NZ306584A

NZ306584A - Milk or dairy products derived from animals and which are free of beta casein

Info

Publication number: NZ306584A
Application number: NZ306584A
Authority: NZ
Inventors: Corran Norman Stuart Mclachlan
Original assignee: Corran Norman Stuart Mclachlan
Priority date: 1995-05-16
Filing date: 1996-05-09
Publication date: 2000-02-28

Abstract

A milk or other dairy product, capable of minimising the onset of disease such as coronary heart disease or enhancing the immune response is derived from animals which are substantially free of the b-casein A1 allele. Bulk milk can be produced by testing for and culling cows who test positive for the b-casein A1 allele, or by producing immunoglobulins and other immune response proteins, in cow's milk from animals not possessing the b-casein A1 allele, or other commercial milk producing animals, to this allele, to counteract the immunosuppressant substances present that are produced from it, in commercial milking cows such as Holsteins, together with its blending with non-treated milk or the recovery of such immunoproteins.

Description

<div class="application article clearfix" id="description"> 1 fntcllectua!.. property OFt-fOF OF n.z 1 1 SEP 2003 received MILK AND MILK PRODUCTS FOR PREVENTING OR TREATING HEART DISEASE FIELD OF THE INVENTION This invention relates to a method of producing milk free of (3-casein A1 by testing genetic material of lactating bovines, selecting bovines based on the results of the testing, and milking 5 -the selected bovines. The invention also relates to milk obtained by that method, and to food products and medicaments which contain or are processed from that milk. BACKGROUND OF THE INVENTION Description of the Background Art 10 It has long been understood that the early lactation mammary secretions of certain species, known as colostrum, contains substances that prevent disease, whilst the. immune system of the young of the species is developing. This is particularly true of the ruminants, such as the Bos family. However the ingestion of colostrum is not essential in the human. These substances were identified as proteins (globulins) with immuno-properties which became known as i: immunoglobulins, (B L Larsen Immunoglobulins of Mammary Secretions in Advanced Dairy Chemistry Volume 1 Proteins. Ed. PF Fox Elsevier ,1992). Immunoglobulins are present in the serum and mammary secretions of all mammalian species as part of the immune defence system of the animal. The immunoglobulins are also known as antibodies and are,produced by the body's immune system in response to the presence of 20 substances called antigens, including a wide range of molecules, bacteria, viruses, cells and particles that do not express specific markers of 'self called histocompatibility antigens. Molecular antigens are largely peptides, proteins and carbohydrates. The classic immune ^ response involves the production of antibodies capable of neutralising these antigens. -2 - intellectual pro ofpjof of nj.; 1 1 SEP 200; RECEIVED The term antigen is now widely used to .indicate any molecule that can be specifically recognised by the adaptive elements of the immune system, that is by both B cells, which produce immunoglobulins and T cells which release substances such as cytokines, (Immunology, 3rd Edition ,Ed. I Roitt, J Brostoff, D Male, Mosby, London, 1993). There are five classes, or isotypes, of immunoglobulins all of which have a similar basic structure, but have differences in their organisational structure as well as the amino acid sequences present and carbohydrate groups present. In addition to the immunoglobulins there are present related immune system proteins. These are known as complement and they are a complex group of proteins which assist the function of antibodies. Their properties are described in the above texts. There are at least 11 proteins in the complement group some of which are expected to be present in milk at the milligram per 100 millilitre level. There are numerous patents that have been filed which seek to: 1. isolate the immunoglobulins present in mammary secretions, particularly colostrum but also including milk and products derived from milk such as whey. Generally the species involved is the domestic cow, Bos taurus, but it may include sheep or goats. 2, produce an "immune milk" or "health food" incorporating the immunoglobulin proteins, either as a result of stimulating the milk producer's immune system by the addition or injection of substances into the animal's body, either once or systematically, which result in an immune response, or by concentrating the small amounts of immunoglobulins that are naturally present in milk -derived products. In the former case the immunoglobulins may be specific responses to the injection of pathogenic bacteria into the milk-producing animals. Examples of these patents include: Japanese patent (1988) JP 63-133941, Hon T, Nishimoto K, Kimura M, Yommazaki N, describes a process in which immunoglobulins are collected by ultrafiltration from whey, the by-product of cheese or casein manufacture. The immunoglobulin content of powder derived from this process was found to contain about ten times the immunoglobulin content of dried human milk. UK Patent Application (1987) GB 2 179 947 Monsan PFE, Thibault PA, Brossad C, Bruvier CSJ describes a process for the extraction of proteins, preferably lactdferrin or -3 - immunoglobulins from whey comprising concentration of the whey using ultrafiltration with a polysulphone membrane (with MW cut-off 25,000-50,000) followed by diafiltration. The retentate is then subject to adsorption of the retained proteins by ion exchange treatment using a weak cationic carboxymethyl resin at pH 5-8.5 and preferably at 7-8; and elution at the same pH. European Patent Application (1984) EP 0 102 831 A1 , Linggood MA, Porter P, Powell JR describes the immunisation of host animals with a range of E. Coli implicated in human gastroenteric disease and the production of immunoglobulins, and a synthetic milk containing the immunoglobulins that are specific responses to the inoculation of the host. UK Patent Application- (1987) 8729031, to R C Bottomley claims the production of a whey protein concentrate rich in immunoglobulins by the use of ultra-filtration through a membrane having a cut-off of 500,000 daltons which retains the immunoglobulins, or by subjecting whey to the action of an anion exchange resin which does not remove immunoglobulins so causing an increase in their concentration in the effluent. European patent application (1989) EP 0 336 694 Beck LR, describes a process for extracting i an anti-inflammatory factor from cow or ewe milk,' taken from animals that have been previously immunised by the administration of bacterial antigens. The anti-inflammatory factor is then extracted from whey that has been subjected to ion-exchange chromatography and molecular sieve chromatography. US patent (1992) 5 106 618 Beck LR, Kotler DP describes the production of a 'hyperimmune'' milk obtained by inoculating a milk-producing animal with a non-protozoan bacterial antigens, collecting the milk from the animal and the pasteurising and concentrating prior to use. US patents (1989) 4 879 110 and (1993) 5 194 255 Beck LR, Stolle RJ, describe a method for inducing the production of a milk anti-hypertensive factor in an animal such as a cow by injecting bacterial antigens into the animal. The anti-hypertension factor is isolated by (1) removing from the milk molecules having a molecular weight greater than 10,000 daltons;(2) fractionating by ion-exchange chromatography the effluent to obtain a negatively charged fraction;(3) fractionating the negatively charged material eluted from the ion-exchange column using molecular sieve chromatography and isolating the hypertensive fraction from the latter step by isoelectric precipitation. office of n.z 1 1 SEP 2003 RECEIVED intellectual property OFRCF OF M2 t 1 SEP 2003 -<• i received Pft YJT t-tjn S > rj v 5 Ta5A US patent (1980) 4,216,236 Mueller HR, Legier CN, Secretin MC, Blonay CN claims the incorporation of soluble proteins obtained from whey using an ultrafiltration step with membranes having a molecular weight cut-off between 1000 and 500,000 incorporating immunoglobulins or to which immunoglobulin powder or concentrate has been added. 5 US patent (1984) 4 490 290 Ganni MM, May K, Porter P, describes the recovery of one or more milk immunoglobulins by passing the milk through a re-usable immunoadsorbent column comprising an insoluble carrier material to which is bound a low-affinity monoclonal antibody specific to the antibody(ies) but not specific to any other common constituent of milk. The bound immunoglobulin(s) are released by eluting the immunosorbent with 4 M MgCl? . 1C Problem Notwithstanding all these patents and the claimed benefits of their products there is a considerable body of evidence that links milk particularly of the Bos taurus, the domestic cow with allergy problems with young children, asthma, chronic immune disorders such as diabetes mellitis, and atherosclerosis. Recent studies have also linked increased consumption of casein 15 with the formation of hepatic tumours in rats, due it appears to a depressed NK cell cytotoxic activity, Bell RC, et al Nutr Cancr 22:151-162,(1994). To date it has' not been possible to identify .any particular fraction or molecule that is responsible for disorders such as atherosclerosis, although the consumption of animal fats and their associated saturated fatty acids have been claimed to either cause, or contribute to, 2.0 coronary heart disease, hypertension and obesity as is set out in most medical texts on these subjects and the Surgeon-General's Report on Nutrition and Health, DHHS Publication No 88-50210 (1988). OBJECT 25 It is an object of this invention to provide a method of producing milk substantially free of p~ easein A1 suitable for use in the prevention or treatment of coronary heart disease, or to at least provide a useful alternative. DEFINITIONS - 5 - "P-casein A1 Allele" is a term used herein in reference to one of the variant forms of the p-casein gene. Expression of the A1 allele results in the production of«"P-casein A1". Where reference is made to the presence of the (3-casein A1 allele in an individual or population it encompasses both homozygous and heterozygous genotypes with respect to that allele. Similarly, where reference is made to the presence of p-casein A1 it encompasses phenotvpes resulting from either a homozygous or heterozygous state with respect to the P-casein A1 allele. The term "Immune milk" is used herein reference to milk obtained from an animal that has been immunised to selectively induce for formation of immunoglobulins and other immune proteins, directed against specific bacterial and/or viral pathogens or other foreign antigens that are known to cause diseases, in its milk, such milk being used to prevent disease, within the milk drinker, by fortifying the body's natural resistance against specific disease-causing antigens. The term "processed dairy product(s)" is used herein to refer to dairy products derived from a source of bulk milk (ie from milk from more than one animal) and includes, but is not limited to: (a) bulk milk used to make cheese whether or not the milk has been pasteurised or sterilised prior to cheese making, (b) milk powder(s), (c) milk fats, (d) milk solids, (e) casein(s), caseinate(s), and casein hydrolysates, (f) pasteurised, sterilised, preserved milks including micro filtered milks, UHT milks. impTrrSTrrrr-""------ NM/tlLEoTUAL PROPERTY office of n.z -6- (g) low fat milks, (h) modified or enhanced milks, (i) ice-cream or other frozen dairy based confections, (J) fermented milk products such as yoghurt or quark, (k) cheeses including full fat, partial de-fatted and fat-free processed che^es, (1) milk whey, (m) food products enriched through the addition of milk products su^fi as soups, (n) milk from which allergenic molecules have been removed, (o) confections such as chocolate, (p) carbonated milk products, including those with added pbfosphate yiciyor citrate, (q) infant formulations which may contain full, partiallyde-fattecj >r nonfat milk together with a number of additional supplements, (r) liquid or powdered drink mixtures, (s) butter, buttermilk, buttermilk powder. STATEMENT OF This invention has a number of aspects. Th^se include the following: - a method of producing milk suitable for use m the treatment or prevention of coronary heart disease from a herd of 1 aerating bovines which milk is substantially free of p- 1 ^ & 2 3 casein A but which containsimy one gr more of P-caseins A , A , B, C, D and E, the method including the steps/of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding (3/fcasein A1; / / / / / / / / • l (ii) selecting bovines which do not have DNA encoding P-casein A to form the ffd of lactating bovines; (m) milking bovines from the herd to give bulk milk; and / (iv) producing milk for sale from the bulk milk; - 6- (g) low fat milks, (h) modified or enhanced milks, (i) ice-cream or other frozen dairy based confections, (j) fermented miik products such as yoghurt or quark, (k) cheeses including full fat, partial de-fatted and fat-free processed cheeses, (1) milk whey, (m) food products enriched through the addition of milk products such as soups, (n) milk from which allergenic molecules have been removed, (o) confections such as chocolate, (p) carbonated milk products, inciuding those with added phosphate and/or citrate, (q) infant formulations which may contain full, partially de-fatted or nonfat milk together with a number of additional supplements, (r) liquid or powdered drink mixtures, (s) butter, buttermilk, buttermilk powder.' This invention has a number of aspects. These include the following: - a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P- method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding P-casein A1; STATEMENT OF INVENTION casein A1 but which contains any one or more of P-caseins A2, A3, B, C, D and E, the (ii) selecting bovines which do not have DNA encoding p-casein A to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; intellf uljai mfopf On /r:i- <» 1 1 SEP 2003 a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of p-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C; (ii) selecting bovines which do not have DNA encoding any one or more of P-caseins A1, B and C to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-casein A1 but which contains P-casein A2, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding P-casein A2; (ii) selecting bovines which do have DNA encoding P-casein A2 to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd lactating bovines which milk is substantially free of P-casein A1 but which contains any one or more of p-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of the lactating bovines for the presence of DNA encoding any one or more of P-caseins A2, A3, D and E; (followed by page 7a) Ill# ■S, /z) -7a- 2 ^ (ii) selecting bovines which do have DNA encoding any one or more of P-cas^ins A , A3, D and E to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk suitable for use in the treatment or prevention of^oronary heart disease from a herd of lactating bovines which milk is substantially free ofp-casein A1 but which contains P-casein A2, the method including the steps of: IMTn i rnfi'i *. iivifclltcttjal propfriy oftpe of |\(2 l/ SEP 2003 / RECEIVED (i) testing genetic material of lactating bovines l or the presence of DNA encoding P-casein A1 and DNA encoding p-casein A2; (ii) separating bovines which have DNA/encoding B/6asein A2 from bovines which have DNA encoding P-casein A1 or which haver DNA encoding both P-casein A1 and P-casein A2 to form the herd/if lactating ^ovines; (iii) milking the bovines which have DNA encoding P-casein A2 from the herd to give the bulk milk; and (iv) producing milk for sale from the Hulk milk; a method of producing mijk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-caseins A1, B and C but wl steps of: C but which contains/ny one or more of P-caseins A2, A3, D and E, the method including the / / / (i) toting genetic/material of the one or more lactating bovines for the presence of )NA encoding any one or more of P-caseins A1, B and C and DNA encoding any / f one or more of P-caseins A2, A3, D and E; / / / / (ii) separating bovines which have DNA encoding any one or more of P-caseins A , B / 2 and C from bovines which have DNA encoding any one or more of P-caseins A , / A3, D and E to form the herd of lactating bovines; (followed by page 8) 7a - intellectual property office of n.z. -1 OCT 2003 RECEIVED (ii) selecting bovines which do have DNA encoding any one or more of P-caseins A , A3, D and E to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-casein A1 but which contains P-casein A2, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding P-casein A1 and DNA encoding P-casein A2; (ii) separating bovines which have DNA encoding P-casein A2 from bovines which have DNA encoding P-casein A1 or which have DNA encoding both P-casein A1 and P-casein A2 to form the herd of lactating bovines; (iii) milking the bovines which have DNA encoding P-casein A2 from the herd to give the bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of P-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C and DNA encoding any one or more of p-caseins A2, A3, D and E; (ii) separating bovines which have DNA encoding any one or more of p-caseins A1, B f 2 and C from bovines which have DNA encoding any one or more of P-caseins A , A3, D and E to form the herd of lactating bovines; (followed by page 8) (iii) milking the bovines which have DNA encoding any one or more of P-caseins A2, A3, D and E from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk; a method of producing milk as defined above in which the lactating bovines tested in step (i) are, or include, Bos taurus bovines; a method as defined above wherein the lactating bovines are Bos taurus bovines; milk containing P-casein but free of P-casein A1 which is the product of a method as defined above; milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of p-casein A1 but which contains any one or more of P-caseins A2, A3, B, C, D and E, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding p-casein A1; (ii) selecting bovines which do not have DNA encoding P-casein A1; and (iii) milking the selected bovines; milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of P-caseins A2, A3, D and E, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C; intellectual property ofrcf of n,z 1 I SEP 2003 (followed by page 8a) received -8a- (ii) selecting bovines which do not have DNA encoding any one or more of p-caseins A1, B and C; and (iii) milking the selected bovines; milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-casein A1 but which contains P-casein A2, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding P-casein A2; (ii) selecting bovines which do have DNA encoding P-casein A2; and (iii) milking the selected bovines; milk suitable for use in the treatment or prevention of coronaiy heart disease obtained from one or more lactating bovines which milk is substantially free of P-casein A1 but which contains any one or more of p-caseins A2, A3, D and E, which milk is a product of a method including the steps of: (i) testing genetic materia! of the one or more lactating bovines for the presence of DNA encoding any one or more of p-caseins A2, A3, D and E; (ii) selecting bovines which do have DNA encoding any one or more of P-caseins A2, A3, D and E; and (iii) milking the selected bovines; (followed by page 9) IKIIM iVTwi'i'Ti "■"* in I ellbcl ual PRQpj :i rrv office- of nz i I SEP 2003 received -9- - milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-casein A1 but which contains P-5 casein A2, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding P-casein A1 and DNA encoding P-casein A2; 10 (ii) separating bovines which have DNA encoding P-casein A2 from bovines which have DNA encoding P-casein A1 or which have DNA encoding both P-casein A1 and P-casein A ; and 15 20 (iii) milking the bovines which have DNA encoding p-casein A2; - milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of P-caseins A2, A3, D and E, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C and DNA encoding any 2 3 one or more of P-caseins A , A , D and E; 25 (li) separating bovines which have DNA encoding any one or more of p-caseins A1, B and C from bovines which have DNA encoding any one or more of p-caseins A2, •i A , D and E; and (iii) milking the bovines which have DNA encoding any one or more of P-caseins A2, 30 A3, D and E; - a product which contains P-casein but is substantially free of P-casein A1 and which contains or is processed from a milk as defined above; 35 (followed by page 9a) intellectual propfrty ofrcf of m.z 1 1 SEP 2003 received . -9a- - a product as defined above which is a food product; 5 - a product as defined above which is a medicine; - the use of a milk as defined above, or of a product as defined above, in the preparation of a medicine; and 10 - the use as defined above in which the medicine is for use in the treatment or prevention of coronary heart disease. The discovery that is the basis of this Invention 15 It has been reported that certain groups of peoples are not subject to the diseases described above, notwithstanding the fact that they consume considerable quantities of milk proteins. These people include the Tibetans, rural Gambians,-the Masai and Samburu people of Kenya. The latter peoples are also found not to suffer from obesity, even in old age, Tne only major difference between the milk consumed by the above people is that it is derived from Zebu, Bos 20 Indicus, and Yak, Bos Mutus. Neither milk contains the casein allele described as [3-casein A1. In addition, people such as the Eskimo do not suffer from diseases such as CHD compared with their dairy product consuming Danish countrymen as is illustrated in Table 1: —- Lnrr,%AL PROPf Hry u 1-1-/0 f)F i\j j (followed by page 10) - 10- Table 1, Age-adjusted differences in morbidity from chronic diseases between Greenland Eskimos and Danes Eskimos/Danes 5 S troke Psoriasis Diabetes Bronchial asthma Malignant disorders Acute myocardial infarction 1/10 2/1 1/20 rare 1/25 1/1 10 Thyrotoxicosis Multiple sclerosis Polyarthritis chronica 0 low rare Acta Med Scand 208: 401-406, (1980) -■< l5 These and other aspects of this invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only with reference to the preferred embodiments, and makes reference also to the following graphs':' Figure I is a graph entitled "The effect of food component on Ischaemic Heart Disease during 1985 for males aged 30-69". This shows the death rate of all ages per 100,000 of population, 20 for a range of countries, based on the consumption of P-casein. Figure 2 is a graph showing the effect of dairy protein consumption on Ischaemic Heart Disease for males aged 30-69 for the year 1985. ✓ Figure 3 is a graph showing the effect of saturated fat consumption on Ischaemic Heart Disease for males aged 30-69 for the year 1985. 25 Figure 4 is a graph showing the effect of red meat consumption on Ischaemic Heart Disease for males aged 30-69 for the year 1985. Figure 1 shows a very strong correlation between the consumption of the food component, identified as p-casein A1 (discussed in more detail below), and the death rate. Whereas the overall dairy protein consumption (Figure 2) does not provide such a strong correlation nor JO does the effect of saturated fat consumption (Figure 3), nor the consumption of red meat (Figure 4) come anywhere close to the very strong correlation with the inventor has identified in relation to the consumption of p-casein A1, both between countries and within countries. In the states of the form West Germany Ischaemic Heart Disease death rates are found to correlate - 12 - directly with the consumption of (3-casein A1 (Table 1A). In this instance the composition of the state dairy herd have remained virtually constant from the 1950's through to the 1980's. Table 1A: CHD nutritional risk factors, Federal Republic of Germany based on S chl eswig-Holstein Saturated Fat Cholesterol Alcohol Gufcohydnites Energy 2-A1 Re! IHD est. Schleswig Holsiein 1.00 1.00 1.00 1.00 LOO 1.0 1.0 Niedersachsen 0.97 0.96 1.00 0.98 0.99 0.92 0.38 Nordrhein Westfalen 0.99 1.02 0.99 1.00 1.02 0.97 1.00 Hessen 0.95 0.96 0.98 0.98 0.98 0.75 0.74 Rheirtiand-Pfaiz 0.95 0.99 ' 1.00 1.02 1.0 0.87 0.78 Saarland 0.94 0.93 0.98 1.01 0.98 0.90 0.88 'aden Wuntenburg 0.93 1.02 1.02 1.05 1.03 0.50 0.72 Bavern * 0.96 0.99 1.22 1.06 1.02 0.50 0.74 DETAILED DESCRIPTION Caseins constitute the majority of the milk proteins. Dairy cattle exhibit genetic polymorphism in their proteins. Tne heterogeneity of the caseins is further complicated by the fact that they are the products of co-dominant allele autosomal genes. Some indication of their number, and 3 the major product fragments into which they are split by proteolytic action of a variety of enzymes, is illustrated by the j3-caseins in Table 2. I INifcLi-EOTUAL PROPFRTY I OFRCE OF IM.Z 1 1 SEP 2003 I RECEIVED 5 15 Tabic 2. The p-casein family of proteins Former nomen. recommended-nomen. source of fragment P-casein A1 p-CN A'-5P .... P-casem A" P-CN A:-5P p-casein AJ p-CN A3-5P p-casein I? P-CN B-5P P-casein C P-CN C-4P .... P-casein D p-CN D-4P P-casein F p-CN E-5P — yi-casein A1 p-CN A1 -1 P(f29-209) p-CN A'-5P Yi-cascin A2 P-CN A2-lP(f29-209) p-CN A2-5P Yi-casein A3 p-CN A3-l P(f29-209) P-CN A3-5P Yi-casein B P-CN B-l P(f29-209) p-CN B-5P Yi-casein A2 P-CN a2 (fl 06-209) P-CN a'-5P or p-CN a2-5p • \ 3 Yrcasein A p-CN A3 (fl 06-209) p-CN A3-5P Yi-casein B P-CN B (fl06-209) P-CN B-5P Yj-casein A £-CN A-'-5P, p-CN A2oPor P-CN A3-5P Yi-cascin B p-CN B .'fl08-209) P-CN B In addition there are a number of proteose peptone components. 2<; \V N l-.iycl Nomenclature of Proteins of C'uw's Milk: Fillh Revision J, Dairy Science 67:1599-163 t. (1984) Most animals are heterozygous. That is their protein composition contains a mixture of the various alleles inherited from the genes of their sire and dam. It appears that the original cow from which the current domesticated species developed contained only the p-casein A2 allele, p-casein a' ditlers from A* in containing the replacement of ammo acid proline^ by a histidine. The corresponding A1 allele is a relatively recent modification. However some animals are 30 homozygous, that is their proteins are of one type only; in the case of p-caseins either A1, A2, A'1, or B, C. D or E. 35 Bovine milk is an important-souist o^t^einT<1nthoti!fernuf?1'(:nts°n5quired by humans and the common domestic cattle specics such as the Holstein have greater quantities of the A1 allele than any other p-casein allele. Approximately 84 percent of the present American dairy herd is estimated to carry this allele. In the graph shown in Figure 1 the consumption of p-casein A1 (and its derived proteolysis products) are plotted against the incidence of ischaemic heart di intellectual property qfficb op n.z - 1 1 SEP 2003 received OF NZ :? jam 2000 RECEIVED WO 96/36239 PCT/NZ96/00039 ( - 23 - Balance Sheets 1979-81 and WHO Trends in Mortality for Selected Causes of Death 1985-1989 and other reported CHD data. In Table 3 the effect of heating milk to -6-3 °C for 20-30 minutes, known as Holder Pasteurisation, is set out together with the corresponding rate of CHD. 5 Table 3. CHD rates following the Introduction of Holder Pasteurisation Population group Holder intra year Angina pectoris(APl) mort. p mill. Cerebral embolism and thrombosis(CET) AP 1 AP2 AP3 A% CET 1 CET 2 CET 3 A % U.K 10 Edinburgh 1923 1925 67,. 92 37.3a 1924 ' 174 236 35.6 Glasgow 1924 1924 56 91 62.5a 1924 77 101 31.2 Dundee 1924 1925 42 64 52.4* 1925 162 188 16.0 i Aberdeen 1926 1926 91 135 48.4a 1927 121 227 87.6 Lanarkshire 1935 1937 188 375 99.5b 1938 153 193 26.1 15 (excluding 1947 1948 685 1185 73.0 1948 298 518 73.8 Glasgow) 1952 1-954 1185 1523 28.5 1954 518 680 31.3 County of Sutherland 1954 1954 963 1710 77.9 1954 610 823 34.9 20 County of Bute' 1956 1956 1610 2848 76.9 1956 955 1398 46.4 London Admin. - County 1925 1925 31 112 261.3° 1926 90 120 33.3 Average increase 81.8 41.6 25 Norway Oslo 1922 1922 3 43 1333,3d not available Columns API and CET1 denote the year of commencement of the sudden rise in the appropriate mortality. . Columns AP2 and CET2 denote the appropriate average mortality fcr the 4 years immediately preceding the year of :ntroduction of pasteurisation. 30 ! .olumns AP3 and CET3 denote the appropriate average mortality for the 4 years immediately succeeding the introduction of pasteurisation. A% represents average increase. 3 Possibly low becausc deaths ascribed to "coronary thrombosis" were not included in International List No. 89 in Scotland until 1931. 35 b Possibly enhanced as deaths ascribed to "coronary thrombosis" were now included in International List No. 94. c Possibly enhanced because (1) after 1927 all deaths ascribed to "coronary thrombosis" were included-unlike those in Scotland-in International list No. 89 and (ii) the large London creameries introduced Holder pasteurisation during this period. tO d Mortality ascribed to the following group of classifications: angina pectoris, infarctus cordis, sclerosis an. coron. cordis. '' A proteolytic enzyme plasmin, which is naturally present in milk, and which is largely associated with the casein, is both increasingly active at higher temperatures and is quite heat 5 stable. At 60 °C it has been demonstrated to have a relatively high rate of conversion of 1" iNTi'LLECTUAL' PROPERTY"" 1 1 office of n.z I 1 1 SEP 2003 i INTELLECTUAL property"! | OFFfOp Of M2 J j 1 1 SEP 2003 I - h - i received I caseins, preferentially P-caseins to a range of proteolysis products. The increased mortality rate, demonstrated in Table 3, as a result of heating of the milk is presumed to be due to the formation of further proteolysis products, in addition to those naturally present, during the heating phase. It is possible however that the specific fragment of P-casein A1 that is entering or effecting the body's immune system which result from an enzyme contained within a psychotropic bacterium, or spore forming bacterium, present in the milk. Both the ratio of p-casein A'/p-casein A2 and the concentration of psychotropic bacteria vary seasonally in milk. This seasonal fluctuation is thought responsible- for part of the seasonal fluctuation in the illnesses that we have noted above. This work is further supported by the results of Bell Rc, Golemboski KA, Dietert RR, and Campbell TC, Nutrition and Cancer 22;(2),151-162,(1994) who found that when Fischer 344 rats were fed diets containing 6 percent and 22 percent casein after being injected with a liver cancer causing substance, aflatoxin, the percentage of animals developing liver cancer increased directly proportional to the increase in casein in the diet. They interpreted the results to suggest that a low protein diet might result in lower suppression of the natural killer cell cytotoxicity activity. With our knowledge we can re-interpret their data to suggest that based on our own observations on the effect of P-casein A1 on immunosuppression in humans, its reduction in the rat's diet reduced cancer formation by a factor of four due to a dose specific effect on the rat's immune system. The preferred forms of this invention comprises the-elimination from milk of P-casein A1 or its proteolysis products, or protein fragments formed in any other way, either 'in vitro' or 'in vivo' and which have immunosuppressant properties, by the use of .immunoglobulins raised against P-casein A1, the removal of P-casein A1 and the inactivation of plasmin and other proteolytic enzymes. The preferred forms.of the invention represents a significant advance over existing treatments for atherosclerosis, and other generally chronic immunosuppressant diseases in that it will prevent their occurrence in the new-born who, when they are genetically susceptible, will in other circumstances develop the diseases as they age. In addition it is believe it will assist in the restoration of organs and cells in those people where the damage to the bodies' organs is not permanent, by removing the source of chronic immune suppression. - 15 - By the term treatment, for the purpose of this invention it is intended that the symptoms of the disorder be ameliorated or completely eliminated or, where genetic typing indicates that an individual is of high risk of developing a disorder, of ensuring that it does not develop. 5 Example 1 In its preferred form the treatment consists of inoculating a milk producing animal, that does not possess the P-casein A1 allele, preferably one that is homozygous for the P-casein A2 allele, preferably one that produces commercially feasible quantities of milk, such as a cow, sheep, goat, or zebu with P-casein A1, or its proteolysis products, or fragments thereof, produced in 10 any other manner, so that antigens to the foreign P-casein A1 protein are produced. These antigens may be produced either alone, or as part of a wider inoculation programme, to produce a milk with an enhanced antigen concentration as has been described in the Art. This antigen enhanced milk is then added to 'normal' milk, or milk, or milk products, whose P-casein A1 content has been reduced, using techniques known to those skilled in the Ait, to counteract the 15 presence of the immune suppressing P-casein A1 derived material. Alternatively the above antigen(s) may be recovered by .-one of the; processes known to the Art and used as a food supplement in its own right either alone or as a food additive. Because the immunoglobulins formed as a result of the inoculation programme are somewhat heat sensitive then care has to exercised with the pasteurisation and handling of the final 10 "'product if a powdeir is required as is described in the existing Art. Alternatively, immunoglobulins and other antigens, are recovered from non p-casein A! containing milk by ultrafiltration, ion exchange chromatography either singly, or in combination, or by use of a suitable immunoadsorbent column, comprising an insoluble carrier material to which is bound a low-affinity monoclonal antibody specific to one or more milk 5 immunoglobulins but not specific to any other common constituent of milk. Such milk may having been derived from an .animal that has been inoculated with a vaccine derived from a bacteria such as E. coli, for example, or which has been inoculated with 'bacterial antigens', as described in the Art. Alternatively, enhanced quantities of antigens are produced as a result of the inoculation, or inoculation programme of P-casein A!-free animals, to provide a milk | INTELLECTUAL PROPERTY! J OFRCE OF N.Z i 1 I SEP 2003 I RFCFIUI=D I - 16 - product with all the claims as described in the prior An. Tnis invention has the advantage over the existing Art that immunosuppressant proteins resulting from the presence of, or, derived from the p-casein A1 allele are eliminated from the final milk, or milk-derived products. Another alternative includes the use of a plasmin inhibitor, such as a protein like aprotonin, or other such inhibitors, known to the Art, which are added, either singly or in mixtures, to the milk, as part of the above invention, to suppress the formation of additional P-casein A1 proteolysis products that would otherwise be formed during processing, and storage, prior to sale. Example 2 A milk or other dairy product according to the invention can be produced by testing individual cows in a dairy herd for the presence of the [3-casein A1 allele, or for the presence of P-casein A1 in milk, and then selectively culling those cows returning a positive result, until the bulk milk produced by the herd is substantially free of P-casein A1. Alternatively, homozygous cattle containing the P-casein A2 allele can be selectively bred so that the p-casein A1 allele is eliminated from the herd. An alternative approach to remove P-casein A1 from bulk milk would involve separating cattle from existing herds which contain the p-casein A1 allele, allowing- the remainder of the herd (which are free of the P-casein A1 allele) to be used for the production of bulk milk or other iairy products, and those cattle containing the A1 allele to be used for the production of products for purposes other than human consumption. Such a segregation process within a herd may be facilitated by the use of ear tags or the like to mark individual animals. Industrial Application The invention provides milk, and products containing or processed from the milk, capable of increasing the health of an individual, or the health of a population. In particular, the invention provides a method of producing milk suitable for use in the treatment or prevention of coronary heart disease including testing genetic material of lactating bovines, selecting bovines on the basis of the test results and milking them. intellectual propf of-rcfr op n.z I 1 SEP 2003 RECEIVED -17- ' The invention applies a method of reducing the onset of coronary heart disease in a human population which derives some of its food intake from milk or other diary products by reducing or substantially eliminating the presence of (3-casein A1 within the diet of that population. 5 ADVANTAGES By reducing or substantially eliminating the presence of P-casein A1 in the diet of humans, it is believed that the immune response of an individual or a population may be enhanced, or immunosuppression reduced, increasing the general well-being of the individual or the population. It is believed that some individuals may be particularly susceptible to the presence 10 f P-casein A1, and it may be possible to develop a test for such susceptible individuals, and to recommend that they reduce or eliminate their consumption of milk or other diaiy products containing P-casein A1. VARIATIONS Recognising that dairy products free of P-casein A1 are desirable it is preferable to ensure that 5 the animal from which the product is derived has been tested for the presence of the P-casein A1 allele or P casein A1 expressed therefrom and subsequent selective breeding programmes (selecting for P-casein A1 negative animals) carried out to eliminate the presence of the {3-casein A1 from the herd. It will be recognised that such testing may be carried out in a number " ways without departing from the scope of the present invention. ) Finally, it will be appreciated that various other alterations and modifications may be made to the foregoing without departing from the spirit or scope of this invention. OPRCP f)F ^2 " 1 I SEP 2003 RECEIVED 18 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS: 1. 5 10 15 2. 20 25 30 3. 35 A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of p-casein A1 but which contains any one or more of p-caseins A2, A3, B, C, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding p-casein A1; (ii) selecting bovines which do not have DNA encoding p-casein A1 to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which miik is substantially free of p-caseins A1, B and C but which contains any one or more of p-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C; (ii) selecting bovines which do not have DNA encoding any one or more of p-caseins A1, B and C to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of p-casein A1 but which contains P-casein A2, the method including the steps of: 19 (i) testing genetic material of lactating bovines for the presence of DNA encoding P-casein A2; (ii) selecting bovines which do have DNA encoding p-casein A2 to form the herd of lactating bovines; (iii) milking bovines from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of p-casein A1 but which contains any one or more of p-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding any one or more of P-caseins A2, A3, D and E; (ii) selecting bovines which do have DNA encoding any one or more of p-caseins A2, A3, D and E to form the herd of lactating bovines; (iii) miiking bovines from the herd to give bulk miik; and (iv) producing milk for sale from the bulk milk. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-casein A1 but which contains p-casein A2, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding P-casein A1 and DNA encoding P-casein A2; 20 (ii) separating bovines which have DNA encoding P-casein A2 from bovines which have DNA encoding p-casein A1 or which have DNA encoding both P-casein A1 and P-casein A2 to form the herd of lactating bovines; (iii) milking bovines which have DNA encoding p-casein A2 from herd to give bulk milk; and (iv) producing milk for sale from the bulk milk. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from a herd of lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of P-caseins A2, A3, D and E, the method including the steps of: (i) testing genetic material of lactating bovines for the presence of DNA encoding any one or more of p-caseins A1, B and C and DNA encoding any one or more of P-caseins A2, A3, D and E; (ii) separating bovines which have DNA encoding any one or more of P-caseins A1, B and C from bovines which have DNA encoding any one or more of p-caseins A2, A3, D and E to form the herd of lactating bovines; (iii) milking bovines which have DNA encoding any one or more of p-caseins A", A3, D and E from the herd to give bulk milk; and (iv) producing milk for sale from the bulk milk. A method of producing milk as claimed in any one of claims 1 to 6 in which the lactating bovines tested in step (i) are, or include, Bos taurus bovines. A method as claimed in claim 7 wherein the lactating bovines are Bos taurus bovines. Milk containing p-casein but free of p-casein A1 which is the product of a method as claimed in any one of claims 1 to 8. 21 Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of (3-casein A1 but which contains any one or more of p-caseins A2, A3, B, C, D and E, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding p-casein A1; (ii) selecting bovines which do not have DNA encoding p-casein A1; and (iii) milking the selected bovines. Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of p-caseins A2, A3, D and E, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding any one or more of p-caseins A1, B and C; (ii) selecting bovines which do not have DNA encoding any one or more of p-caseins A1, B and C; and (iii) milking the selected bovines. Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-casein A1 but which contains p-casein A2, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding P-casein A2; (ii) selecting bovines which do have DNA encoding p-casein A2; and intellectual property qfrof of n.z 1 1 SEP 2003 22 (iii) milking the selected bovines. Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of p-casein A1 but which contains any one or more of P-caseins A2, A3, D and E, which milk is a product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding any one or more of p-caseins A2, A3, D and E; (ii) selecting bovines which do have DNA encoding any one or more of p-caseins A2, A3, D and E; and (iii) milking the selected bovines. Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of 0-casein A1 but which contains P-casein A2, which milk is the product of a method including the steps of: (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding P-casein A1 and DNA encoding P-casein A"; (ii) separating bovines which have DNA encoding p-casein A2 from bovines which have DNA encoding p-casein A1 or which have DNA encoding both P-casein A1 and p-casein A2; and (iii) milking the bovines which have DNA encoding p-casein A2. 23 15. Milk suitable for use in the treatment or prevention of coronary heart disease obtained from one or more lactating bovines which milk is substantially free of P-caseins A1, B and C but which contains any one or more of P-caseins A2, A3, D and E, which milk is the product of a method including the steps of: 5 (i) testing genetic material of the one or more lactating bovines for the presence of DNA encoding any one or more of P-caseins A1, B and C and DNA encoding any one or more of p-caseins A2, A3, D and E; 10 (ii) separating bovines which have DNA encoding any one or more of p-caseins A1, B and C from bovines which have DNA encoding any one or more of P-caseins A2, A3, D and E; and (iii) milking the bovines which have DNA encoding any one or more of p-caseins 15 A2, A3, DandE. 16. A product which contains p-casein but is substantially free of P-casein A1 and which contains or is processed from a milk as claimed in any one of claims 10 to 15. 20 17. A product as claimed in claim 16 which is a food product. 18. A product as claimed in claim 16 which is a medicine. 19. The use of a milk as claimed in any one of claims 6 to 15, or of a product as claimed 25 in claim 16, in the preparation of a medicine. 20. The use of claim 19 in which the medicine is for use in the treatment or prevention of coronary heart disease. intellectual property office of hi 1 1 SEP 2003 </div>