A1 and A2 Milk: Truth vs Hype

Milk is nutrient-rich, white liquid food produced by the mammary glands of mammals. Interspecies consumption of milk is not uncommon, particularly among humans, many of whom consume the milk of other mammals. Milk contains a significant source of energy, protein, and micronutrients such as calcium, magnesium, phosphorus and vitamin B 12. It positively associated with a variety of health conditions. However, in the past few years, bovine milk comes under the scrutiny of its possible role in some non-communicable disorders. Milk is divided into two groups A1 and A2. A1 milk produces BCM-7 on its digestion which is believed to be involved in Autism, Diabetes-type 1, Sudden death Syndrome in infants, Ulcerative colitis, Cardiac problems, Obesity, Arteriosclerosis, Intolerance bloating, Mental disorders (Parkinson, Schizophrenia) and other non-communicable disorders (Parashar et al., 2015).


India is the number one producer of milk in the world and most of the Indian cattle breeds are A2 in nature. It seems that A1 milk is not a major problem for India. Despite being the largest producer of milk in the world (187.7 Million Tonnes per annum-DAHD&F, 2019). Milk production is still not able to fulfill the growing demands. To increase the milk production government of India has taken several initiatives and one of the areas is the cross-breeding program in bovines. Crossbreds of bovine have definitely contributed to increasing the overall milk production, but at the same time, the A1 allele has also entered their genome, creating concerns of production of BCM-7 from their milk after human consumption.


History

Interest in the distinction between A1 and A2 beta-casein proteins began in the early 1990s via epidemiological research and animal studies initially conducted by scientists in New Zealand, which found correlations between the prevalence of milk with A1 beta-casein proteins in some countries and the prevalence of various chronic diseases. Cow milk is about 87 percent water and 13 percent solids, a combination of fat, carbohydrates in the form of lactose, minerals, and protein. About 30 to 35 percent of the casein is beta-casein, of which there are several varieties, determined by the genes of the cow. The most common milk protein beta-casein has 2 variants are A1 and A2 protein. Scientists believe the difference originated as a mutation that occurred between 5000 and 10,000 years ago as cattle were being taken north into Europe.


The sole difference is that one of the 209 amino acids that make up the beta-casein proteins, a proline, occurs at position 67 in the chain of amino acids that make up the A2 beta-casein, while in A1 beta-casein, a histidine occurs at that position (proline at position 67 was replaced by histidine). The mutation subsequently spreading widely throughout herds in the Western world through breeding(Parashar et al., 2015).


A1 and A2 milk

β-casein accounts for 24-28% of total milk protein. The molecular weight of β-casein is 24 kilo Dalton. It comprises of 209 amino acids. At least 12 variants of this protein are known. Each variant differs from other variants in terms of amino acid substitution at the fixed position. For example, β- casein A1 has histidine amino acid at 67th position while β- casein A2 has proline at same position.β-casein protein is coded by chromosome number 6 of the bovine genome. Histidine is coded by CAT (cytosineadenosine-thymine) bases whereas proline is coded by CCT (cytosine-cytosine-thymine); here due to mutation adenosine is replaced by cytosine. Molecular biology methods can determine this difference of single nucleotide in the β-casein gene and can identify individual animals whether they carry the gene for β-casein A1 or β-casein A2 or both. β-casein protein is co-dominantly expressed in the A1A2 genotype where allele A1 and A2 are equally expressed. β-casein A1 is the result of a genetic mutation in cattle, believed to have occurred about 5000 years ago in Anatolia, Turkey (Ng-Kwai-Hang et al., 2003).


Milking animals have been screened for β-casein A1 and β-casein A2 in many countries. There is a difference in the occurrence of β-casein A1 or β-casein A2 amongst species, breeds, and geographical locations. The results are denoted in terms of frequency of occurrence. The frequency for the A2 allele in Guernsey, Brown Swiss, Jersey, Holstein, Ayrshire, and Red Danish bovine breeds is about 96-98%, 66-70%, 50-63%, 44-53%,40-49%, and 23% respectively. There is a difference in the occurrence of β-casein A1 or β-casein A2 amongst species, breeds, and geographical locations. The A2 cow breeds include Sahiwal, Gir, Red Sindhi, Tharpakar, Rathi, Kankrej, Ongole, Hariana and others. The milk delivered by all the desi cows is of the A2 variety.

However, India went on to hybrid its native desi cows with the European species of Jerseys and Holstein Friesian which delivered A1 milk. In hydrolyzed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk.



(Woodford, 2009)

Betacasomorphin-7 (BCM-7)

β-casein A1 and A2 respond differently towards proteolytic degradation by enzymes of the human gut. This is possible because of one amino acid difference at the 67th position in β-casein. A1 β-casein has histidine at 67th position while A2 β-casein has proline. The difference in the amino acid at the 67th position results in differences in the susceptibility of a peptide bond between amino acids 66 and 67. The peptide bond between isoleucine and histidine (A1 milk) is cleaved by elastase, while the bond between isoleucine and proline (A2 milk) is not hydrolyzed.


The digested product contained a seven amino acid long peptide having sequence Tyr60-Pro61-Phe62-Pro63-Gly64- Pro65-Ile66 and is referred to as beta casomorphin-7 or popularly called BCM-7 which is not formed on the digestion of β-casein A2.BCM-7 can pass through the intestinal barrier in neonates as they have increased permeability for improved nutrient absorption. However intestinal permeability reduces as the age progresses. Also in adults with compromised digestive health or conditions such as celiac disease, stomach ulcers or autism have increased intestinal permeability which means that BCM-7 can enter the bloodstream more easily in adults also.


Biological effects of BCM-7


Effect of A1 milk consumption on human health

  • Ecological studies conducted in nineteen countries (U.K, Finland, Ireland, Sweden, Denmark, France, Germany, Iceland, Norway, Austria, Switzerland, USA, Japan, Israel, Australia, New Zealand, Hungary, Venezuela, and Canada) revealed that there was a strong relation between consumption of β- casein A1 and incidence of type 1 diabetes mellitus (DM1).

  • Ecological analysis conducted in 22 countries has also shown a close relationship between cardiovascular disease and consumption of A1 milk.

  • Epidemiological studies presented in one of the patent applications reveal that ‘statistically relevant’ correlation exists between β-casein A1 consumption and aggravation of neurological disorders such as autism and schizophrenia.

Role in neurological disorders

Opioid receptors are found in the brain and classified into three groups, μ (MOP), κ (KOP) and δ (DOP). MOP or μ receptor is found in the brain, immune cells, endocrine glands and to some extent in the intestine. From guinea pig ileum assay (GPI) it was known that bovine milk has an opioid effect and it acts most probably through the MOP receptor. Bovine BCM-7 has a much higher affinity for MOP receptors as compared to human milk BCM-7 (YPFVEPI). Also BCM-7 level in human milk reduces drastically after 2 to 4 months of delivery of a newborn, reducing the overall effect of opioid peptides such as human BCM-7. This kind of reduction in BCM-7 level occurs in bovine milk is still unknown.


Bovine milk proteins also produce opioid antagonists like casoxin A, B, and C during its digestion. But overall maximum theoretical yield (mg/g protein) of opioid agonist can be higher as compared to the opioid antagonist (Meisel et al., 2000). BCM-7 has also shown an increase in the expression of genes responsible for producing inflammatory enzymes such as myeloperoxidase that could further increase the expression of MOP receptors in the intestine. The overall process further enhances the action of this peptide. However, information regarding a similar type of action is not available for the brain. Both autism and schizophrenia are mental disorders and it is believed that the symptoms of these disorders can be reversed by changes in food habits.

PC: Authors


Patients suffering from autism and schizophrenia (leaky gut syndrome) have shown a large amount of BCM-7 in their urine. Such patients have shown dramatic improvements in their symptoms following a gluten-free and casein-free diet (GFCF). However, the diet failed to fully cure these disorders. It is believed that mercury toxicity inactivates cysteine amino acid present in the active site of the DPP (IV) enzyme which is required during the digestion of casein. In autistic and schizophrenic patients it has been seen that they not only have a high level of casomorphin in their blood samples but also have a high level of gliadorphin peptide (gliadorphin comes from gluten digestion) in their serum.This proves that DPP (IV) normal activity is very important for protein digestion (milk protein or wheat protein), and its impaired function could be involved in mental disorders. In neonates high level of mercury toxicity could arise through vaccination, mother’s dental amalgams or other environmental factors. Further, high intestinal permeability; lack of breastfeeding & immature BBB (blood-brain barrier) enhances the movement of BCM-7 from gut to the brain which shows its effect. Gluten and casein both are very similar peptide and their digestion process is also similar. By making a change in food habits, reduction in pollution can eliminate these disorders.


Role in type 1 diabetes

Type 1 diabetes is an autoimmune disorder in which beta cells of the pancreas are destroyed by the immune system of the host. This disorder mostly affects children and requires daily insulin injection to control glucose levels. The potential role of cows’ milk in diabetes is still debated and there is no consensus on the diabetogenicity of individual milk proteins.


It is believed that BCM-7 suppresses the body’s immune system that may enhance the survival of pathogens such as enteroviruses or bacteria (Mycobacterium avium). These pathogens are ultimately involved in the trigger of type1 diabetes-like symptoms (Dai, Y.D et al., 2009). The second theory says autoantibody generation against beta cells. According to this theory some of the peptide sequences of β-casein which are produced during digestion, mimic the sequence of GLUT-2 transporter (involved in glucose transportation in the cell). In this response, T cells recognize them as an antigen and activate B cells for the production of antibodies. Antibodies not only targets the digested segment of the β-casein product but also to insulin-producing beta cells, causing type 1 diabetes. This data is not conclusive in blaming A1 milk for diabetes. Also, it was clear from the above experiment that the genetic aspect of diabetes making some of the animals prone to diabetes than others at the same diet, wheat-containing diet is more diabetogenic as compared to milk protein (casein). Further soy protein can be good for health. A1 milk is not diabetogenic (Beales et al., 2002).


Role in cardiovascular disease (CVD)

It is believed that BCM-7 could be involved in the oxidation process of low-density lipoprotein (LDL). The oxidized form of LDL is engulfed by macrophages by specific receptors present on their surface. These macrophages ultimately converted into foam cells and start the process of atherosclerosis in heart. It is long before known that a high-fat diet can increase the oxidation process in LDL (Suzukawa et al. 1996). However, food containing fish (a rich source of omega-3 fatty acids) or bovine whey protein is believed to be inhibitors of oxidation of LDL. Lactoferrin of bovine whey protein prevents the accumulation of cholesterol in macrophages by oxidized LDL. Also, an omega-3 fatty acid with a supplement of vitamin E or C is very helpful in inhibiting the oxidation of LDL.


Tailford et al., 2003 published a paper where it was shown that A1 milk is more responsible in causing atherosclerosis as compared to A2 milk. In the rabbit model, an artificial injury was made in the carotid artery of animals and was fed on A1 and A2 milk diet respectively. After 6 weeks of the diet, it was found that rabbits fed on the A1 milk diet had thicker fatty streak on the injured area as compared to animals fed on A2 milk. However, data is not very conclusive as to the formation of foam cells in fatty streak during tissue repair and in the atherosclerosis process are two different events.

Methods for typing animals (A1/A2) and detection of BCM-7


It is difficult to ascertain the presence of the β-casein A1 or β-casein A2 variant in milk. Thus, efforts have been made to identify alleles in β-casein from DNA isolated from animals. DNA from blood, hair, skin and even milk somatic cell is isolated from bovine. Some of these methods have acquired commercial dimensions and even patented.


1.Allele-specific PCR (AS-PCR)

This method is based on this concept for the typing of animals uses DNA from bovine blood and finds single nucleotide polymorphisms (SNPs) and mutations (Keating et al., 2008).


2.Amplification created restriction site PCR (ACRS-PCR)

This method for typing animals using DNA from bovine blood using the concept of amplification created restriction site (ACRSPCR). It is quite similar to allele PCR (Raies et al., 2012).


3.Single strand conformation polymorphism PCR (SSCP-PCR)

This method for typing of animals using DNA from bovine blood. The method gives information about whether the bovine will produce A1 or A2 milk (Barroso et al., 1999).


4.Taq Man method

In this method dsDNA is isolated from somatic cells of bovine milk; ssDNA probe (18 bases long) is made specifically for A1 and A2 beta-casein gene that contains at 5’ end fluorescent agent and 3’ end a quencher (Manga et al., 2010)


5.Typing of animals based on the presence or absence of BCM-7

The method requires isolation of β-casein from milk, in vitro digestion by gastrointestinal enzymes and separation of generated peptides by the HPLC-MS method (De Noni, 2008).


6.The detection method of BCM-7 in urine/blood

RIA/ELISA methods are described for assaying BCM-7 in urine or plasma (Sokolov et al., 2014).


Conclusion

In the crossbreeding program, Indian bovine was crossed with an exotic European breed. Crossbreeding program has created several high yields producing variety but at the same time they are producing A1 milk which is a matter of concern. In the last 20 to 30 years several studies were carried on A1 milk and its effect on human health. But animal and human studies did not given substantial evidence of A1 milk for its role in these non-communicable disorders (diabetes, autism and others). That is why we are still consuming A1 milk and it is not banned. It seems that early consumption could have an indirect role in theses disorder along with other environmental factors and food habits. In All in all, controversy on A1 milk is an issue that needs to be addressed but its harmful effect has yet to be proved with scientific research. This requires more of animal trials and generation of data on human subjects to get any conclusion on it. The recent emergence of several A2 milk dairy industry players in india is an indication of consumers’ willingness to pay a premium for perceived better quality and safer milk. Instead of headlong A2 milk, the dairy industry players could make available to this premium segment by offering better value proposition such as high protein milk, lactose free milk, adulterant or antibiotic free milk, certified organic milk etc.


Authors

ICAR-Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India.

College of Veterinary and Animal Sciences-KVASU, Mannuthy-680651, Kerala, India. Madras Veterinary College-TANUVAS, Vepery-600 007, Tamilnadu, India.


References

Barroso, A., Dunner, S. and Cañón, J., 1999. use of PCR-single-strand conformation polymorphism analysis for detection of bovine β-casein variants A1, A2, A3, and B. Journal of animal science, 77(10), pp.2629-2632.

Beales, P., Elliott, R., Flohe, S., Hill, J., Kolb, H., Pozzilli, P., Wang, G.S., Wasmuth, H. and Scott, F., 2002. A multi-centre, blinded international trial of the effect of A 1 and A 2 β-casein variants on diabetes incidence in two rodent models of spontaneous type I diabetes. Diabetologia, 45(9), pp.1240-1246.

Dai, Y.D., Marrero, I.G., Gros, P., Zaghouani, H., Wicker, L.S. and Sercarz, E.E., 2009. Slc11a1 enhances the autoimmune diabetogenic T-cell response by altering processing and presentation of pancreatic islet antigens. Diabetes, 58(1), pp.156-164.

De Noni, I., 2008. Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas. Food Chemistry, 110(4), pp.897-903.

Keating, A.F., Smith, T.J., Ross, R.P. and Cairns, M.T., 2008. A note on the evaluation of a beta-casein variant in bovine breeds by allele-specific PCR and relevance to β-casomorphin. Irish Journal of Agricultural and Food Research, pp.99-104.

Manga, I. and Dvořák, J., 2010. TaqMan allelic discrimination assay for A1 and A2 alleles of the bovine CSN2 gene. Czech Journal of Animal Science, 55(8), pp.307-312.

Meisel, H. and Fitzgerald, R.J., 2000. Opioid peptides encrypted in intact milk protein sequences. British Journal of Nutrition, 84(S1), pp.27-31.

Ng-Kwai-Hang, K.F. and Grosclaude, F., 2003. Genetic polymorphism of milk proteins. In Advanced Dairy Chemistry—1 Proteins (pp. 739-816). Springer, Boston, MA.

Parashar, A. and Saini, R.K., 2015. A1 milk and its controversy-a review. International Journal of Bioassays, 4(12), pp.4611-4619.

Raies, H, R Kapila, UK Shandilya, AK Dang, S Kapila 2012. “Detection of A1 and A2 genetic variants of beta-casein in Indian crossbred cattle by PCR-ACRS.” MilchwissenschaftMilk Science International. 67.4 : 396-398.

Sokolov, O., Kost, N., Andreeva, O., Korneeva, E., Meshavkin, V., Tarakanova, Y., Dadayan, A., Zolotarev, Y., Grachev, S., Mikheeva, I. and Varlamov, O., 2014. Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity. Peptides, 56, pp.68-71.

Suzukawa, M., Abbey, M., Clifton, P. and Nestel, P.J., 1996. Enhanced capacity of n-3 fatty acid-enriched macrophages to oxidize low density lipoprotein mechanisms and effects of antioxidant vitamins. Atherosclerosis, 124(2), pp.157-169.

Tailford, K.A., Berry, C.L., Thomas, A.C. and Campbell, J.H., 2003. A casein variant in cow's milk is atherogenic. Atherosclerosis, 170(1), pp.13-19.

Woodford, K.B., 2009. Devil in the Milk: Illness, health and politics of A1 and A2 milk. Chelsea Green Publishing.

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