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Health implications, science behind A1 and A2 milk

Friday, 16 August, 2013, 08 : 00 AM [IST]

Laxmana Naik, Kiran Lata, Rajan Sharma, Y S Rajput and Abhishek

Introduction
Milk has been known as nature’s single most complete food. In addition to supplying healthy nutrients, inclusion of milk in the diet has been positively associated with a variety of health attributes.

However, in spite of many health benefits, the consumption of cow’s milk has come under scrutiny for its reported links to the risk of chronic diseases. In the last few decades, some health risk factors associated with cow milk protein; specifically ß-casein has been a major discussion. Digestion of this specific type of casein has been found to produce an opioid peptide, which can be very problematic for humans as well as animals.

In cow milk protein, there are two entirely natural genetic variants of ß-caseins; called A1 and A2 types and the respective milk is referred as A1 or A2 milk. These two milk proteins differ by a single amino acid in their polypeptide chain. A1 milk has histidine at 67th position and A2 milk has proline at same position. It is assumed that, after ingestion of A1 type milk, a hydrolysed peptide releases in the digestive system. This peptide exhibits a strong opioid activity, shown to promote heart disease, affects regulation of insulin formation, causes allergic responses, and affects the human immune response; this is also linked to type-1 diabetes and many other health risk factors. In contrast A2 milk has no such side-effects, assumed that, it is safe for consumption and regarded as good type of milk.

Cardiac link
In 1900s, scientists believed that introduction of pasteurisation, homogenisation and other processing techniques; led to the increased incidence of cardiovascular disease (CVD) and other health problems (McLachan, 2001).

Subsequently during the last three decades, increased use of bovine growth hormones and antibiotics for commercial dairy farming, was pinpointed at by consumers, as major health risk (Forman, 2004). Changing selection targets in the last few decades has resulted in changes in bovine breed composition in most European countries (EFSA Scientific Report, 2009). It is likely that these changes in breed composition have had an impact on milk composition, including protein variants.

In the mid of 1970s, Australian dairy herds switched from using Jersey cows to Holsteins, at that time few dairy farmers families noticed some adverse effects on their health issues. Research suggests that A1 ß-caseins may be associated with this serious health conditions and significant relationship was established between consumption of A1 milk and the incidence of type-1 diabetes (Elliott et al, 1999), autism, schizophrenia CVD, neurological disorders and sudden infant death syndrome. (McLachan, 2001; Laugesen and Elliott, 2003; Bell et al, 2006; Muntoni, 2006; Truswell, 2006; Woodford, 2009). With these above evident, a strong hypothesis was correlated upon consumption of A1 type milk on health-related issues to humans. At present, A1 and A2 type milk is emerged as one of the hot debating topic among the consumer worldwide.

Chemistry of A1 and A2 type milk
Bovine milk contains six major proteins. Out of these, four are part of the casein system and the other is whey protein (Table 1). Caseins group usually makes up about 80% of the total milk protein. The ß-casein is one of the major class of casein, represents approximately 25-35% of the total milk casein. The polypeptide chain of ß-casein, which is made up of 209 amino acid residues and this ß-casein gene has 12 known genetic variants. Most common forms of ß-casein genes in dairy cattle breeds are A1 and A2 (Aschaffenburg, 1961; Farrell et al, 2004; Kaminski et al, 2007; Keating et al, 2008). The basic difference between these two natural genetic variants is in their amino acid at 67th position in their polypeptide, which is histidine in A1 and proline in A2 type ß-casein. Milk that contains A1 ß-casein is known as A1 type milk, whereas milk that is of A2 ß-casein is called A2 type milk.

Milk from Jersey cows, Guernsey cows, camels, sheep, buffalo, yaks, donkeys, goats and Asian cows including humans, naturally contains mostly A2 type, whereas; milk from cows such as Holsteins and Friesians produce mostly A1 type. Milk produced in Australia, some European country and New Zealand is generally mix of A1 and A2 milks (Truswell, 2006; Kaminski et al, 2007; Woodford, 2007; Woodford, 2008). Most of native Indian cows and buffalo fall in the category of A2 type milk (De et al, 2011).

Naturally all milk was A2 type, until a mutation affecting in some species in New Zealand, Australia and other Western countries. It is believed that inbreeding and thousands of years of domestication have resulted in A1 type. This mutation in the DNA sequence coding for the ß-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) to proline (CCT) at amino acid position 67 of the protein (De et al, 2011). This single amino acid difference between the A1 and A2 type of ß-casein is due to the naturally occurring "single nucleotide polymorphism" (SNP) in the DNA of ß-casein genes of the cow.

Theoretically, this difference in amino acid sequence suggests a conformational difference in the secondary structure (folding of the amino acid chain) of the expressed protein, which subsequently may have ramifications on the physical properties of the respective casein micelles, further affecting digestion and properties of the caseins. It has been demonstrated that this one amino acid difference affects the proteolytic processing of the ß-casein in the mammalian gut. In vitro studies have shown that, the bioactive peptide ß- casomorphin-7 (BCM-7) is yields (Jinsmaa and Yoshikawa, 1999) by the successive gastrointestinal proteolytic digestion of ß-casein A1, but such cases were not seen in ß-casein A2 type milk, resulting in differential levels of bioactivities of the digested material.

Milking animals have been screened for ß-casein A1 and ß-casein A2 in many countries. There is difference in occurrence of ß-casein A1 or ß-casein A2 amongst species, breeds, and geographical locations. The results are denoted in terms of frequency of occurrence. This is compiled in report published by European Food Safety Authority (EFSA) in year 2009. As per this report, frequency for 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. It is believed that initially all animals were of A2 type. About 5,000 years back, mutation occurred in European taurine (Bos taurus) cattle probably somewhere close to Anatolia (Turkey) on the way to Europe from the domestication centre.

A limited work has been carried out in India for screening of milk animals for variants of ß-casein. In studies conducted at National Bureau of Animal Genetic Resource, Karnal, it has been observed that out of 231 buffaloes (8 breeds of river buffalo), all were of A2 type. Also, the allele frequency of A2 in 15 breeds of Zebu cattle (total of 618 animals screened) was 98.7%.

Another study conducted at National Dairy Research Institute, Karnal, revealed that all the indigenous animals from Sahiwal (total of 15 animals screened) and Tharparkar (total of 14 animals screened) breeds were of A2 type. However, in cross-bred (Karan Fries and Karan Swiss; total of 38 animals screened), the A2 allele frequency was 79-89%. Fresh or unprocessed milk obtained from healthy cow does not contain such opioid peptides. By contrast, there are substantial bodies of evidence indicating that different proteolytic systems involved in fermented milk or cheese manufacture can potentially hydrolyse ß-casein to BCM-7 or other BCMs and further degrade these peptides to shorter-chain peptides and even amino acids (EFSA Scientific Report, 2009).

Health implications
Genetic difference between A1 and A2 ß-caseins results in releasing its own, unique set of bioactive peptides on digestion by intestinal gut enzymes. Many benefits are thought to be due to the bioactives that are released from the ß-caseins on digestion. Therefore, it is the digestion or breakdown products of ß-casein, which have been shown to display the effects on the body. When ß-caseins are digested; they produce a range of protein fragments or “peptides,” which have been identified as having biological activity or bioactivity as shown in the Figure 1.

The one amino acid difference at position 67 of the ß-casein affects how they are liberated from the polypeptide chain. In the gut, digestive enzymes hydrolyse the ß-casein A1 peptide chain next to the histidine amino acid resulting in the release of highly bioactive peptide comprising the sequence of Tyr60- Pro61- Phe62- Pro63- Gly64- Pro65- Ile66 (as shown in Figure 1), called as BCM-7 (Noni, 2008; EFSA Scientific Report, 2009; Woodford, 2009).

Possibly owing to its morphine like behaviour, this released BCM-7 has longer half-life period relative to other casomorphins, which exhibits strong opioid activity and shows neurological impairment. It may also bind to the opiate receptors expressed by cells of digestive system, which can result in delayed gastrointestinal transit time and leads to constipation. BCM-7 can cross the blood-brain barrier easily, affects human immune responses, cause allergic responses, affects the regulation of insulin formation and also linked to type-1 diabetes (Merriman, 2009). Such liberated peptide weakly holds on to opiate receptors and found in significant quantity in the blood and urine of these animals, further BCM-7 has been reported to catalyse the oxidation of low density lipoprotein (LDL) cholesterol and promotes heart disease (Allison and Clarke, 2006).

The pattern
In the case of the variants containing proline (A2 milk), the enzymatic hydrolysis of the Ile66-Pro67 bond does not occur or occurs at a very low rate (EFSA Scientific Report, 2009). The unique pattern of bioactive peptides released from hydrolysis of A2 milk provides high degree of protection to the consumer. Solitary possible mechanism is that the proline has a strong bond to BCM-7; so that essentially no BCM-7 is found in the urine, blood or GI tract. Stepwise release of bioactive peptides from A2 ß-casein offers immune modulator peptides, antihypertensive and enzyme linked neurological inhibitory peptides, and no such above stated adverse health effects reported upon A2 milk consumption. Populations, which consume milk containing high levels of ß-casein A2 variant, have a lower incidence of cardiovascular disease and type-1 diabetes. Furthermore, consumption of milk with the A2 variant may be associated with less severe symptoms of autism and schizophrenia (Bell et al., 2006).

In the scientific report of EFSA (2009), they have done extensive review of scientific literature on A1 and A2 milk. In this report a cause-effect relationship between the oral intake of BCM-7 or related peptides and aetiology or course of any suggested non-communicable diseases cannot be established. Consequently, a formal risk assessment of food-derived peptides such as BCM-7 has not been suggested in their final recommendation.

Testing methods
The concept of A1 and A2 milk is being exploited commercially and many companies are promoting A2 milk at premium rates. Thus, to avoid dupery, the requirement of an analytical method is imminent to distinguish A2 milk from A1 milk. The A2 Corporation has developed a DNA test kit to distinguish animal secreting type for A1 and A2 milk. In this test, a hair sample is plucked from the cow's tail and then analysed to determine whether the animal is of A1 or A2 or a combination. Another reliable and more sensitive allelic discrimination assay using TaqMan fluorogenic probes was developed to detect single nucleotide substitution characterising the A1/A2 alleles of the CSN2 gene; as it is the most frequent in a number of dairy cattle breed, this method was validated using DNA samples of known genotypes with different concentrations and the results were compared by employing artificially created restriction site (ACRS)-PCR assay (Manga and vorak, 2010).

An innovative PCR-based test method has been successfully developed (De et al, 2011) to detect the A1 or A2 ß-casein variant forms in the cattle and buffalo milk at NDRI. All the above stated test methods differentiate either A1 or A2 milch animal, but as such, there is no direct test available to confirm the milk at the consumption stage to assure consumer confidence.

Conclusion
The A1 and A2 hypothesis is both intriguing and potentially very important for public health, the risk to benefit ratio associated with this hypothesis for any aged group is most debated issue. Most of the above-mentioned diseases are multi-factorial; also depends on the change in lifestyle, geographical, socio-economic and other environmental factors.

Since, milk is the vital food for infants and other vulnerable groups, many milk components offer protective role, but in some health insinuation cases as attributed by A1 milk; then it is the matter of choice. People may wish to reduce or eliminate A1 ß-casein from their diet as a precautionary measure; it is straightforward to switch to A2 type milk consumption. The shift in paradigm is observed in Australia, New Zealand and many other markets where A2 milk and milk products are accessible at premium price. Whether there is a definite health benefit to milk containing the A2 genetic variant is unknown and requires further investigation. A1 A2 milk case is rare in the field of dietary supplements and nutrition to find such compelling epidemiogical data that exist showing a significant relationship between a food and disease risk. Despite these findings, strong relationships do not always prove causality. Nonetheless, the results of the numerous studies deserve further investigation.

References
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Aschaffenburg, R., 1961. Inherited casein variants in cow’s milk, Nature, 192: 431- 432.

Bell, S. J., Grochoski, G.T. and Clarke, A. J. 2006. Health implications of milk containing ß-casein with the A2 genetic variant, Critical Reviews in Food Science and Nutrition, 46(1): 93-100.

De, S Kishore, C.M., Mukherjee, A. and Kaur, R. 2011. Typing of milk for A1 and A2 ß Casein, In: Chemical analysis of value-added dairy products and their quality assurance. NDRI Karnal Pub, 204-205.

EFSA Scientific Report. 2009. Scientific report of European Food Safety Authority (EFSA): Review of the potential health impact of ß-casomorphins and related peptides, 231: 1-107.

Elliott, R. B., Harris, D. P., Hill, J. P., Bibby, N. J. and H. E. Wasmuth. 1999. Type I (Insulin-Dependent) Diabetes mellitus and cow milk: Casein variant consumption. Diabetologia, 42:292-296.

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Forman, J.Q. 2004. Should I be worried about bovine growth hormones in milk? The Boston Globe. C2.

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Kaminski, S., Cieslinska, A. and Kostyra, E. 2007. Polymorphism of bovine ß- casein and its potential effect on human health. Journal of Applied Genetics, 48:189- 198.

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(Naik, Lata and Sharma are from division of dairy chemistry; and Rajput and Abhishek are from division of animal biochemistry, National Dairy Research Institute, Karnal. They can be reached at rajansharma21@gmail.com)