Buffalo ghee comes from A2 milk, because water buffaloes have shown only the A2 form of beta-casein in every genotyping study we found: 657 buffaloes of four breeds in Brazil and 250 Azi-Kheli buffaloes in Pakistan, all A2A2, none carrying A1. The label still says little about the ghee, which FSSAI defines by fat and moisture, not protein.
Search results on this question contradict each other. This article settles it from published sequencing and genotyping papers, then explains why the answer matters for buffalo milk and barely at all for buffalo ghee.
At a glance
- A1 and A2 beta-casein differ at one position in the protein chain: EFSA's 2009 review states that position 67 carries histidine in A1 and proline in A2.
- A 2021 Brazilian study genotyped 657 water buffaloes of four breeds and found every one A2A2; its authors conclude that the A1 allele does not exist in the species.
- A 2023 Pakistani study sequenced the gene in 250 Azi-Kheli buffaloes and found only the A2 type, with no proline-to-histidine change at position 67.
- Cattle are mixed: an ICAR study of about 4,000 animals found the A2 allele at a frequency of 0.95 in Indian cattle, while surveys in EFSA's breed table put A1 at 0.36 in Canadian and 0.72 in Irish Holstein-Friesians.
- FSSAI's ghee standard requires at least 99.5 per cent milk fat and at most 0.5 per cent moisture, leaving at most 0.5 per cent for protein and everything else, and has no A1 or A2 parameter.
Is buffalo milk A1 or A2?
A2, on every genetic study we could find. Two independent teams, in Brazil and Pakistan, genotyped a total of 907 water buffaloes (657 + 250) and found the A2 version of the beta-casein gene in every animal, with no A1. A separate laboratory analysis of buffalo mozzarella found only the A2 protein in the cheese.
The terms come from cattle. Beta-casein is one of the main milk proteins, and EFSA's 2009 review states the difference plainly: "In β-casein A1 and B variants histidine occurs in position 67, whereas in β-casein A2 proline is present in the same position." Our article on what A2 milk is covers the cattle side and the human trials. Water buffalo (Bubalus bubalis) is a different species, so the only way to know which form it carries is to read its gene or its protein.
What do the buffalo genotyping studies show?
Three kinds of evidence point the same way. A DNA study of 657 buffaloes found only A2A2 genotypes, a sequencing study of 250 buffaloes found no histidine at position 67, and a protein analysis of buffalo cheese detected only the A2 variant. None of the three found A1.
The largest, from the Federal University of Bahia in Brazil (2021), genotyped 657 buffaloes of four breeds. In the abstract's words, "all animals carried the A2A2 genotype, i.e., allele A1 does not exist in the buffalo species. Thus, all milk products of buffaloes are naturally A2."
The second, from the University of Agriculture Peshawar (2023), took blood from 250 Azi-Kheli buffaloes and sequenced position 67 of exon 7, the site of the A1 and A2 difference. The animals were "homozygous for only A2 type variant", and "the amino acid change (proline to histadine) on 67 position of exon 7 was not found". Three other new variants turned up elsewhere in the exon; none is the A1 change.
The third looks at the protein. A 2017 University of Melbourne study of buffalo and cow mozzarella "detected the presence of only β-casein variant A2" in the buffalo cheeses, against "both β-casein variants A1 and A2" in the bovine cheese, by liquid chromatography mass spectrometry.
We also searched PubMed for the opposite finding: "buffalo beta-casein A1 variant" returned four papers and "Bubalus bubalis beta-casein polymorphism" twenty-one. None reported histidine at position 67.
Why do some websites say buffalo milk has A1 beta-casein?
We found no sequencing or genotyping study that supports the claim. One likely source of confusion is that buffalo beta-casein variants have their own names, and one Indian paper used the label "A1" for a buffalo allele that is not the cattle A1.
That paper, from the National Dairy Research Institute at Karnal (2013), screened exon VII of the buffalo gene and found a predominant allele it calls B, at 85 per cent and always homozygous, and a rare allele it calls A1, found only in heterozygous animals. Sequencing "revealed three nucleotide substitutions at codon 68, 151 and 193 of exon VII". The abstract reports no change at codon 67, where cattle A1 differs from A2. That buffalo "A1" is a differently named variant, not evidence of histidine at position 67, and the claim that buffalo milk "contains A1" may be a slip from one naming system into another.
How does buffalo compare with cow and goat?
Of the three animals, cattle are the only one in which both A1 and A2 are found, and the mix varies by breed and region. Buffalo and goat beta-casein carry proline at position 67 in every study we read. The table gives each study's size; ten animals and 4,000 do not carry equal weight.
| Animal | Beta-casein at position 67 | Evidence and its size | Source |
|---|---|---|---|
| Cattle in India, native breeds (Bos indicus) predominating | Mostly A2; A1 in some animals | About 4,000 animals of three cattle types plus breeding bulls: A2 allele 0.95 in Indian cattle; A2-type haplotype 0.941 across native breeds | ICAR-NBAGR, 2022 |
| Western dairy cattle (Bos taurus) | Mixed A1 and A2, by breed and country | Breed surveys compiled by EFSA: Holstein-Friesian A1 at 0.36 (920 animals, Canada) and 0.72 (696, Ireland); Guernsey A2 at 0.98 (196, USA) | EFSA, 2009 |
| Water buffalo (Bubalus bubalis) | A2 only (proline) | 657 buffaloes of four breeds, all A2A2; 250 Azi-Kheli buffaloes, all A2, no histidine at 67; only A2 protein in buffalo mozzarella | de Oliveira et al., 2021; Khan et al., 2023; Nguyen et al., 2017 |
| Goat | A2-type (proline) | Beta-casein sequenced by HPLC-MS/MS in French Alpine, Nubian and Creole goats, 10 per breed; proline at 67 in all three | Muñoz-Salinas et al., 2022 |
A note on cattle. The ICAR-National Bureau of Animal Genetic Resources study found enough A1A2 breeding bulls to warn that sire lines should be screened "so as to drift the herd towards A2": Indian breed is a strong hint, not a guarantee for one cow. EFSA, for its part, notes that "Guernsey cattle almost exclusively appear to have the β-casein A2 genotype".
The goat study, from the Autonomous University of Querétaro in Mexico, is small and sequenced the protein rather than genotyping animals. We found no equivalent paper for sheep: a PubMed search for "sheep beta-casein A1 A2 variant" returned two papers on laboratory methods, neither reporting a sheep genotype.
Does the A2 label matter for buffalo ghee?
Very little, for two reasons. Buffalo milk is A2 by nature, so the label adds nothing a buyer did not already have. And A1 and A2 describe a protein, while ghee is almost all fat. FSSAI defines ghee by fat and moisture, and its standard contains no A1 or A2 parameter at all.
FSSAI's standard for milk fat products (regulation 2.1.8) describes ghee as a fatty product derived from milk "by means of processes which result in almost total removal of water and milk solids-not-fat", and sets it at a minimum of 99.5 per cent milk fat and a maximum of 0.5 per cent moisture. If at least 99.5 per cent of the jar is fat, protein, water and everything else together can be at most 0.5 per cent (100 − 99.5 = 0.5), and beta-casein is only one of the proteins in that remainder. Our article on how much protein ghee has traces where the rest goes.
The standard says nothing about which animal the milk came from: FSSAI defines milk as "the normal mammary secretion derived from complete milking of healthy milch animal", and the ghee standard's raw material is simply "milk and products obtained from milk". What differs is the milk going in: FSSAI's milk standard sets buffalo milk at a minimum of 5.0 per cent fat against 3.2 per cent for cow milk. How the two ghees compare in colour, yield and taste is set out in cow ghee versus buffalo ghee.
Nor can a test on the ghee settle the question. Every method we found for telling A1 from A2 reads the animal's DNA or the beta-casein in milk or cheese, and FSSAI's ghee parameters cover moisture, milk fat, free fatty acids, fat constants and the absence of β-sitosterol, not protein variants. A ghee lab report cannot show A2 status, whatever the animal; see whether a lab can prove ghee is A2.
We make A2 bilona ghee from Badri cattle, not buffaloes, and we would still rather you knew this: in cattle, A2 is a statement about the herd; in buffalo, about the species; in ghee of either kind, about a protein that has almost entirely left the jar.
What should you look for on a buffalo ghee label instead?
Since "A2" is a given for pure buffalo milk, the useful questions are about what else is in the jar and how it was made:
- Buffalo milk only, or mixed? FSSAI's "mixed milk" is any combination of species-identified milks. Of cow, buffalo and goat milk, only cow milk has been shown to carry A1 beta-casein, so the species question is the real A2 question.
- Does it meet the ghee standard? Look for a batch report with moisture at or below 0.5 per cent, milk fat at or above 99.5 per cent and β-sitosterol absent.
- How was it made? FSSAI says ghee "has especially developed flavour and physical structure as a result of its method of manufacturing"; method is what separates one ghee from another once the standard is met.
- Is the A2 claim doing any work? On buffalo ghee it is not. Treat it as neutral: neither a warning sign nor a reason to pay more.
None of this is health advice. The studies here show which protein variant an animal carries, not what it does in people. If milk or ghee troubles you, follow your doctor's advice.
The short version
Buffalo ghee is made from A2 milk, because every genotyping study we found shows water buffaloes carrying only the A2 form of beta-casein: 907 animals in Brazil and Pakistan, all A2A2, with proline rather than histidine at position 67. Claims that buffalo milk contains A1 have no sequencing study behind them; one Indian paper's buffalo "A1" is a differently named variant with changes at codons 68, 151 and 193, not 67. In cattle the mix is real and varies by breed, and the small goat evidence also points to A2. FSSAI requires ghee to be at least 99.5 per cent milk fat and sets no A1 or A2 test, so the label on buffalo ghee tells you almost nothing the species had not already settled.
Sources
Further reading behind the figures above. Himdhara's own numbers — yields, batch results — come from our production records and the batch reports on the lab reports page.
- de Oliveira LSM, Alves JS, Bastos MS, et al. (2021). Water buffaloes (Bubalus bubalis) only have A2A2 genotype for beta-casein. Tropical Animal Health and Production.
- Khan K, Suhail SM, Khan R, et al. (2023). Genetic polymorphism of Β-casein gene and its association with milk production and composition in Azi-Kheli buffalo. Tropical Animal Health and Production.
- Nguyen HTH, Ong L, Lopez C, Kentish SE, Gras SL (2017). Microstructure and physicochemical properties reveal differences between high moisture buffalo and bovine Mozzarella cheeses. Food Research International.
- P V V, Brahma B, Kaur R, Datta TK, Goswami SL, De S (2013). Characterization of β-casein gene in Indian riverine buffalo. Gene.
- Mukesh M, Swami S, Bhakhri G, et al. (2022). Demographic pattern of A1/A2 beta casein variants indicates conservation of A2 type haplotype across native cattle breeds (Bos indicus) of India. 3 Biotech.
- European Food Safety Authority (2009). Review of the potential health impact of β-casomorphins and related peptides. EFSA Scientific Report 231.
- Muñoz-Salinas F, Andrade-Montemayor HM, De la Torre-Carbot K, et al. (2022). Comparative Analysis of the Protein Composition of Goat Milk from French Alpine, Nubian, and Creole Breeds and Holstein Friesian Cow Milk: Implications for Early Infant Nutrition. Animals.
- Food Safety and Standards Authority of India. Food Safety and Standards (Food Products Standards and Food Additives) Regulations, 2011: Chapter 2.1, Dairy products and analogues (2.1.1, 2.1.2 and 2.1.8). FSSAI.