AU2004206984B2

AU2004206984B2 - Bovine genotype testing for beta-caseins

Info

Publication number: AU2004206984B2
Application number: AU2004206984A
Authority: AU
Inventors: Corran Norman Stuart Mclachlan
Original assignee: A2 Corp Ltd
Current assignee: A2 Milk Co Ltd
Priority date: 1995-05-16
Filing date: 2004-09-01
Publication date: 2008-04-17
Anticipated expiration: 2016-05-09
Also published as: AU4266100A; AU2008203187A1; AU2004206984A1

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Corran Norman Stuart McLachlan Actual Inventor/s: Corran Norman Stuart McLachlan Address for Service is: SHELSTON IP Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: FOOD PRODUCT AND PROCESS Details of Original Application No. 42661/00 dated 23 Jun 2000 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 34645AUP02 500422390_1 .DOC/5844 00 -la- BOVINE GENOTYPE TESTING FOR BETA-CASEINS SFIELD OF THE INVENTION C 5 This invention relates to a method of producing milk free of P-casein A' by testing genetic material of lactating bovines, selecting bovines based on the results of the testing, and milking 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.

CN BACKGROUND OF THE INVENTION SDescription of the Background Art All references, including any patents or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in Australia or in any other country.

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-propertics which became known as immunoglobulins, (B L Larson Immunoglobulins of Mammary Secretions in Advanced Dairy Chemistry Volume I Proteins. Ed. PF Fox Elsevier. 1992).

Imnmunoglohulins are present in the scrum and nmammanry secrelions of all mammnalian specics a;s part of the i mmune d(efence of the ani mal. The i nImunoglubulins are also known ;is antibodics and are produced by the body's imnimune systeml in response to the presence of 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.

N; d l I t30--C a rs '7 1C? -;999' P7 1 AU 2\6 A2\ N P7 12A6 ALi 2 .nn els -oc 13 03 OF 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 Sdescribed 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 n aturally 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, Hori 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 lactoferrin or 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 A' 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 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.

00 -4- 0 O 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 E membranes having a molecular weight cut-off between 1000 and 500,000 incorporating Simmunoglobulins or to which immunoglobulin powder or concentrate has been added.

US patent (1984) 4 490 290 Ganni MM, May K, Porter P, describes the recovery of one or fmore milk immunoglobulins by passing the milk through a re-usable immunoadsorbent column 0 O comprising an insoluble carrier material to which is bound a low-affinity monoclonal antibody O specific to the antibody(ies) but not specific to any other common constituent of milk. The bound immunoglobulih(s) are released by eluting the immunosorbent with 4 M MgC 2 C 10 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 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, 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).

DEFINITIONS

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, C< 5 the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

00 "p-casein A' Allele" is a term used herein in reference to one of the variant forms of the O 10 P-casein gene. Expression of the A' allele results in the production of "P-casein Where reference is made to the presence of the 1-casein A' allele in an individual or Spopulation it encompasses both homozygous and heterozygous genotypes with respect to that allele. Similarly, where reference is made to the presence of P-casein A' it encompasses phenotypes resulting from either a homozygous or heterozygous state with respect to the p-casein A' allele.

The term "Immune milk" is used herein in 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.

This invention is applicable to all products derived from cattle (live or dead) which products are substantially free of 3-casein A or contains immune response proteins (including immunoglobulins) to P-casein This includes meat (including offal) blood and blood products (such as black pudding), casein, gelatine, milk and other dairy products, as well as manufactured products containing some or all of the foregoing examples (including whiteners for beverages that include some milk solids).

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: milk used to make cheese whether or not the milk has been pasteurised or sterilised prior to cheese making, milk powder(s), milk fats, milk solids, casein(s), caseinate(s), and casein hydrolysates, pasteurised, sterilised, preserved milks including microfiltered milks, UHT milks.

It He It-- lncs e'r 7 1 OV 7 .1)9q' P-,1286 AU 2 Scis- P7 1 26 AUt 2 ,L-t ,11- 13 i8/07 00

O

0 low fat milk, modified or enhanced milks t ice cream or other frozen dairy based confections fermented milk products such as yoghurt or quark e 5 cheeses including full fat, partial de-fatted and fat-free processed cheeses milk whey food products enriched through the addition of milk products such as soups 00 milk from which allergenic molecules have been removed confections such as chocolate

\O

carbonated milk products, including those with added phosphate and/or citrate infant formulations which may contain full, partially de-fatted or nonfat milk Stogether with a number of additional supplements liquid or powdered drink mixtures butter, buttermilk, buttermilk powder.

STATEMENT OF INVENTION In aspects of the invention there is provided: a method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A' but which contains any one or more of P-caseins A 2

A

3 B, C, D and E, the method including the step of: testing the cow for the presence of the p-casein A' allele: a method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A' but which contains any one or more of 3-caseins A 2

A

3 B, C, D and E, the method including the step of: testing genetic material of the cow for the presence of DNA or RNA encoding p-casein A'; a method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of p-casein A' but which contains p-casein A 2 the method including the step of: testing the cow for the presence of the P-casein A' allele and the P-casein

A

2 allele; a method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A but which contains P-casein A the method including the step of: testing genetic material of the cow for the presence of DNA or RNA encoding P-casein A' and DNA or RNA encoding P-casein A 2 a method of forming a herd of bovine cows which are capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of p-casein A' but which contains any one or more of 3rl \Me I bo-* Cases,, Pat- \7 1 0- 1999\ 7126 AU 2\Spec is\ P71286 AU.2 amencLTFeL:scioc 13/03 09 00 O caseins A 2 A B, C, D and E, the method including the steps of: testing cows for the presence of the P-casein A' allele; and (ii) selecting cows which do not have the P-casein A' allele to form the herd; Sa method of forming a herd of bovine cows which are capable of producing

C

c 5 milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A' but which contains any one or more of Pcaseins A 2 A B, C, D and E, the method including the steps of: 00 testing genetic material of cows for the presence of DNA or RNA CA encoding p-casein and (ii) selecting cows which do not have DNA or RNA encoding P-casein A' to form the herd; Sa method of forming a herd of bovine cows which are capable of producing Smilk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A' but which contains P-casein A the method including the steps of: testing cows for the presence of the P-casein A' allele and the P-casein A' allele; and (ii) selecting cows which have the P-casein A 2 allele but do not have the 3casein A' allele to form the herd; a method of forming a herd of bovine cows which are capable of producing milk suitable for use in the treatment or prevention of coronary heart disease which milk is substantially free of P-casein A' but which contains P-casein A 2 the method including the steps of: testing genetic material of cows for the presence of DNA or RNA encoding P-casein A' and DNA or RNA encoding P-casein A 2 and (ii) selecting cows which have DNA or RNA encoding P-casein A 2 but do not have DNA or RNA encoding p-casein A' to form the herd; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows which milk is substantially free of p-casein A' but which contains any one or more of 3-caseins A 2 A B, C, D and E, the method including the steps of: testing cows for the presence of the 3-casein A' allele; (ii) selecting cows which do not have the P-casein A allele; and (iii) milking the selected cows; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows which milk is substantially free of P-casein A' but which contains any one or more of p-caseins A A B, C, D and E, the method including the steps of: testing genetic material of cows for the presence of DNA or RNA encoding P-casein A; (ii) selecting cows which do not have DNA or RNA encoding P-casein A'; and \Mlbourt,.eCases\ PAL L MC 1?9) 1286, AU, 2\ Spec. 1286 AU, 2 amendmer.LSc1oC 13/03,O 00 -8-

O

O (iii) milking the selected cows; a method of producing milk suitable for use in the treatment or prevention of t coronary heart disease from one or more bovine cows which milk is substantially free Sof P-casein A' but which contains P-casein A 2 the method including the steps of: c 5 testing cows for the presence of the p-casein A' allele and the p-casein

A

2 allele; (ii) selecting cows which have the P-casein A 2 allele but do not have the 3casein A' allele; and S(iii) milking the selected cows;

NO

a method of producing milk suitable for use in the treatment or prevention of C coronary heart disease from one or more bovine cows which milk is substantially free O of p-casein A but which contains p-casein A 2 the method including the steps of: testing genetic material of cows for the presence of DNA or RNA encoding p-casein A' and DNA or RNA encoding p-casein A 2 (ii) selecting cows which have DNA or RNA encoding p-casein A 2 but do not have DNA or RNA encoding p-casein and (iii) milking the selected cows; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows which milk is substantially free of p-casein A' but which contains any one or more of P-caseins A 2

A

3 B, C, D and E, the method including the steps of: breeding one or more bovine bulls that do not have the P-casein A' allele with bovine cows that do not have the P-casein A' allele to give progeny cows which do not have the P-casein A' allele; and (ii) milking the progeny cows; a method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows which milk is substantially free of P-casein A' but which contains any one or more of P-caseins A 2

A

3 B, C, D and E, the method including the steps of: breeding one or more bovine bulls that do not have DNA encoding for Pcasein A' with bovine cows that do not have DNA encoding for P-casein A' to give progeny cows which do not have DNA encoding P-casein and (ii) milking the progeny cows.

\Ple I bourt,.o aenZ I 0-7199 2R6 SAU. amend-eLs (1,c 13;03/O 00

O

The discovery that is the basis of this Invention SIt 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, 00 even in old age. The only major difference between the milk consumed by the above people is that it is derived from Zebu, Bos Indicus, and Yak, Bos Mutus. Neither milk 0 10 contains the casein allele described as P-casein In addition, people such as the c Eskimo do not suffer from diseases such as CHD compared with their dairy product 0consuming Danish countrymen as is illustrated in Table 1: N \Melbo-e\ C 5es\ P\tePl\7 1000-71 999\P71286 AU 2\Specis\P?1286 AU.2 amenden-doc 1),O3,Of/ Table 1. Age-adjusted differences in morbidity from chronic diseases between Greenland Eskimos and Danes Eskimos/Danes Acute myocardial infarction 1/10 Stroke 2/1 Psoriasis 1/20 Diabetes rare Bronchial asthma 1/25 Malignant disorders 1/l Thyrotoxicosis rare Multiple sclerosis 0 Polyarthritis chronica low Acta Med Scand 208: 401-406, (1980) 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 1 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, for a range of countries, based on the consumption of 1-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.

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 3-casein A' (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 does the effect of saturated fat consumption (Figure 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 both between countries and within countries. In the states of the form West Germany Ischaemic Heart Disease death rates are found to correlate -11directly with the consumption of 1-casein A' (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 Schleswig-Holstein Saturated Fal Cholesterol Alcohol Carbohydrates Energy P-A Rcl IHD sc.

Schleswig Holstein 1.00 1.00 1.00 1.00 1.00 1.0 Niedersachsen 0.97 0.96 1.00 0.98 0.99 0.92 0.88 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 Rheinland-Pfalz 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 Baden Wurtenburg 0.93 1.02 1.02 1.05 1.03 0.50 0.72 Bayern 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. The 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 the major product fragments into which they are split by proteolytic action of a variety of enzymes, is illustrated by the P-caseins in Table 2.

-12.

Tal h -casein afl f-C A 3 O3-casein B I O-CN O-casein A 2 P-CN CA-P J3-casein D 3 O-CN D-P iO -casein B f3-CN B-51? 7,-casein AC f-CN AC-IPf1? 29 7,-casein AD P-CN A-lP~f92 -casein AE O-CN A-IP~t92 y,-caseii B' f-CN A- IP(f29-209) y2-casein A 2 O-CN A' (f1 06-209) y2-casein A 3 O-CN A 3 (f106-209) y2-casein B P-CN B (f],06-209) y3-caseifl A B-CN A (f 108R-209)~ source of fragment source of fragment J3-CN f3-CN A 2 -51? J3-CN A 3 f3-CN f3-CN A'1-5P or O3-CN A-P O-CN A 3 f3-CN 13-CN A'-5P, 1-CN A 2 -5P or 13-CN A 3 f3-CN B y3-casein B O-CN B (fl 08-209) In addition there are a numbe r of proteose peone compnn nrt.

W N Eigel Nomenclature of Proteins of Cow's Milk: Fifth Revis .ion 1. Dairy Science 67:1599-1631, (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 A 2 allele.

f3-casein A' differs from A 2 in containing the following additional. amino acids proline 34 and histadine 6 5 The corresponding A' allele is a relatively recent modification. However some animals are homozygous, that is their proteins are of one type only; in the case of j 3 -caseins either A, .A 3 or B, C, D or E.

Bovine milk is an important source of proteins and other nutrients required by humans and the common domestic cattle species such as the Holstein have greater quantit ies of the A' allele than any other O-casein. allele. Approximately 84 percent of the present American dairy herd is estimated to carry this allele.

In the graph shown in Figure I the consumption of f3-casein A' (and its derived proteolysis products) are plotted against the incidence of ischaemic heart disease based on FAO'Food -13- 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 63 °C for 20-30 minutes, known as Holder Pasteurisation, is set out together with the corresponding rate of CHD.

Table 3.

Population group_

U.K

Edinburgh Glasgow Dundee Aberdeen Lanarkshire (excluding Glasgow) County of Sutherland County of

.T

CHD rates following the Introduction of Holder Pasteurisation

I

rolder intro. Angina pectons(AP1) Cerebral embolism Ad Ih~ vear mortn omill r millru LuuM oUU iJc AP I AP2 AP3 A% CET 1 CET 2 CET 3 A% 1923 1924 1924 1926 1935 1947 1952 1925 1924 1925 1926 1937 1948 1954 67 56 42 91 188 685 1185 92 91 64 135 375 1185 1523 37.3a 62.5a 52.4a 48.4a 9 9 .5b 73.0 28.5 1924 1924 1925 1927 1938 1948 1954 174 77 162 121 153 298 518 236 101 188 227 193 518 680 35.6 31.2 16.0 87.6 26.1 73.8 31.3 1954 1954 963 1710 77.9 Bute 1 London Admin.

County 1 .956 1956 1610 2848 76.9 1954 610 823 34.9 1956 955 1398 46.4 1926 90 120 33.3 41.6 925 1925 31 .1 Norway Oslo Average increase 1922 1922 3 112 261.3' 81.8 43 1333.3 d not available Columns API and CETI denote the year of commencement of the sudden rise in the appropriate mortality.

Columns AP2 and CET2 denote the appropriate average mortality for the 4 years immediately preceding the year of introduction of pasteurisation.

Columns AP3 and CET3 denote the appropriate average mortality for the 4 years immediately succeeding the introduction of pasteurisation.

A% represents average increase.

S Possibly low because deaths ascribed to "coronary thrombosis" were not included in International List No. 89 in Scotland until 1931.

b Possibly enhanced as deaths ascribed to "coronary thrombosis" were now included in International List No. 94.

S Possibly enhanced because 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.

d Mortality ascribed to the following group of classifications: angina pectoris. infarctus cordis, sclerosis art. 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 stable. At 60 °C it has been demonstrated to have a relatively high rate of conversion of 14caseins, 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 A' 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'/pcasein 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 Shave 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 0-casein A' on immunosuppression in humans, its reduction in the rat's diet reduced cancer formation by a factor of four due to a dose specific 2 effect on the rat's immune system.

The preferred forms of this invention comprises the elimination from milk of P-casein A' 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 the removal of P-casein A' 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.

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.

Example 1 In its preferred form the treatment consists of inoculating a milk producing animal, that does not possess the P-casein A! allele, preferably one that is homozygous for the P-casein

A

2 allele, preferably one that produces commercially feasible quantities of milk, such as a cow, sheep, goat, or zebu with P-casein or its proteolysis products, or fragments thereof, produced in any other manner, so that antigens to the foreign 0-casein A' 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

A'

content has been reduced, using techniques known to those skilled in the Art, to counteract the presence of the immune suppressing 3-casein A' 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 product if a powder is required as is described in the existing Art.

Alternatively, immunoglobulins and other antigens, are recovered from non. -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 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 -16product with all the claims as described in the prior Art. This invention has the advantage over the existing Art that immunosuppressant proteins resulting from the presence of, or, derived from the P-casein A' 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 A' 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 P-casein A' allele, or for the presence of 3-casein A' in milk, and then selectively culling those cows returning a positive result, until the bulk milk produced by the herd is substantially free of 0-casein Alternatively, homozygous cattle containing the p-casein A 2 allele can be selectively bred so that the p-casein A' allele is eliminated from the herd.

An alternative approach to remove P-casein A' from bulk milk would involve separating cattle from existing herds which contain the P-casein A' allele, allowing the remainder of the herd (which are free of the P-casein A" allele) to be used for the production of bulk milk or other dairy products, and those cattle containing the A' 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 a useful food product capable of increasing the health of an individual, or the health of a population. In one aspect the invention provides a method of enhancing the immune response of an individual or a population who or which derives some of his/her/its food intake from milk or other diary products, by reducing or substantially eliminating the presence of P-casein A' in the diet of that individual or that population.

17- In a particularly preferred form, 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 p-casein A' within the diet of that population.

ADVANTAGES

By reducing or substantially eliminating the presence of p-casein A' 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 of p-casein 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 diary products containing P-casein A'.

VARIATIONS

Recognising that dairy products free of P-casein A' are desirable it is preferable to ensure that the animal from which the product is derived has been tested for the presence of the -casein A' allele or p casein A' expressed therefrom and subsequent selective breeding programmes (selecting for P-casein A' negative animals) carried out to eliminate the presence of the 3casein A' from the herd. It will be recognised that such testing may be carried out in a number of ways without departing from the scope of the present invention.

Without departing from the scope of this invention an alternative approach to remove the 3casein A' allele from a herd may be carried out. Such an approach may include the screening of sperm to be used for the purpose of artificial insemination for the presence or. absence of the P-casein A' allele and selecting against those sperm which contain this allele.

In addition to the methods of removing P-casein A' (or the P-casein A' allele), from meat products, milk and "processed dairy products" that have been disclosed herein it would be within the scope of this invention to use a number of alternative methods. Such alternative methods may involve the removal of P-casein A' from milk products via ultrafiltration -18techniques or by utilising a non-toxic chemical or enzymatic process to remove or inactivate 3casein A'.

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.

Claims

1. A method of identifying a bovine cow capable of producing milk suitable for Suse in the treatment or prevention of coronary heart disease, which milk is substantially c 5 free of P-casein A' but which contains any one or more of 3-caseins A 2 A 3 B, C, D and E, the method comprising the step of: testing the cow for the presence of the P-casein A' allele. 00 CA 2. A method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially c free of P-casein A' but which contains any one or more of p-caseins A 2 A 3 B, C, D Sand E, the method comprising the step of: testing genetic material of the cow for the presence of DNA or RNA encoding p-casein A.

3. A method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially free of p-casein A' but which contains p-casein A 2 the method comprising the step of: testing the cow for the presence of the P-casein A' allele and the P-casein A 2 allele.

4. A method of identifying a bovine cow capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially free of P-casein A' but which contains P-casein A 2 the method comprising the step of: testing genetic material of the cow for the presence of DNA or RNA encoding p-casein A' and DNA or RNA encoding P-casein A 2 A method of forming a herd of bovine cows which are capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially free of P-casein A' but which contains any one or more of P- caseins A 2 A B, C, D and E, the method comprising the steps of: testing cows for the presence of the p-casein A' allele; and (ii) selecting cows which do not have the p-casein A' allele to form the herd.

6. A method of forming a herd of bovine cows which are capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially free of P-casein A' but which contains any one or more of P- caseins A 2 A B, C, D and E, the method comprising the steps of: testing genetic material of cows for the presence of DNA or RNA encoding p-casein and (ii) selecting cows which do not have DNA or RNA encoding P-casein A' to form the herd. N: \MeIbOne Cses\ Patent\ 7 1000-71999\p72R6, AU 2\S;ecis\P7 1286 AU.2 amenc.nents doc 1303/OR 00 -20- 20

7. A method of forming a herd of bovine cows which are capable of producing milk suitable for use in the treatment or prevention of coronary heart disease, which G milk is substantially free of P-casein A' but which contains P-casein A 2 the method c 5 comprising the steps of: testing cows for the presence of the p-casein A' allele and the p-casein A 2 allele; and 00 (ii) selecting cows which have the p-casein A 2 allele but do not have the 3- Scasein A' allele to form the herd.

8. A method of forming a herd of bovine cows which are capable of producing O milk suitable for use in the treatment or prevention of coronary heart disease, which milk is substantially free of p-casein A' but which contains B-casein A 2 the method comprising the steps of: testing genetic material of cows for the presence of DNA or RNA encoding 0-casein A' and DNA or RNA encoding P-casein A 2 and (ii) selecting cows which have DNA or RNA encoding p-casein A 2 but do not have DNA or RNA encoding p-casein A' to form the herd.

9. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of P-casein A' but which contains any one or more of P-caseins A 2 A 3 B, C, D and E, the method comprising the steps of: testing cows for the presence of the P-casein A allele; (ii) selecting cows which do not have the -casein A' allele; and (iii) milking the selected cows. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of p-casein A' but which contains any one or more of p-caseins A A 3 B, C, D and E, the method comprising the steps of: testing genetic material of cows for the presence of DNA or RNA encoding P-casein A'; (ii) selecting cows which do not have DNA or RNA encoding P-casein A'; and (iii) milking the selected cows. S1. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of P-casein A' but which contains p-casein A 2 the method comprising the steps of: testing cows for the presence of the p-casein A' allele and the p-casein A 2 allele: N. \Melbourne \C-s\PacenC\7 100-7 191), P712q6 LI \Spe-\P712A6 ALI 2 -e-lien,-sdIoc 13/O3/OF 00 -21- O (ii) selecting cows which have the p-casein A allele but do not have the P- C, casein A allele; and t (iii) milking the selected cows. c 5 12. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of p-casein A' but which contains P-casein A 2 the method comprising the steps of: 00 testing genetic material of cows for the presence of DNA or RNA encoding p-casein Al and DNA or RNA encoding p-casein A 2 \O (ii) selecting cows which have DNA or RNA encoding P-casein A 2 but do not have DNA or RNA encoding P-casein A and S(iii) milking the selected cows.

13. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of p-casein A but which contains any one or more of 3-caseins A 2 A B, C, D and E, the method comprising the steps of: breeding one or more bovine bulls that do not have the P-casein A' allele with bovine cows that do not have the P-casein A' allele to give progeny cows which do not have the P-casein A' allele; and (ii) milking the progeny cows.

14. A method of producing milk suitable for use in the treatment or prevention of coronary heart disease from one or more bovine cows, which milk is substantially free of p-casein A' but which contains any one or more of P-caseins A2, A, B, C, D and E, the method comprising the steps of: breeding one or more bovine bulls that do not have DNA encoding for i- casein A' with bovine cows that do not have DNA encoding for P-casein A' to give progeny cows which do not have DNA encoding P-casein and (ii) milking the progeny cows. A method as claimed in claim 13, comprising the step of testing DNA or RNA from cells obtained from the one or more bovine bulls for the presence of the p-casein A' allele.

16. A method as claimed in claim 14, comprising the step of testing DNA or RNA from cells obtained from the one or more bovine bulls for the presence of DNA or RNA encoding P-casein A'.

17. A method as claimed in claim 13, comprising the step of testing DNA or RNA from cells obtained from each of the bovine cows for the presence of the p-casein A' allele. N \Me!bourne\Cases\Patent \710001999\P71286AU, 2\Specis\P71286AU 2 amendments cc 13/03/Ot r 00 -22- O O

18. A method as claimed in claim 14, comprising the step of testing DNA or RNA t from cells obtained from each of the bovine cows for the presence of DNA or RNA encoding P-casein A'.

19. A method as claimed in claim 13, comprising the step of testing DNA or RNA from cells obtained from the one or more bovine bulls for the presence of the P-casein 00 A 2 allele and the p-casein A' allele. NO O 10 20. A method as claimed in claim 14, comprising the step of testing DNA or RNA Lfrom cells obtained from the one or more bovine bulls for the presence of DNA or RNA O encoding P-casein A 2 and DNA or RNA encoding P-casein A'.

21. A method as claimed in claim 13, comprising the step of testing DNA or RNA from cells obtained from the one or more bovine cows for the presence of the p-casein A 2 allele and the p-casein A' allele.

22. A method as claimed in claim 14, comprising the step of testing DNA or RNA from cells obtained from the one or more bovine cows for the presence of DNA or RNA encoding P-casein A 2 and DNA or RNA encoding P-casein A.

23. A method as claimed in any one of claims 13 to 22, comprising the step of testing milk obtained from each of the bovine cows for the presence of P-casein A'.

24. A method as claimed in any one of claims 2,4, 6, 8, 10, 12, 15, 16, 19 and where the genetic material, DNA or RNA obtained from the one or more bovine bulls is obtained from semen, blood, hair, or skin of the one or more bovine bulls. A method as claimed in any one of claims 2, 4, 6, 8, 10, 12, 17, 18, 21 and 22, where the genetic material, DNA or RNA obtained from each of the bovine cows is obtained from blood, hair, or skin of the bovine cows.

26. A method as claimed in any one of claims 13 to 25. where the breeding of the one or more bovine bulls with the bovine cows is by natural insemination or is by artificial insemination.

27. A method as claimed in any one of claims 1 to 26, where the one or more bovine cows are, or include, Bos taurus bovines.

28. A method as claimed in any one of claims 13 to 26, where the one or more bovine bulls are, or include, Bos taurus bovines. N: \r~e I bure\C-es\ ter) t \7 1000-7 .1999\P712R6 AU. 2\Sp 12.q6 AUl 2 amendencs.doc 13/03,OR 00 -23- O O 29. A method as claimed in any one of claims 1 to 28, where the P-casein contained in the milk produced comprises P-casein A 2 in an amount greater than c by weight of the P-caseins in the milk. c 5 30. A method as claimed in claim 13, where the P-casein contained in the milk produced comprises P-casein A2 in an amount approximately 100% by weight of the P- caseins in the milk. 00

31. Milk containing P-casein but free of P-casein A which is the product of a method as claimed in any one of claims 9 to S32. A product which contains or is processed from milk as claimed in claim 31.

33. A product as claimed in claim 32 which is a food product.

34. The use of milk in the treatment or prevention of coronary heart disease, which milk is substantially free of P-casein A' and has been obtained from a bovine cow identified according to the method of any one of claims 1 to 4, obtained from one or more cows from a herd formed according to the method of any one of claims 5 to 8, or obtained according to the method of any one of claims 9 to 26. A method of treatment of prevention of coronary heart disease, comprising a) reducing or substantially eliminating the presence of p-casein A' in the diet of a subject suffering from or at risk of developing coronary heart disease, or b) replacing milk or milk products in the diet of the subject with milk which is substantially free of P-casein A' and which has been obtained from a bovine cow identified according to the method of any one of claims I to 4, obtained from one or more cows from a herd formed according to the method of any one of claims 5 to 8, or obtained according to the method of any one of claims 9 to 26.

36. A method as claimed in any one of claims I to 14, milk as claimed in claim 31, a milk product as claimed in claim 32 or claim 33, and a use as claimed in claim 34, substantially as herein described with reference to any of the examples. N:\tli-lbourne\Cases\Patent\71000-71999\P?12 AU 2\Spcis\P71286 AU.2 imendmeris.<soc 13.03 08