Patent Application: US-36967903-A

Abstract:
a milk or other dairy product , capable of minimizing the onset of disease such as coronary heart disease or enhancing the immune response is derived from animals which are substantially free of the β - casein a 1 allele . bulk milk can be produced by testing for and culling cows who test positive for the β - casein a 1 allele , or by producing immunoglobulins and other immune response proteins , in cow &# 39 ; s milk from animals not possessing the β - casein a 1 allele , or other commercial milk producing animals , to this allele , to counteract the immnunosuppressant 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:
this invention is applicable to milk , and all products processed from that milk , which milk is substantially free of β - casein a 1 . as used herein , the term “ treatment ” in relation to coronary heart disease means at least a reduction in the risk of a coronary heart disease event occurring in a human . the terms “ treat ” and “ treating ” have equivalent meanings . coronary heart disease means any disease or disorder relating to the coronary heart system and includes atherosclerosis and ischaemic heart disease . the term “ bos taurus ” refers to any cow whose pedigree from its three prior generations is 50 % or more of bos taurus origin . the term “ β - casein a 1 allele ” is a term used with reference to one of the variant forms of the β - casein gene . expression of the a 1 allele results in the production of the β - casein a 1 protein . where reference is made to the presence of the β - casein a 1 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 β - casein a 1 , it encompasses phenotypes resulting from either a homozygous or heterozygous state with respect to the β - casein a 1 allele . an example of an animal which is heterozygous for β - casein is a β - casein a 1 a 2 bovine . some animals are homozygous , for example bovines that are a 1 a 1 for β - casein and those that are a 2 a 2 for β - casein . a β - casein a 2 a 2 bovine is capable of producing only the β - casein a 2 protein . genetic variation within a species is due at least in part to differences in the dna sequence . while there are relatively few such differences in relation to the number of dna bases or the size of the genome , they can have a major impact as is evident in the genetic heterogeneity of the human and bovine populations . for example , in bovines , a mutation in the dna sequence coding for the β - casein protein at nucleotide position 200 has resulted in the replacement of a cytidine base with an adenine base . thus , the triplet codon affected by this change codes for histidine ( cat ) rather than for proline ( cct ) at amino acid position 67 of the protein . thus , the histidine at position 67 results in the cow producing β - casein a 1 while the proline results in the cow producing β - casein a 2 ( note : the preceding discussion assumes that the ancestral bovid expressed β - casein a 2 and that there are no other dna variations at other positions on the dna sequence ). the term “ substantially ” as used in the expression “ substantially free of β - casein a 1 ” reflects that it cannot be said with 100 % certainty that a sample of milk is absolutely free of β - casein a 1 . on rare occasions , and despite all efforts to ensure that a herd is β - casein a 1 free , an animal capable of producing β - casein a 1 in its milk could present itself in the herd because of a genetic mutation or because of human error . herds are formed by the genotype testing of animals and then selecting the desired individuals . all such testing is subject to human error . the phrase “ substantially free of β - casein a 1 ” is therefore intended to account for this . without the word “ substantially ”, the phrase would be unduly limiting . as used herein , the term “ ldl ” means low density lipoprotein , “ hdl ” means high density lipoprotein , and “ vldl ” means very low density lipoprotein . as used herein , the term “ triglyceride ” means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules . as used herein , the term “ cardiovascular disease ” is any disease of the blood vessels of the circulation system caused by , facilitated by or shown to be linked to abnormally high concentrations of lipids in the vessels . as used herein the term “ arteriosclerosis ” is a degeneration of the walls of the arteries due to the formation of foam cells and aortic streaks which narrow the arteries . this limits blood circulation and predisposes an individual to thrombosis . as used herein , the term “ atherosclerosis ” is a disease of the arteries in which fatty plaques develop on the inner walls , with eventual obstruction of blood flow . as used herein , the term “ apolipoprotein b ” or “ apoprotein b ” or “ apo b ” refers to a specific protein component of plasma lipoproteins involved in lipid and cholesterol transport , most notably ldl and vldl . cholesterol synthesised de novo is transported from the liver and intestine to peripheral tissues in the form of lipoproteins . most of the apolipoprotein b is secreted into the circulatory system associated with vldl . as used herein , the term “ hypercholesterolemia ” means elevated levels of circulating total cholesterol , ldl - cholesterol and vldl - cholesterol as per the guidelines of the expert panel report of the national cholesterol educational program ( ncep ) of detection , evaluation of treatment of high cholesterol in adults ( arch . int . med . ( 1988 ) 148 , 36 – 39 ). as used herein , the term “ hyperlipidemia ” or “ hyperlipemia ” is a condition where the blood lipid parameters are elevated in the blood . this condition manifests an abnormally high concentration of fats . the lipids fractions in the circulating blood are , total cholesterol , ldls , vldls , and triglycerides . as used herein , the term “ lipoprotein ”, such as in vldl , ldl and hdl , refers to a group of proteins found in the serum , plasma and lymph which are important for lipid transport . the chemical composition of each lipoprotein differs in that the hdl has a higher proportion of protein virsus lipid , whereas the vldl has a lower proportion of protein versus lipid . as used herein , the term “ xanthomatosis ” is a disease evidence by a yellowish swelling or plaques in the skin resulting from deposits of fat . the presence of xanthomas are usually accompanied by raised blood cholesterol levels . as used herein , the term “ gras ” means “ generally regarded as safe ” with respect to food additives . the products processed from milk that form part of this invention are derived from a source of bulk milk ( i . e . milk from more than one animal ) and include , but are not limited to : ( a ) bulk milk ( b ) bulk milk used to make cheese whether or not the milk has been pasteurised , sterilised or otherwise treated to reduce the the population of microbes prior to cheese making , ( c ) milk powders , ( d ) milk solids , ( e ) caseins , caseinates , and casein hydrolysates , ( f ) pasteurised , sterilised , preserved milks including microfiltered milks , uht milks , ( 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 cheeses , ( l ) milk whey , ( m ) food products enriched through the addition of milk products such as soups , ( n ) milk from which potentially allergenic molecules have been removed , ( o ) confections such as chocolate , ( p ) carbonated milk products , including 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 , and ( s ) buttermilk and buttermilk powder . it has been reported that certain human population groups exhibit a relatively low incidence of coronary heart disease and certain other diseases , notwithstanding the fact that they consume considerable quantities of milk and milk proteins . these people include the tibetans , rural gambians , and the masai and samburu people of kenya . the inventor has identified the fact that a major difference between these population groups and other similar population groups is that the milk consumed by the above people is derived from bos indicus bovines ( e . g . the zebu breed ) and from the yak ( bos grunniens ). such milk does not contain β - casein a 1 . in addition , a comparative study in denmark of the causes of morbidity in the greenland eskimo population and the predominant danes , shows very large relative differences that are suggestive of differences in life - style risk factors . one notable difference is that the danes are large consumers of dairy products whereas the eskimos are not . the differences in morbidity are illustrated in table 1 below . a further comparison has been carried out using data from the states of the former west germany . in this case , the coronary heart disease death rates have been found to correlate strongly with the relative regional average consumption of β - casein a 1 ( table 2 ). in this instance , the composition of the individual state dairy herds remained virtually constant from the 1950 &# 39 ; s through to the 1980 &# 39 ; s . the data show a remarkable relationship between the relative incidence of ischaemic heart disease and the relative average consumption of β - casein a 1 across the 8 states . this is in marked contrast to the relatively poor relationships between the incidence of ischaemic heart disease and the recognised listed dietary risk factors . a regression relationship between ischaemic heart disease and fat intake was conducted and was shown to be not significant ( p & lt ; 0 . 0684 , r 2 = 0 . 20 ). however , the regression between ischaemic heart disease and the intake of β - casein a 1 was highly significant ( p & lt ; 0 . 0001 , r 2 = 0 . 71 ). the regression relationships are : the multiple regression relationship was then generated . in this case , the inclusion of both fat intake and β - casein a 1 intake did not improve the relationship over that with β - casein a 1 alone . the regression relationship is : the analyses of the relationships between various dietary factors and ischaemic heart disease outlined in this document indicate the potential importance of the β - casein variant as a risk factor for heart disease . the difference between the two casein variants is only one amino acid . this suggests that the products of proteolysis of these variants may be linked to the identified risk factor . some indication of the number , and the major product fragments into which they are split by proteolytic action of a variety of enzymes , is illustrated for the β - caseins in table 3 . bovine milk is an important source of proteins and other nutrients required by humans . a high proportion of the common domestic cattle breeds , such as the holstein , express the β - casein a 1 allele . for example , it is estimated that in the late 1980s more than 70 % of the californian dairy herd carried the a 1 allele . as noted previously , the β - casein a 1 variant is of particular interest and therefore , considering its contribution to milk consumed by the human population in many parts of the world , the proteolysis products of β - casein a 1 are of particular interest . in the graph shown in fig1 , the incidence of ischaemic heart disease is plotted against the estimated average consumption of β - casein a 1 ( and its derived proteolysis products ). fig1 shows a very strong correlation between the consumption of β - casein a 1 and death rate from ischaemic heart disease . in contrast , the correlation with the consumption of dairy protein ( fig2 ) is much lower . neither saturated fat consumption ( fig3 ) nor the consumption of red meat ( fig4 ) show the strong correlation which the inventor has identified in relation to the consumption of β - casein a 1 , both between countries and within countries . the single amino acid difference between the two predominant β - casein variants has highlighted the potential role of a difference in the proteolysis products from different β - casein variants as potential risk factors for coronary heart disease . therefore , the potential impact of pasteurisation is of interest , as prolonged heating is a factor that is known to influence proteolysis and the monomer to micelle ratio which is known to affect digestion of β - casein . in particular , this relates to the more severe forms of heat treatment that were used in the early years of pasteurisation ( e . g . holder pasteurisation which heats milk to 63 ° c . and holds it for 20 – 30 minutes ). hence the impact of the introduction of holder pasteurisation on the death rate from coronary heart disease is of interest . the inventor has examined the available data and the results of the analyses are presented in table 4 . the analyses reveal a very marked and sudden increase in the death rate from coronary heart disease in the four years after the introduction of holder pasteurisation . such data would suggest the presence of a novel risk factor associated with pasteurisation . it is the inventor &# 39 ; s contention that this risk factor may be associated with a derivative of β - casein a 1 ( for example , a proteolysis product ). it is possible , however , that a specific fragment or fragments of β - casein a 1 affect the body &# 39 ; s immune system as a result of their immunosuppressant properties . by reducing or substantially eliminating the presence of β - casein a 1 in the diet of an individual , it is believed that its immune response may be enhanced , or immunosuppression reduced , thereby improving the general well - being of the individual . it is believed that some individuals may be particularly susceptible to the presence of β - casein a 1 , and it may be possible to develop a test for such susceptible individuals , and to recommend that they reduce or eliminate the consumption of milk or other dairy products containing β - casein a 1 . in humans , low density lipoprotein ( ldl ) oxidation is considered to be a primary step in the evolution of artherosclerotic damage ( steinberg et . al ., 1989 ). analysis of protein oxidation products isolated from atherosclerotic lesions implicates the tyrosyl radical ( a reactive nitrogen species ) and hypochlorous acid in ldl oxidation ( heinecke et . al ., 1999 ). in addition , it has been found ( torreilles and guerin , 1995 ) that β - casomorphin - 7 ( and truncated forms , e . g . β - casomorphins 5 and 6 ) could promote peroxidase - dependent oxidation of human ldls . the reaction is independent of free metal ions but requires casein - derived peptides with tyrosyl end residues . this implies that the tyrosyl ending peptide is a diffusable catalyst that conveys oxidising potential from the active site of the heme enzyme to ldl lipids . casomorphin - 7 is a potential source of a tyrosyl radical and has been shown to cause ldl peroxidation . casomorphin - 7 is produced from β - casein a 1 , but not from β - casein a 2 ( jinsmaa et . al ., 1999 ). hypercholesterolemia is a condition with elevated levels of circulating total cholesterol , ( low density lipoprotein ) ldl - cholesterol and ( very low density lipoprotein ) vldl - cholesterol . in particular , high levels of ldl and vldl are positively associated with coronary arteriosclerosis while high levels of hdl are negative risk factors . the role of ldl oxidation has gained much attention in the literature . it is well documented that modified ldl , or more specifically oxidised ldl , has an increased ability to bind endothelial and smooth muscle cell walls thus promoting further injury by continued oxidation within the lumen of arteries . furthermore , oxidised ldl is known to invoke a range of physiological responses directly involved in the atherosclerotic process . these responses include the stimulation of the secretion of mediators of inflammatory response ( e . g . interleukin - 1 , interleukin - 6 , tumour necrosis factor alpha and macrophage colony stimulating factor ) which are chemo - attractants for macrophages which recognise the oxidised ldl within the subendothelial space and actively engulf them , subsequently turning into foam cells . the foam cells then aggregate to form fatty streaks . such fatty streaks are the first identifiable characteristic lesion of advanced atherosclerosis . hyperlipidemia also predisposes one to coronary heart disease , as well as to cancer and obesity . hyperlipidemia is one of the high risk factors useful in the early diagnosis of these life threatening diseases . hyperlipidemia is a condition where the blood lipid parameters are elevated . the lipids fractions in the circulating blood are total cholesterol , ldl , vldl , and triglycerides . safe levels of these according to the american heart association guidelines are represented below . active treatment by diet modifications and drugs are necessary to reduce the risk of fatality when the levels are abnormal . as with hypercholesterolemia , hyperlipidemia results from diet , heredity , lifestyle , environment , familial diseases , or stress . the condition may be inherited or may be secondary to another disorder , such as systemic lupus erythematosus ( sle ), hypothyroidism , nephrotic syndrome , cushing &# 39 ; s syndrome , diabetes mellitus , obesity , alcoholism , corticosteroid therapy or estrogen therapy . hypercholesterolemia and hyperlipidemia are also major causes of atherosclerosis . atherosclerosis is responsible for more deaths in the u . s . than any other single condition . atherosclerotic heart disease involving the coronary arteries is the most common single cause of death , accounting for one third of all deaths . atherosclerotic interference with blood supply to the brain ( causing stroke ) is the third most common cause of death ( after cancer ). atherosclerosis also causes a great deal of serious illness by reducing the blood flow in other major arteries , such as those to the kidneys , the legs and the intestines . atherosclerosis is a cardiovascular condition that occurs as a result of narrowing down , or stenosis , of the arterial walls . the narrowing is due to the formation of plaques ( raised patches ) or streaks in the inner lining of the arteries . these plaques consist of oxidised - ldl , monocytes , macrophages , foam cells , damaged muscle cells , fibrous tissue , clumps of blood platelets , cholesterol , and sometimes calcium . they tend to form in regions of turbulent blood flow and are found most often in people with high concentrations of cholesterol in the bloodstream . the number and thickness of plaques increases with age , causing loss of the smooth lining of the blood vessels and encouraging the formation of thrombi ( blood clots ). it is not uncommon for fragments of thrombi to break off and form emboli , which travel through the bloodstream and block smaller vessels ( e . g . coronary arteries ) leading to ischaemic damage of tissue , or ischaemic heart disease . medication is not a satisfactory treatment for ischaemic heart disease because much of the damage to the artery walls has already been done . anticoagulant drugs have been used to try to minimise secondary clotting and embolus formation , but these have little or no effect on the progress of the disease . vasodilator drugs are used to provide symptom relief , but are of no curative value . surgical treatment is available for certain high - risk situations . balloon angioplasty can open up narrowed vessels and promote an unproved blood supply . the blood supply to the heart muscle , following coronary vessel blockage or damage , can also be restored through a vein graft bypass . large atheromas and calcified arterial obstructions can be removed by endarterectomy , and entire segments of diseased peripheral vessels can be replaced by woven plastic tube grafts . in some instances the reduction of hypercholesterolemia can be achieved by modification of the diet and / or use of drugs thereby minimizing the risk of fatality from the disease . reduction of serum cholesterol in humans has been achieved by consumption of dietary plant fibre and other effective components of foods . however , there remains a need for a safe and effective treatment for the above conditions . the inventors have surprisingly found that consumption of β - casein a 2 reduces serum cholesterol levels , reduces circulating triglyceride levels , reduces ldl concentrations and thus ldl : hdl ratios , reduces serum levels of apolipoprotein b and is atheroprotective . these effects observed by the inventors mean that the consumption of β - casein a 2 is beneficial for the prevention or treatment of a variety of diseases or disorders including hypercholesterolemia , hyperlipidemia , and atherosclerosis . the finding is supported by experimental studies where ten groups of nz white rabbits were fed ad libitum with controlled diets ( see example 3 ). recognising that dairy products free from β - casein a 1 are desirable , it is preferable to ensure that the animal from which the product is derived has been tested for the presence of the β - casein a 1 allele . subsequent separation of the bovines into separate herds and / or selective breeding programmes ( selecting for β - casein a 1 negative animals ) can be carried out to eliminate the presence of the β - casein a 1 from the herd . while the subject of u . s . patent application ser . no . 09 / 906 , 807 is the selection of bovine cows for milking based on genotyping of the cows for the presence of dna or rna encoding β - casein a 1 , or other β - caseins , this invention is directed to the breeding of cows with bulls to give progeny cows that produce milk free of the β - casein a 1 protein . if both parents of a cow are known to have no dna encoding for β - casein a 1 , then the cow will not have the ability to produce β - casein a 1 in its milk . determination of whether the parents have dna encoding for β - casein a 1 may be by a variety of methods . one method is by genotyping a bull and breeding it with a cow known to have no dna encoding for β - casein a 1 . an alternative method is by genotyping a cow and breeding it with a bull known to have no dna encoding for β - casein a 1 . a preferred method of the invention is the genotyping of a bovine bull to establish that it has no dna encoding for β - casein a 1 and then breeding that bull with bovine cows known ( by genotyping or any other method ) to have no dna encoding for β - casein a 1 . the progeny cows can then milked to give milk that is free of β - casein a 1 . preferably the milk contains only β - casein a 2 of the possible β - casein variants . the breeding may be by natural or artificial insemination . artificial insemination is anticipated to be the prevalent method . any known method for the genotyping of bovines may be used . such methods can be specific for dna or rna encoding either β - casein a 1 or β - casein a 2 . however , general methods which do not specifically test for dna or rna encoding β - casein a 1 , but additionally test for dna or rna encoding other β - caseins , may also be used to form a herd of bovines which do not produce β - casein a 1 or produce only β - casein a 2 of the possible β - casein variants in their milk . for the avoidance of any doubt , any reference to dna in the methodology of this invention is intended to include cdna ( which is dna derived from rna ). for example , it is known that β - casein a 1 has histidine at position 67 of the protein whereas β - casein a 2 has proline at the same position . this is due to the presence of an adenine nucleotide at position 200 of the β - casein dna . this produces the triplet codon that specifies histidine ( cat ) rather than proline ( cct ). a test which identifies the codon that will specify histidine at position 67 of the β - casein protein can therefore be used to exclude bovines which produce β - casein a 1 in their milk . similarly , a test which identifies the codon that will specify proline at position 67 of the β - casein protein can therefore be used to select bovines which produce β - casein a 2 ( or β - caseins a 3 , d or e ) in their milk . while a test for animals that are homozygous for the presence of cct ( that codes for proline ) at codon 67 of an animal &# 39 ; s β - casein gene does not unequivically show whether or not the animal is homozygous for the β - casein a 2 allele , the test can show that an animal does not possess any of the alleles for β - casein a 1 , b and c . such a test does not need to be any more specific because culling animals negative for the test will mean the elimination of β - casein a 1 producing animals from the herd . it is also known that β - caseins b , c and f , in addition to β - casein a 1 , have histidine at position 67 . also , β - caseins a 3 , d and e , in addition to β - casein a 2 , have proline at position 67 . therefore , a test which distinguishes between the codons that specify proline and histidine at position 67 will also distinguish between β - caseins a 1 , b , c and f on the one hand and β - caseins a 2 , a 3 , d and e on the other hand . for example , while a test for the presence of cat ( histidine ) or absence of cct ( proline ) in one or other or both of an animal &# 39 ; s alleles at codon 67 of its β - casein gene does not unequivocally show whether or not the animal contains the β - casein a 1 allele , the test can show that an animal may contain one or more of the alleles for β - casein a 1 , b , c and f . such a test does not need to be any more specific because culling animals positive for the test ( i . e . absence of the proline codon in at least one allele ) will mean the elimination of β - casein a 1 producing animals from the herd . a dna or rna test which gives positive identification for animals homozygous for cct ( proline ) at codon 67 can therefore be used to ascertain whether a particular bovine does not possess a β - casein a 1 allele , whether homozygous or heterozygous . thus , bovines which do possess the cct ( proline ) at codon 67 at one or both alleles can therefore be culled from a herd to give a herd which is free of the β - casein a 1 allele . milk obtained from that herd therefore cannot contain β - casein a 1 . where it is known that the genetic makeup of the herd is such that the only possible alleles possessed by the individuals are for β - caseins a 1 and a 2 , the culling from the herd of those bovines positive for histidine at position 67 gives a herd where each individual is homozygous for the β - casein a 2 allele . such a herd will produce milk possessing only β - casein a 2 . the determination of whether the genotype at codon 67 of the β - casein gene is cct ( proline ) or cat ( histidine ) can be made by many different methods that are available and which could be used to assay for this single nucleotide polymorphism ( snp ). the methods include dna sequencing , sscp ( single stranded conformation polymorphism ), allele specific amplification , and assays designed using proprietary chemistries such as taqman ™ ( pe biosystems ), invader ™ ( third wave technologies ), snapshot ™ ( pe biosystems ), pyrosequencing ™ ( pyrosequencing ab ), sniper ™ ( pharmacia ), and dna chips ( hybridisation or primer extension chips ). the preferred method should have the ability to function well with rapidly extracted impure dna . high test throughput (& gt ; 1000 of samples per day ) at low cost is desirable . since the preferred objective is to identify bovines that are homozygous for the β - casein a 2 allele , the unequivocal positive identification of animals homozygous for cct at codon 67 is preferred , rather than simply the absence of a result in a test for the alternative cat codon . two examples of practical methods for the large scale genotyping of bovines are : a manual acrs ( amplification created restriction site ) method which can be conducted easily in any molecular genetics laboratory and requires no specialist equipment or devices . the method can be easily scaled up to analyse hundreds of samples per day . a highly automated method such as the sequenom ™ primer extension and mass spectrometry system which is capable of analysing thousands of samples per day . the aim of the acrs method is to create an amplicon in which only one allele of an snp will form a restriction site . the restriction site is created by site directed mutagenesis in the amplification step . a dde1 restriction site can be created when the nucleotides ct are present at nucleotide 200 and 201 ( positions 2 and 3 of codon 67 ) of the β - casein gene . this would positively identify the presence of the cct ( proline ) codon . in example 1 below , the 3 ′ section of the casein dde2 primer has a mismatch at its penultimate nucleotide ( fig5 ). this is important as it creates a dde1 restriction site in the a 2 amplicon only ( shown in italics in fig5 ). in fig5 , codon 67 in each template is in bold lowercase . the template is reversed to present the primer in the usual 5 ′– 3 ′ orientation . the mismatch base is underlined . variations of the test could include modification of the sequence of the 5 ′ end of the casein dde2 primer or 5 ′ extension of the casein dde2 primer with a nucleotide sequence homologous to the β - casein template or 5 ′ extension of the casein dde2 primer with nucleotides which are not homologous to the β - casein sequence . the second primer for the acrs is less critical and many compatible primers could be used . the primer known as casein4 5 ′- ccttctttccaggatgaactccagg - 3 ′ ( seq id no : 2 ) has been found to be the most effective . pcr amplification with this pair of primers produces a 121 base pair fragment in all β - casein alleles . however , the definitive diagnostic step is that only alleles with ct at positions 200 and 201 ( i . e . specifying amino acid 67 of the β - casein ) can be cut with the restriction enzyme dde1 . this produces distinctive 86 - and 35 - base pair fragments . the first step of the primer extension method is pcr amplification of the region of the β - casein gene containing codon 67 . in example 2 below , a 319 bp fragment ( shown in fig7 ), was amplified . in fig7 , the primer regions are shown underlined . alternate bases of the snp are shown bracketed . the post pcr product is cleaned with a sap reaction to remove unincorporated dntps . an extension primer complementary to the bold itallicised sequence is added to the cleaned product along with an extension mixture containing dda , ddc , ddt and dg . the following size extension products are obtained : if codon 67 of β - casein is cat , a 20 bp , 6209 . 10 dalton product is obtained , whereas if the sequence is cct , a 23 bp , 7205 . 70 dalton product is obtained . these products can be clearly distinguished and separated from possible contaminants by maldi - tof mass spectrometry . the results of the genotype testing obtained from either method are then used to select bovines positively identified as having cct ( proline ) at position 67 at both alleles . such bovines cannot produce β - casein a 1 in their milk . the selected bovines are kept in a separate herd and are milked separately . ideally the milk from that separate herd is kept separate from other milk which may contain β - casein a 1 . the selected bovines may be uniquely identified ( e . g . alternatives include ear - tagging with a unique tag , or use of an electronic tag or use of a specific tag that identifies the bovine as being free of the β - casein a 1 allele or branding for future identification ). the selected bovines are milked to give milk free of β - casein a 1 . preferably , the milk is phenotype tested to confirm that the milk is substantially free of β - casein a 1 . a bulk quantity of milk from the selected bovines may then be processed into one or more milk products , such as fresh milk , cheeses , yoghurts , milk powders etc . with the objective of investigating any positive effects of the consumption of β - casein a 2 , rather than the positive effects of avoiding β - casein a 1 , rabbit feeding studies were undertaken and are described in example 3 . the results were found to be consistent with the epidemiology studies described above and , further , indicate that the consumption of β - casein a 2 is beneficial . in rabbit groups 3 – 8 , three concentrations of β - casein a 1 and a 2 ( 5 %, 10 % and 20 %) in the diet were tested , with whey protein added such that a total of 20 % milk protein was present in each diet ( i . e . 15 %, 10 %, 0 % added whey protein respectively ). cholesterol ( 0 . 5 %) was also added to these diets to ensure measurable lesions . as controls , group 1 rabbits were fed 20 % whey protein and group 2 rabbits were fed 20 % whey protein plus 0 . 5 % cholesterol . groups 9 and 10 were fed 10 % β - casein a 1 ( or a 2 ) plus 10 % whey only . the rabbits on the specially prepared diet ate only an average of 30 . 05 ± 0 . 97 grams per day . not surprisingly , almost all rabbits lost weight over the 6 week experimental period , with a 5 . 62 % average weight loss . this did not affect their overall health and the rabbits were alert , responsive and showed no signs of distress . this is consistent with an earlier study which found that , with rabbits fed a semisynthetic diet containing casein , the feed intake and weight gain was reduced by half as the diet was not readily accepted , although the rabbits still appeared healthy . rabbits placed on a high level of dietary casein ( 50 %) failed to gain weight during the course of that study . 10 % β - casein a 2 with no added cholesterol ( group 10 ) produced a significantly lower serum cholesterol level at t = 6 weeks than all other groups including whey only ( group 1 ) and 10 % β - casein a 1 ( group 9 ). the β - casein a 1 group had serum cholesterol levels higher than the whey only group . this highlights the necessity to examine the effect of the specific β - caseins rather than mixtures , and is the first time that the differential effects of β - casein variants on serum cholesterol levels have been described . the mean serum cholesterol level in rabbits fed 20 % β - casein a 1 alone ( group 9 ) was higher than those in group 1 ( whey only ) animals . thus , β - casein a 1 may be slightly more atherogenic than whey protein . adding 0 . 5 % cholesterol to all diets increased the serum cholesterol levels at least 3 - fold , which was much greater than the effect of a 1 β - casein compared with whey alone . there was no significant difference between the serum cholesterol levels of rabbits fed β - casein a 1 or a 2 in the presence of added dietary cholesterol . the level of serum ldl in group 10 ( β - casein a 2 ) was significantly lower than that in groups 9 ( β - casein a 1 ) or 1 ( whey only ), all with no dietary cholesterol . the level of ldl for group 9 was higher than that for group 1 , but not significantly . in the presence of added dietary cholesterol , serum ldl was significantly elevated in all groups ( 2 – 8 ) and these were not significantly different from each other . a similar pattern occurred for serum hdl except that group 10 was not significantly lower than group 1 . the anti - atherogenic potential of β - casein a 2 was further highlighted by the ratios of ldl to hdl . there was a much greater difference in serum ldl levels between groups 9 ( β - casein a 1 ) and 10 ( β - casein a 2 ) than in serum hdl levels . thus the ldl to hdl ratios were significantly less for β - casein a 2 fed animals than for β - casein a 1 fed animals . indeed , the ldl to hdl ratio of group 10 was significantly lower than all other groups , including group 1 ( 20 % whey alone ). there was no significant difference between all groups for triglycerides or homocysteine . thus , dietary β - casein a 2 , in the absence of a cholesterol - enriched diet , leads to lower serum cholesterol levels ( total , ldl and hdl ) than both β - casein a 1 and whey . it was found that both β - casein a 1 and a 2 led to fatty streaks compared with 20 % whey , but β - casein a 2 produced less extensive lesions than β - casein a 1 . indeed , 10 % β - casein a 1 without dietary cholesterol produced about the same amount of lesions as 10 % β - casein a 2 with added dietary cholesterol . the thickness of fatty streaks in the aortic arch of rabbits fed β - casein a 2 , either with or without dietary cholesterol , was zero or else very close to zero . in contrast , the thickness of aortic arch fatty streaks in rabbits fed β - casein a 1 was significantly higher . thus , β - casein a 1 is more atherogenic than both whey and the a 2 casein variant . in the presence of 0 . 5 % dietary cholesterol , only 20 % β - casein a 2 produced a smaller surface area of aorta covered by fatty streaks than 20 % whey . however , the thickness of the fatty streaks in the aortic arch was significantly smaller for both 20 % and 5 % □- casein a 2 - fed animals compared with whey ( 0 . 03 ± 0 . 015 ), with a low ( but not significant ) value for 10 % β - casein a 2 fed animals ( 0 . 01 ± 0 . 008 ). balloon injury of arteries combined with a cholesterol - enriched diet is known to produce lesions in rabbits that closely resemble advanced human atheroma . there was no significant difference in intima to media ratio of the balloon injured right carotid artery of rabbits fed β - casein a 1 or a 2 either with or without dietary cholesterol . however , groups fed β - casein a 2 ( both with and without added cholesterol ) had smaller neointimal thickenings than their β - casein a 1 fed counterparts . animals fed β - casein a 2 generally had slightly thinner intimas than those fed whey ( both with and without dietary cholesterol ). these results , combined with the fact that 10 % a 2 with no added cholesterol produced a significantly lower serum cholesterol level than all other groups including whey only and 10 % β - casein a 1 , demonstrate for the first time that β - casein a 2 has an atheroprotective effect while β - casein a 1 is atherogenic . the invention is further described with reference to the following examples . however , it is to be appreciated that the invention is not limited to the examples . at least 10 hairs were pulled from the end of the tail switch of a cow so that the hook - shaped follicles were retained on the end of the removed hairs . this was achieved easily by pulling the tail hairs upward while holding the rest of the switch down . if the tail has been docked , longer hairs from the end of the docked tail or other locations on the body may be substituted . tail hairs are preferred . five hair follicles from one cow were cut into a sterile 1 . 5 ml microfuge tube . solution a ( 200 μl ) was added to the tube and the tube placed in a boiling water bath for 15 minutes . the tube was removed and solution b ( 200 μl ) added followed by mixing . solution a ( 200 mm naoh ) solution b ( 100 mm tris - hcl , ph 8 . 5 with an extra 200 mm hcl )— prepared by combining 1 m tris - hcl , ph 8 . 5 ( 10 ml ) with conc . hcl ( 1 . 67 ml ) and making up to 100 ml with distilled water . crude dna extract ( 1 . 5 μl ) from hair follicles ( prepared as above ) or dna ( 20 – 50 ng ) ( prepared by another method ) was transferred to a well of a 96 - well pcr plate . pcr cocktail ( 20 μl ) was added to the well . the well was overlayed with mineral oil and centrifuged briefly to remove air bubbles . pcr was carried out on an mj research ptc200 ( hot bonnet ) using the following protocol : following pcr , restriction enzyme cocktail ( 10 μl ) was added and the mixture incubated at 37 ° c . overnight . the restriction enzyme cocktail was prepared according to the following : the amplification product ( 10 μl ) was analysed by electrophoresis ( 80v , 1 hour ) in ethidium bromide stained agarose gel ( 3 %, 1 × tbe ). fig6 shows the results of 20 samples analysed by the procedure outlined above . a size standard ladder was loaded in position 0 . the 100 bp band is identified in fig6 . the negative control ( no dna ) was loaded in position 20 . samples homozygous for ct at positions 2 and 3 of codon 67 of the β - casein gene result in a single 86 bp band when cut by dde1 . this is shown in load positions 1 , 2 , 10 , 11 , 12 , 13 , 14 and 17 . samples not containing ct at positions 2 and 3 of codon 67 of the β - casein gene are not cut by dde1 , leaving a single 121 bp band . this is shown in load positions 4 , 5 , 7 and 9 . heterozygous samples result in both cut ( 86 bp ) and uncut ( 121 bp ) bands . this is shown in load positions 3 , 6 , 8 , 15 , 16 , 18 and 19 . because of heteroduplex formation , the uncut band ( 121 bp ) is expected to be more intense than the cut band ( 86 bp ). dna extracts from hair follicles were prepared using the method described in example 1 . alternatively , genomic dna isolated by other methods can be used at a concentration at about 2 . 5 ng / μl . a dna sample ( 1 μl ) from each of 96 animals was placed into a 96 well pcr microtitre plate ( or alternatively , from each of 384 animals into a 384 well pcr plate ). for the 96 well plate , a cocktail of the following reagents was prepared in a 1 . 5 ml microtube . the cocktail ( 4 μl ) was added to each well in the plate with a repeating pipette . the following sap solution was prepared in a 1 . 5 ml microtube : the solution was mixed well and centrifuged for ten seconds at 5000 rpm . sap solution ( 2 μl ) was transferred to each well of the plate containing the samples . the plate was incubated using a thermocycler at 37 ° c . for 20 minutes , 85 ° c . for 5 minutes , and then holding at 4 ° c . the following extension cocktail was prepared in a 1 . 5 μl microtube : the extension cocktail ( 2 μl ) was added to each well of the sample plate . the plate was sealed and thermocycled as follows : prior to mass spectrometry the samples were cleaned using spectroclean and then analysed using maldi - tof ms . the profiles obtained for homozygous and heterozygous animals for the cct and cat snps are shown in fig8 . the location of the primer , analyte a and analyte c extension products are shown . sixty new zealand white / lop cross rabbits of both sexes ( 16 – 24 weeks old ) were obtained from the university of queensland central animal breeding house . at time = 0 , the right carotid artery of all rabbits was balloon de - endothelialised . the rabbits were then randomly divided into 10 groups and fed the specified diets for 6 weeks . blood samples were taken at t = 0 , 3 and 6 weeks for analysis of serum constituents . at t = 6 weeks , all animals were sacrificed and tissue samples taken . a summary of the study protocol is shown in table 1 . at time = 0 , all 60 rabbits were anaesthetised with a mixture of ketamine ( 30 mg / kg ) and xylazine ( 7 mg / kg ) i . m . and maintained on halothane ( laser animal health , qld , australia ). an incision was made through skin and fascia , and blunt dissection and incision through the stylohyoid muscle exposed the upper part of the right common carotid artery . the left , uninjured common carotid artery served as a contralateral control . side branches were temporarily ligated with 5 / 0 silk ( davis & amp ; geck , danbury , conn ., usa ) and a 2 french ( 2f ) fogarty embolectomy catheter ( baxter healthcare corporation , irvine , calif ., usa ) was introduced through an arteriotomy made in the superior thyroid artery , a branch of the external carotid artery ( fig2 . 2 ). the catheter was advanced 10 cm ( to the aortic arch ), the balloon inflated with 0 . 2 ml of saline and the catheter withdrawn in order to deendothelialise the artery . the injury procedure was performed three times in each animal to ensure complete removal of the endothelium . after injury , the catheter was removed , the superior thyroid artery ligated with 5 / 0 silk and the wound closed using 2 / 0 dexon braided sutures ( sherwood medical , st louis , mo ., usa ). a broad - spectrum antibiotic ( 5 mg / kg baytril , bayer australia ltd , pymble , australia ) containing 50 mg / ml enrofloxacin was administered sub - cutaneously at the completion of the procedure . the rabbits were monitored post operatively and returned to their cages when fully conscious . for three days post - surgery each animal was gavaged with approximately 170 mg of paracetamol ( smithkline beecham international , ermington , australia ) and subcutaneous administration of antibiotics was continued . the study involved 10 diets , the compositions of which are shown in table 2 . essentially , each diet contained 20 % total milk protein , but differed in the proportion of casein protein to whey protein . diets of groups 1 , 9 and 10 contained no further additives , while diets of groups 2 , 3 , 4 , 5 , 6 , 7 and 8 were supplemented with 0 . 5 % cholesterol . there were 6 rabbits in each group and the diet was continued for 6 weeks . it was found that it took several days for the rabbits to become accustomed to their new diets , which differed from their normal pellets in colour , smell , texture and weight . pellets fed in the new diets were white , while normal pellets are brown . thus , for five days prior to surgery , all rabbits were weaned onto their respective diets by mixing increasing amounts of the new pellets ( 25 %, 50 %, 75 %, 100 %) with normal rabbit pellets ( to total 200 grams ). at t = 0 , only new pellets ( 200 grams per day ) were offered ( exceptions , see below ) for the following 6 weeks and water was available ad libitum . it is known that rabbits eat 90 – 130 grams of pellets per day . however , 200 grams was required to fill the hopper such that the animals could reach the food . a fresh 200 grams of pellets was provided daily and the amount of pellets left uneaten by each rabbit was determined by weighing each day . several measures were employed to encourage the rabbits to eat the new diet . firstly , a small amount of aniseed oil was dropped onto the back of the food hoppers to stimulate the rabbits &# 39 ; appetites . secondly , if a rabbit was still not eating its new diet or its weight loss was approaching 10 % of original body weight , normal food was added to the diet at 25 , 50 or 75 % mixtures . when this occurred , the white diet pellets were carefully separated from the brown normal pellets and weighed each day to determine the actual amount of experimental diet consumed . finally , if the weight loss continued , the rabbits were offered 200 grams of normal food per day until their body weight increased . all rabbits were weighed weekly , commencing the day of surgery , until the week of sacrifice . blood was collected from each rabbit prior to surgery , at the commencement of diet and at t = 3 and 6 weeks . each rabbit was wrapped securely and the fur covering the central ear artery was shaved . a 24 gauge catheter ( becton diskinson pty ltd , nsw , australia ) was inserted and 5 ml of blood was collected into sterile tubes . the blood was allowed to clot and was centrifuged at 4500 rpm for 10 minutes . the serum supernatant was extracted and analysed for total serum cholesterol , triglycerides , hdl , ldl and homocysteine . six weeks after injury , 1000 i . u . heparin ( david bull laboratories , mulgrave , victoria ) in 1 ml saline were injected into an ear vein of each rabbit . five minutes later , the rabbits were euthanased with an overdose of pentobarbitone ( 325 mg / ml lethabarb , ( virbac australia pty ltd , victoria )). an incision was made from the chin to pelvis and continued to the hindlimbs and forelimbs . the peritoneal cavity was then carefully incised to expose internal organs and the rib cage was cut away to expose the heart . the left ventricle was cannulated with a wide gauge needle attached to tubing which was in turn connected to a perfusion apparatus . a small section of the inferior vena cava was cleared and severed . the circulatory system was flushed with 1 litre of dulbecco &# 39 ; s phosphate buffered saline ( dpbs ) before arterial perfusion fixation was performed with 1 liter of 4 % formaldehyde ( asia pacific speciality chemicals ltd , nsw , australia ) in dpbs at 100 mm hg . the entire aorta , from the iliac bifurcation to the aortic arch , was exposed and removed . the right ( injured ) and the left ( control ) common carotid arteries were fully exposed and removed . all vessels were cleaned of fat and adherent connective tissue before being placed into tubes with 4 % formaldehyde in dpbs . the aortic arch was removed for fixation and histological sectioning . the remainder of the aorta ( approximately 16 cm ) was opened longitudinally and cut into four segments . each segment was sewn ( lumen side up ) onto small pieces of clear plastic to ensure they remained flat . the segments were then stained en face in a saturated solution of the lipophilic dye oil red o to enable determination of luminal surface area covered by fatty streaks . images of each aortic segment were scanned using desk scan ii ( version 2 . 31a , hewlett - packard , u . s . a .) and the total lesion area was measured using the imagej imaging software ( version 1 . 23y , national institute of health , u . s . a .). this program compares the intensity of the lesions ( stained bright red ) against the rest of the arterial surface ( white to pale pink ). the results were expressed as the percentage of surface area covered by lesions . three segments of approximately 3 – 4 mm in length were cut from each aortic arch and placed in dpbs for two washes of 30 minutes each . the segments were then dehydrated in increasing concentrations of ethanol , i . e . 70 % ethanol for 30 minutes , then 95 % ethanol for 30 minutes and finally , 100 % ethanol for three washes of 30 minutes each . they were then placed in the first of two infiltration resin solutions ( technovit 7100 , heraeus , germany ) and left on a rotator overnight . the segments were then transferred to the second resin solution and were again left on a rotator overnight . each segment was then carefully placed upright into embedding moulds , held in place by a minimal amount of superglue at the base of the mould . the same resin mixture used for infiltration was mixed with a hardener and this was pipetted into the mould until all segments were completely covered . the plastic was left to polymerise at room temperature for two hours before being placed into a 37 ° c . oven overnight . the plastic blocks were then gently removed from their moulds and mounted onto histoblocks , using technovit 3040 ( heraeus , germany ) as glue . each block was securely fitted to the microtome ( lkb bromma , germany ) and trimmed by cutting approximately 60 – 70 sections of 10 μm each . these were discarded and the microtome was set to cut 5 μm sections . these sections were collected with forceps and stretched in distilled water held at room temperature . they were then collected onto glass slides and dried for approximately 15 minutes at 60 ° c . the sections were then stained for 10 minutes with toluidine blue before being washed four times with tap water and allowed to air dry . four and five segments , each approximately 3 – 4 mm in length , were cut from the left ( control ) and the right ( ballooned ) carotid arteries respectively . a different number of sections was used in the control and the experimental vessels , to avoid confusion between tissues during embedding and sectioning . the segments were dehydrated , embedded , sectioned and stained using the method previously described for the aortic arch segments . all morphometric analyses of the intima to media ratios ( i : m ) of the ballooned and control carotid arteries and the aortic arch were perfomed using the mocha image analysis system ( version 1 . 1 , jandel scientific , ca ., u . s . a .). measurements were taken to determine the size ( in pixels ) of both the intima and media layers in all segments of each artery . using an excel ( microsoft corporation , n . y ., u . s . a .) spreadsheet , the ratio of intima to media was calculated and recorded for each of the three aortic arch segments per rabbit , the four control carotid segments and the five ballooned carotid segments . all statistical analyses were performed using the sigmastat ( jandel scientific , ca ., u . s . a .) statistical software package . comparison of data from the morphometric analyses were carried out with one - way multiple anovas , using the “ kruskal - wallis ” analysis of variance on ranks test . extreme outliers were identified using tukey &# 39 ; s outlier test . in all statistical analyses a p value of less than 0 . 05 was considered significant . the information has been presented graphically using microsoft excel ( microsoft corporation , n . y ., u . s . a .). all data has been reported as mean ± standard error of mean . rabbits were weaned onto their respective new diets for a few days prior to t = 0 , and some animals whose weight loss approached 10 % had their diet supplemented with normal pellets for a few days . thus it was essential to measure the amount of new pellets eaten by each animal per day by providing a known weight ( 200 grams ) of fresh pellets each morning and weighing the uneaten portion the following morning immediately prior to offering 200 grams of fresh pellets . where a mixture of new pellets ( white ) and normal pellets ( brown ) was offered , the two were separated prior to weighing . the weight of the white pellets eaten by each rabbit varied between rabbits . on group averages , the weight of pellets consumed only varied by 12 grams per day over the six week experimental period and ranged from 23 . 2 grams to 35 . 3 grams per day ( mean 30 . 48 ± 0 . 97 grams ). there was no significant difference in the amount of diet pellets eaten between any of the groups . the addition of 0 . 5 % cholesterol to the diets of groups 2 – 8 did not influence the amount eaten , nor did the type or amount of casein . thus , while the amount of food consumed by the rabbits was relatively low ( mean 30 . 48 grams white pellets per day , mean 10 . 22 grams brown pellets per day , total mean 40 . 72 grams per day ) resulting in some weight loss , the amount of the new diet consumed in each group was relatively constant and thus results between groups can be compared . almost all rabbits lost weight over 6 weeks , with only group 9 ( 10 % a 1 without added cholesterol ) recording an average weight gain of 2 . 15 ± 1 . 2 %. group 10 rabbits ( 10 % a 2 without added cholesterol ) showed only a small weight loss of − 0 . 92 ± 0 . 97 %. group 1 rabbits on 20 % whey only ( no added cholesterol ) lost 4 . 5 ± 6 % of their original body weight over the 6 week study , while the remaining groups 2 – 8 ( all with added cholesterol ) lost mean 7 . 57 ± 4 . 72 % of their original body weight . the greatest mean weight loss was by group 6 . however this was entirely due to one rabbit ( k6 ) that had a large starting weight of 3 . 81 kg . this rabbit ate an average of 21 . 58 grams of pellets per day , approximately 9 grams per day less than the mean of all rabbits ( 30 . 50 ). k6 showed no sign of illness and at the time of sacrifice and all organs appeared normal with the exception of a very small liver of normal colour . the remaining five rabbits in this group lost less than 5 % of their original body weight . there was no apparent relationship between proportion or type of casein on weight change . at week 6 cholesterol levels of almost all groups were significantly different from group 1 ( 20 % whey only ). interestingly , the cholesterol level in group 9 ( a 1 ) was higher than in group 1 , but this was not significant . also , the cholesterol level in group 8 ( 20 % a 2 with cholesterol ) was approximately half of that produced from 20 % whey with added cholesterol ( group 2 ), although this too was not significant . group 9 serum cholesterol was significantly lower than all groups that had dietary cholesterol ( groups 2 – 8 ). group 10 ( a 2 ) serum cholesterol was significantly lower than in group 1 , and indeed was significantly lower than that in all groups , including group 9 . the results indicate that addition to 0 . 5 % cholesterol to the diet has caused an increase in serum cholesterol levels irrespective of the amount of whey in the diet and both the amounts and types of casein . also , in the absence of dietary cholesterol , a 2 produced lower serum cholesterol levels than both a 1 and whey . by week 6 triglyceride levels from groups 5 ( 20 % a 1 ) and 9 ( 10 % a 1 without added cholesterol ) had increased from week 0 and groups 4 ( 10 % a 1 ) and 10 ( 10 % a 2 without added cholesterol ) had shown no change . the serum triglyceride levels for all other groups had fallen from those at week 0 . in all groups with added dietary cholesterol , serum ldl levels were significantly higher than group 1 ( whey alone ) or groups 9 ( a 1 ) and 10 ( a 2 ) with no dietary cholesterol . group 9 ( a 1 with no dietary cholesterol ) was not significantly different from group 1 , but group 10 ( a 2 with no dietary cholesterol ) was significantly lower . also group 10 ldl levels were significantly lower than those for group 9 . all groups with added dietary cholesterol ( groups 2 – 8 ) were not significantly different from each other . thus , serum ldl levels followed the same pattern as serum cholesterol and indicate that addition of dietary cholesterol has increased serum ldl levels in all appropriate groups , while the a 2 casein variant in the absence of dietary cholesterol , produced lower serum ldl than either a 1 or whey alone . serum hdl followed the same pattern as serum ldl with added dietary cholesterol ( groups 2 – 8 ) causing significantly elevated levels than those with no added cholesterol ( groups 1 , 9 and 10 ). group 9 was not significantly different from group 1 , but was significantly higher than group 10 , which in turn was significantly lower than all groups except group 1 . β - casein a 2 without dietary cholesterol produced a lowered serum hdl level than a 1 . the ldl to hdl ratios were determined by dividing the individual rabbit &# 39 ; s serum ldl levels by the same rabbit &# 39 ; s serum hdl levels . the mean ldl : hdl for each group was then determined . it was found that the ldl : hdl of group 10 ( 10 % β - casein a 2 alone ) was significantly lower than all other groups including whey alone and β - casein a 1 alone . the ldl to hdl ratios of all groups with dietary cholesterol ( groups 2 – 8 ) were not significantly different from each other . as with the serum ldl and hdl levels , there was a trend for higher levels with higher doses of β - casein a 1 and lower levels with higher doses of β - casein a 2 . by week 6 , serum homocysteine levels had changed only slightly from those at week 0 with no significant difference demonstrated between the experimental groups . group 1 rabbits fed 20 % whey had 0 % aortic surface area covered by plaque . all other groups had some plaque , including those from group 10 , although this was not significantly higher than group 1 . only groups 1 and 10 were significantly lower than group 3 , which was also considerably higher than groups 2 – 9 , however , variation between rabbits within this group was great , with a range between 2 . 8 and 18 . 4 %. three rabbits were excluded from these calculations as they were deemed extreme outliers . group 1 thoracic aorta had 0 % of the surface area covered by fatty streaks . all other groups had some lesions , including groups 9 ( a 1 ) and 10 ( a 2 ) with no dietary cholesterol . groups 9 and 10 were not significantly different from each other . for both a 1 and a 2 with dietary cholesterol , there was a trend to lower percent lesions with increasing concentration of casein variant , but this was not significant . group 3 was considerably higher than all others , but not significantly different . of the six rabbits in this group , three had a high percentage of surface area covered by plaque and three had low values . the abdominal aorta isolated from animals fed a 2 in the absence of dietary cholesterol ( group 10 ) had only minimal surface area covered by plaque and was not significantly different from group 1 ( whey only ) which had 0 % covered by plaque . group 10 was significantly lower than all other groups including group 9 ( a 1 , no dietary cholesterol ). group 3 ( 5 % a 1 with dietary cholesterol ) was significantly higher than group 2 with 20 % whey and dietary cholesterol . thus , both casein variants without dietary cholesterol produce more extensive fatty streaks than whey alone but a 2 is less atherogenic than a 1 . in addition , a 1 is more atherogenic than a 2 in the presence of dietary cholesterol . with 20 % whey alone ( group 1 ) there was no intimal thickening of the aortic arch . similarly , in the presence of 10 % a 2 with no dietary cholesterol ( group 10 ) or with dietary cholesterol and 5 % or 20 % a 2 ( groups 6 and 8 ), the mean intima to media ratio was 0 . group 7 ( 10 % a 2 with dietary cholesterol ) had a mean intima to media ratio of only 0 . 01 , with four of the six rabbits having 0 %. all a 1 groups ( both with and without dietary cholesterol ) had higher intima to media ratios than groups fed a 2 casein and had intima to media ratios not significantly different from group 2 ( 20 % whey with 0 . 5 % cholesterol ). indeed , 10 % a 1 in the absence of dietary cholesterol produced the same intima to media ratio as 20 % whey with 0 . 5 % whey with 0 . 5 % dietary cholesterol ( 0 . 03 ). thus , in relation to the thickness of fatty streak lesions in the aortic arch , a 1 casein , even in the absence of dietary cholesterol , produces lesions similar to that produced by 0 . 5 % cholesterol . in contrast a 2 , both in the presence and absence of dietary cholesterol , appears to be highly atheroprotective . balloon catheter injury to the right carotid artery t = 0 induced a hyperplastic neointimal thickening in all rabbits by 6 week . measurement of intima to media ratios showed there was a slight , but not significant increase in neointimal thickening in group 2 rabbits ( 20 % whey with 0 . 5 % cholesterol ) compared with group 1 ( 20 % whey alone ). likewise , the groups fed 5 , 10 and 20 % a 1 plus 0 . 5 % cholesterol had thicker , but not significantly different neointimas than group 1 . groups fed 5 , 10 and 20 % a 2 plus 0 . 5 % cholesterol all had thinner neointimas than their a 1 counterparts , but again this was not significant . similarly , in the absence of dietary cholesterol the intima to media ratio in a 2 fed animals ( group 10 ) was lower than in the a 1 - fed group 9 . there was no neointimal thickening observed in all of the control vessels . the invention provides a useful food product capable of increasing the health of an individual , or the health of a population . the invention relates to a method of preventing or treating coronary heart disease in a human population which derives some of its food intake from milk or other dairy products by reducing or substantially eliminating the presence of β - casein a 1 within the diet of that population . 1 . aleandri , r ., buttazzoni , l . g ., schneider , j . c ., caroli , a ., and davoli , r . 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