M., et al., Genes Nutr ., 2007, 2(2):209-223). It was found that colon MPO activity increased in beta-casein A1-fed rats compared to beta-casein A2-fed rats indicating an increased level of neutrophil cells in beta-casein A1-fed rats, which is in turn an indicator of inflammatory response. The effect was not observed in rats treated with naloxone (a known opioid receptor antagonist), demonstrating that the effect is mediated through the interaction of BCM-7 with ⁇ -opiate receptors.
  • naloxone a known opioid receptor antagonist
  • Example 3 relates to effects on MPO activity in the jejunum. The results indicate that there was no observable increase in inflammation in the small intestine on consumption of beta-casein A1. This contrasts with Example 2 which showed inflammation in the colon. The result of Example 3 confirms that the inflammation induced by consumption of beta-casein A1 is localised in the colon and not throughout the intestine.
  • Example 4 relates to concentrations of serum amyloid A (SAA), which is a protein secreted, primarily by the liver, during the acute phase of inflammation (Uhler, C. M. and Whitehead, A. S., Eur. J. Biochem., 1999, 265(2):501-523). SAA levels are an indicator of systemic inflammation. Example 4 shows that there were no significant differences in plasma SAA concentration between any of the treatment groups. In other words, there was no clear evidence of systemic inflammation from this study.
  • SAA serum amyloid A
  • Example 5 which relates to histology studies, reflects that the inflammation in the colon is subtle and may be subclinical in how it presents. Despite MPO data confirming an inflammatory response of the tissue, this was not visually apparent as evidenced by the lack of significance stemming from wide ranging data sets.
  • BCM-7 can counter inflammation.
  • BCM-7 reduces oxidative stress in kidneys (Zhang, W., et al., http://www.ncbi.nlm.nih.gov/pubmed/23658831). Oxidative stress is an inflammatory effect.
  • the reported finding that BCM-7 reduces this inflammatory effect points to the unpredictability of BCM-7 as a factor in health related physiological processes.
  • Example 6 describes a trial to examine whether beta-casein A1 containing milk has an effect on gastrointestinal symptoms relative to beta-casein A2 containing milk.
  • the compromised water absorption function leading to softer faeces is evidence of a higher level of bowel inflammation when beta-casein A1 is consumed relative to beta-casein A2.
  • BCM-7 from beta-casein A1
  • inflammation indicators such as cysteine and glutathione (GSH) levels.
  • ROS Reactive Oxygen Species
  • IBD An association between Reactive Oxygen Species (ROS), which are chemically reactive molecules containing oxygen such as oxygen ions and peroxides, and IBD can be inferred from the evidence that increased ROS levels and decreased antioxidant levels are major contributing factors to pathogenic mechanisms in IBD, Crohn's disease and ulcerative colitis. ROS can also potentiate immune reactions in IBD by elevating inflammatory leukocytes. Further, inflammation and inflammatory disease progression are also associated with defects in mucosal antioxidant defences, especially mucosal GSH levels. Drugs with antioxidant properties are reported to show beneficial effects in the treatment of IBD.
  • Blood levels of cysteine and selenium are critical for the support and maintenance of GSH synthesis, and these in turn are dependent upon the absorption of the food-derived sulfur-containing amino acids cysteine and methionine from the gastrointestinal (GI) tract via the different transporters on the intestinal epithelial border surface.
  • GI gastrointestinal
  • Se essential trace element selenium
  • GPX2 glutathione peroxide
  • GPX1 glutathione peroxide
  • EAAT3 excitatory amino acid transporter 3, EAAC1
  • EAAC1 is selective for cysteine transport and was initially cloned from GI epithelial cells.
  • EAAT3 is most prominently expressed in the small intestine, especially in the terminal ileum, the highest levels being in crypt cells and lower villus regions. This is the major site for multipotent stem cells supporting the epithelial lining of the gut.
  • EAAT3-dependent absorption of cysteine with the local and systemic consequences of lower GSH levels.
  • SAM S-adenosylmethionine
  • Adaptive epigenetic responses to changes in redox status are likely to play a critical role in various diseases, especially those that can be traced to interference with antioxidant homeostasis such as inflammation of the GI tract generally, and more specifically IBD and Crohn's disease.
  • Example 7 indicates that BCM-7 can inhibit the uptake of cysteine in a concentration-dependent manner with an efficacy order of morphine greater than BCM-7, and IC50 values of 0.16 and 1.31 nM in neuronal cells and 6.38 and 15.95 nM, respectively, in GI epithelial cells ( FIG. 5 ). Inhibition of cysteine uptake was fully developed at 30 minutes and was sustained through 48 hours of morphine or BCM-7 exposure ( FIG. 6 ), indicating a long term chronic effect on the cysteine uptake after single exposure to BCM-7. These effects were ⁇ -opioid receptor mediated as indicated by the blockade in the presence of a selective ⁇ -antagonist and not a delta opioid receptor.
  • Example 8 indicates that BCM-7 and morphine caused time-dependent decreases in cysteine and GSH levels.
  • the ratio of SAM/SAH (methylation capacity) was also affected by BCM-7 treatment at different time points ( FIG. 7 ).
  • BCM-7 induces a reduction in major intracellular antioxidant levels, specifically GSH levels, and has the potential to lead to inflammatory change via the oxidative-stress signalling pathway.
  • the changes mediated in the SAM levels could induce epigenetic changes in genes responsible for inflammatory pathways.
  • Example 9 illustrates that treatment with the prototypical opioid morphine resulted in 7,592 differentially expressed transcripts (DETs), while BCM-7 treatment yielded 1,467 DETs. 501 DETs were shared by both morphine and BCM-7. Additionally, this result indicates the diseases and disorders that were found to be significantly associated with the observed changes in global DNA methylation. BCM-7 differentially methylated promoter transcripts (DMTs) were associated with inflammatory disease (Table 5). Hence, the acute effects of BCM-7 on GSH antioxidant levels and oxidative stress essentially translate to chronic adaptive gene expression changes. The genes which are altered epigenetically under the influence of BCM-7 are directly involved in regulating cellular function as well as inflammatory disease pathways, and the genes transcriptionally regulated are also involved in cellular functions and redox homeostasis (Table 5).
  • Example 10 shows the DNA methylation changes in the genes responsible for mediating the inflammatory response under the influence of BCM-7.
  • Cytokines such as NFkappaB and interleukins such as IL1 ( FIG. 8 ) are altered in their epigenetic status after BCM-7 exposure.
  • the changes in the redox status mediated the long term changes in the epigenetic status of the cytokines. This would serve as a memory of the molecular insults, potentially contributing to long term chronic changes and inflammatory responses in diseases such as IBD.
  • the altered epigenetic status is evident from Example 10. Therefore, BCM-7 not only alters MPO activity, as evident from the beta-casein A1 feeding studies, but it also alters the epigenetic status of the MPO gene ( FIG. 9 ).
  • the milk of cows can be tested for the relative proportions of beta-casein A1 and beta-casein A2.
  • cows can be genetically tested for their ability to produce milk containing beta-casein A1 or beta-casein A2 or a combination of both.
  • the invention has distinct advantages over existing techniques or methods for treating or preventing inflammatory bowel conditions. Most existing techniques or methods rely on medical intervention through pharmaceuticals, stress management or dietary modifications, many of which often have limited or no real success.
  • the present invention provides a solution that is comparatively easy to manage, i.e. converting to a non-beta-casein A1 diet through replacement of “regular” (beta-casein A1 containing) milk in the diet with milk that is known to be high in beta-casein A2, preferably where all beta-casein present is beta-casein A2.
  • the invention is considerably less costly than pharmaceutical intervention, and also avoids any need for wholesale dietary modifications such as the avoidance of dairy products or other common food products.
  • the protein component of the diets were derived from skim milk (for the A1 and A2 diets) and on egg white (for the non-milk protein control diet), and were balanced for energy and macronutrient composition (see Table 1).
  • rats received either naloxone or saline (control) via intra-peritoneal injection, and were then orally gavaged with a non-digestible tracer, titanium dioxide. Faecal and urine samples were collected at 7 time points over the following 24 hours, and stored at ⁇ 20° C. (faecal) or ⁇ 80° C. (urine) until they were analysed.
  • Colon tissue from the rats fed according to Example 1 was quantified for myeloperoxidase (MPO) activity based on an established method (Grisham, M. B., et al., Methods Enzymol ., 1990, 186:729-742).
  • Colon tissue (50 mg) was homogenised, partitioned via centrifugation, ruptured by ultrasonic probe and subjected to a freeze-thaw cycle.
  • Endogenous MPO catalyses H 2 O 2 -dependent oxidation of 3,3′,5,5′-tetramethyl-benzidine substrate measured colourimetrically at 562 nm.
  • Activity was normalised by a bicinchoninic acid (BCA) (Smith, P. K., et al., Anal. Biochem ., 1985, 150(1):76-85) protein determination for the same homogenate. The results are shown in FIG. 1 .
  • BCA bicinchoninic acid
  • tissue samples taken from the jejunum were analysed.
  • the methodology used was the same as for Example 2.
  • the results are shown in Table 2 and FIG. 2 .
  • SAA levels were measured in plasma taken from the rats fed according to Example 1. Measurements were made using a commercially available ELISA kit (Tridelta Development Limited, Maynooth, Ireland). The results are shown in Table 3 and FIG. 3 .
  • the procedure used for measuring change in morphology in the gastrointestinal tract of rats is based on histological evaluation of haemoxylin and eosin stained tissue sections under a light microscope (Knoch, B. et al., J. Nutrigenet. Nutrigenomics , 2009, 2(1):9-28 and Barnett, M. P., BMC Immunol ., 2010, 11:39).
  • This method was applied to sections derived from colon tissue of rats fed either the 100% A1 or 100% A2 diets to determine the effects of the beta-casein variants on intestinal inflammation. The results are shown in FIG. 4 .
  • Exclusion criteria were: (1) milk allergy; 2) medically diagnosed lactose intolerance; 3) pregnancy and lactation; 4) cardiovascular events in the last 6 months; 5) opioid consumption; 6) antibiotic treatment in the previous 8 weeks; and 7) immunosuppressive medication or anti-inflammatory drugs in the 4 weeks prior to screening.
  • Participants began with a 14-day dairy washout period (where dairy milk was replaced with supplied rice milk) before being randomised to 2 weeks on either A1 milk or A2 milk. After completing the first arm of the study, participants completed a second 2 week washout before crossing over to the other milk intervention.
  • Participants were instructed to replace all dairy milk with the supplied rice milk for the 14-day washout periods and to avoid all other dairy foods for the duration of the study. They were provided with information relating to hidden sources of dairy, such as biscuits and chocolate, and were provided with a list of dairy free alternatives. Participants were also supplied with enough rice milk (So Natural Rice Milk, Freedom Foods, Australia) to consume 750 mL rice milk/day, for each of the 14-day washout periods. Rice milk was supplied in 1 litre ultra-high temperature (UHT) packages and had the following nutrition profile per 100 mL: energy 212 kJ, total protein 0.3 g, total fat 1.0 g and carbohydrate 10.4 g.
  • UHT ultra-high temperature
  • Both A1 milk and A2 milk were supplied to participants in 1 litre UHT packages and had the following nutrition profile per 100 mL: energy 18910, total protein 3.1 g, total fat 2.5 g and carbohydrate (including lactose) 5.2 g. Participants recorded the volume of milk consumed per day on compliance calendars. Compliance was calculated as a percentage by dividing the milk volume consumed by the expected milk volume consumption each day and multiplied by 100.
  • A1 milk and A2 milk were provided in identical plain packaging so participants were blinded to the milk intervention they were receiving.
  • Radiolabelled [ 35 S]-cysteine uptake assay was performed in Caco-2-GI epithelial cells and neuronal cells, in the presence of BCM-7 released from A1 milk, and compared against untreated controls as well as morphine; a prototypical opioid receptor agonist. Pre-treatment in cells was performed for different time points for 30 min, 4, 24 and 48 h as described previously (Trivedi M., et al.; Mol. Pharm ., 2014). Briefly, SH-SY5Y human neuronal cells and Caco-2 Gut epithelial cells were plated in six-well plates and were pretreated with drugs and incubated for various times prior to measuring uptake.
  • HBSS HBSS containing [ 35 S]-cysteine, (1 ⁇ Ci/1 mL), 10 ⁇ M unlabelled cysteine and 100 ⁇ M DTT, and the cells were incubated for 5 min.
  • the [ 35 S]-cysteine/HBSS mixture was aspirated and treatment was terminated by two washes with ice-cold HBSS.
  • Cells were then lysed with 600 ⁇ L of dH 2 O, scraped, collected in 1.5 mL microcentrifuge tubes, and sonicated for 10 s.
  • Example 7 investigated whether decreases in cysteine uptake as observed in Example 7 could potentially translate into GSH changes and affect antioxidant levels.
  • the intracellular levels of GSH were measured with BCM-7 as well as morphine for different times (30 min, 4 h, and 24 h) with the use of HPLC and an electrochemical gradient detection method used previously (Hodgson et al., J. Alzh. Dis . 2013, Trivedi M., et al., Mol. Pharm . 2014). Briefly, SH-SY5Y neuronal cells were grown to confluence in ⁇ -MEM. Media was aspirated and the cells were washed twice with 1 mL of ice cold HBSS.
  • HBSS was aspirated and 0.6 mL ice cold dH 2 O was added to the cells.
  • the cells were scraped from the flask/dish and suspended in dH 2 O.
  • the cell suspension was sonicated for 15 s on ice and 100 ⁇ L of the suspension was used to determine the protein content.
  • the remaining lysate was added to a microcentrifuge tube and an equal volume of 0.4 N perchloric acid was added, followed by incubation on ice for 5 min. Samples were centrifuged at 5,000 ⁇ g and the supernatant transferred to new microcentrifuge tubes. 100 ⁇ L of sample was added to a conical micro-autosampler vial and kept at 4° C. in the autosampler cooling tray. 10 ⁇ L of this sample was injected into the HPLC system.
  • the column was then equilibrated with 5% B for 12 min prior to the next run. Temperature was maintained at 27° C.
  • the electrochemical detector was an ESA CoulArray with BDD Analytical cell Model 5040 and the operating potential was set at 1500 mV. Sample concentrations were determined from the peak areas of metabolites using standard calibration curves and ESA-supplied HPLC software. Sample concentrations were normalised against protein content. In some cases samples were diluted in mobile phase as needed or up to 50 ⁇ l of sample was injected to assure that thiol levels were within the range of the standard curve. The results are shown in FIG. 7 .
  • MBD methyl-CpG binding domain
  • MBD-seq protein-enriched genome sequencing
  • genomic DNA was extracted from samples with the Easy DNA kit (Invitrogen K1800-01) using the appropriate protocol for cell lines. Fragmentation was performed on Covaris S2 with the following settings: duty cycle 10%, intensity 5, 200 cycles per burst during 200 sec. Fragments were obtained having an average length of 200 bp.
  • the power mode is frequency sweeping, temperature 6-8° C., water level 12. A maximum of 5 ⁇ g was loaded in 130 ⁇ l Tris-EDTA in a microtube with AFA intensifier. For samples with less DNA input (down to 500 ng) the DNA was diluted 1:5 in TrisEDTA. DNA with an input from 5-3 ⁇ g was analysed on the Agilent 2100 using a DNA 1000 chip.
  • DNA with an input lower than 3 ⁇ g was concentrated in a rotary evaporator to 25 ⁇ l and the fragment distribution was checked on a high sensitivity DNA chip.
  • Methylated DNA was captured using the MethylCap kit (Diagenode, Belgium). The yield was typically between 0.5 and 8 ng of total captured DNA. Fragments were subsequently sequenced using an Illumina Genome Analyzer II. The concentrations of fragmented and captured DNA were determined on a Fluostar Optima plate reader with the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen P7589) at 480/520 nm.
  • a DNA Sample Prep Master Mix Set 1 (NEB E6040) was used in combination with a Multiplexing Sample Preparation Oligo Kit (96 samples, Illumina PE-400-1001). The entire fragmented DNA was utilised and followed the NEB protocols, using the multiplexing sequencing adapters provided in the Multiplexing Sample Preparation Oligo Kit. Size selection of the library was carried out on a 2% agarose gel (Low Range Ultra Agarose Biorad 161-3107). A 1Kb Plus ladder (Invitrogen 10787-018) was used and a gel was run at 120 V for 2 hrs. A fragment of 300 bps+/ ⁇ 50 bps was excised and eluted on a Qiagen Gel Extraction Kit column (Qiagen 28704) and eluted in 23 ⁇ l EB.
  • the Illumina library amplification index protocol was used with the following alterations: 22 ⁇ l DNA was used and performed 21 cycles run.
  • the sample was purified on a Qiaquick PCR Purification column (Qiagen 28101) and eluted in 50 ⁇ l EB, 1:5 diluted, concentrated in a rotary evaporator to 10 ⁇ l. 1 ⁇ l was applied to a Agilent 2100 HS DNA chip and the concentration was determined by smear analysis on the Agilent 2100.
  • the samples were diluted to 10 nM. After denaturation with NaOH the samples were diluted to 16 pM.
  • the Paired-End flow cell was prepared according to the Cluster Station User Guide. Sequencing was performed according to the HiSeq user guide (performing a Multiplexed PE Run), with 2 ⁇ 51 cycles for the paired end runs.
  • RNA from each sample was labelled with fluorescent dye (Cy3; Amersham Biosciences Corp, Piscataway, N.J.) using the Low RNA Input Linear Amplification Labelling kit (Agilent Technologies, Palo Alto, Calif.) following the manufacturer's protocol.
  • the amount and quality of the fluorescently labelled cRNA was assessed using a NanoDrop ND-1000 spectrophotometer and an Agilent Bioanalyzer. According to manufacturer's specifications, 1.6 mg of Cy3-labeled cRNA was hybridized to the Agilent Human Whole Genome Oligo Microarray (Agilent Technologies, Inc., Palo Alto, Calif.) for 17 hrs prior to washing and scanning. Data was extracted from scanned images using Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, Calif.).
  • DMTs differentially methylated promoter transcripts
  • DETs differentially expressed transcripts
  • FDR ⁇ 0.1 false discovery rate
  • Transcripts included both genes and non-coding RNAs that were differentially methylated/transcribed.
  • the epigenetic changes as well as the transcription changes induced by BCM-7 in specific biological or functionally relevant pathways were evaluated using the Ingenuity Pathway Analysis (IPA) tool and pathways exhibiting the highest impact were identified. The results are shown in Table 5.
  • IPA Ingenuity Pathway Analysis
  • FIGS. 8 and 9 show the effect of BCM-7 on the DNA methylation changes intragenic or near the promoter region of these genes, namely MPO, IL1R, IL10 and NFKappaB-activating protein.
  • CpG denotes the normal level of CpG islands (high frequency of cytosine-guanine sites) noted in that specific gene locus.
  • Control indicates non-treated controls.

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