Beta-casein variants & digestive outcomes

Beta-casein variants & digestive wellbeing

Lactose intolerance

Lactose intolerance has been described as one of the most common intolerance responses to cows’ milk (1), with an estimated prevalence of around 10% in Caucasian Australians (2).

However, for a proportion of adults who report intolerance symptoms following milk intake, lactose intolerance is not the cause, as cases of perceived lactose intolerance are more common than its prevalence in adults (3-5). For instance, in a group of 406 randomly recruited men and women (mean age 27 years), 20.2% reported abdominal discomfort following dairy intake, but only 6.4% had lactose intolerance diagnosed by a physician (6).

Other natural components in milk may be responsible for stimulating some of the remaining gastrointestinal intolerance responses and indeed there is recent evidence to support that cows’ milk A1 beta-casein protein type may also be involved in some people (7).

A1 and A2 Beta-casein milk proteins

Beta-casein is a cows’ milk protein that makes up around 30% of the total protein contained in cows’ milk and may stimulate effects beyond nutrition, due to the release of biologically active peptides on digestion (8).

Beta-casein may be present as one of two major genetic variants: A1 and A2 (9). A2 is recognised as the original beta-casein genetic variant because it existed before a mutation caused the appearance of A1 beta-casein in some European herds a few thousand years ago (10, 11).

In Australia, regular cows’ milk contains a mix of A1 and A2 beta-casein types. In contrast, the cows’ milk known commonly as A2 milk comes from cows which have the genes to produce only the A2 type of beta-casein (Figure 1).

A1 beta-casein content in ordinary cows’ milk

In Australia, where dairy cows are of northern European ancestry, the relative proportions of the co-dominant A1 to A2 beta-casein genes in cows are typically 1:1, which then produce the same ratio of A1 to A2 beta-casein in their milk (Figure 1A). This tends to be lower in breeds from Southern Europe and this ratio depends on the specific breeding history of the dominant breeds. In Australia, most cows’ milk available commercially contains a mix of A1 and A2 beta-casein. The exception to this is milk produced by dairy cows genotyped and identified to carry only the un-mutated A2 beta-casein expressing gene on both sides of the chromosome (i.e. A2 milk). These dairy cows produce milk containing only the A2 beta-casein type and not the A1 (Figure 1B).


Figure 1: A) A1:A2 beta-casein gene carrying cow, which produces milk containing a 50:50 A1:A2 beta-casein mix. B) A2:A2 beta-casein gene carrying cow, which produces A2 beta-casein but no A1 beta-casein in her milk.

Cows’ milk A1 beta-casein is different to cows’ milk A2 beta-casein and other mammalian beta-caseins. A1 beta-casein has a histidine at position 67 on the 209 amino acid protein chain. Cows’ milk A2 beta-casein, human milk, goat milk, sheep milk and other species’ milk have a proline at their equivalent positions on their beta-casein protein chains, making them ‘A2 like’ (12-14). Due to this amino acid variation, A1 beta-casein releases the bioactive opioid peptide beta-casomorphin-7 (BCM-7) upon normal enzymatic digestion (15-19) (Figure 2).


Figure 2: A1 and A2 proteins showing the amino acid difference at position 67, representing the release of BCM-7. Figure adapted from reference (20)

In contrast, A2 beta-casein releases much less and probably minimal amounts of BCM-7 under normal gut conditions (15-18). BCM-7 was characterised as a casein-derived opioid peptide more than three decades ago (21-24). BCM-7 is a mu-opioid receptor ligand (17, 24) and mu-opioid receptors are expressed widely throughout human physiology, including the gastrointestinal tract (25).

BCM-7 release

BCM-7 has been detected following the simulated gastrointestinal digestion of a variety of infant formula and milk products (16, 26). BCM-7 has also been detected in the jejunal effluents of humans fed 30 grams of casein in amounts compatible with a biological action (27). This confirmed the identification around 30 years earlier of BCM-7 materials in the aspirated small intestinal contents of healthy male adults following milk intake using the ELISA assay technique (28). Following the incomplete digestion of A1 beta-casein, the maximal theoretical release of BCM-7 from 1 cup of milk containing 2-3 grams of A1 beta-casein is between 66 to 100 mg (15, 20) (Figure 3).

Figure 3: Beta-casein content and potential BCM-7 release per 250mL of milk. Figure adapted from reference (20).

A1 Beta-casein and gastrointestinal effects

Two recent animal studies have investigated cows’ milk A1 versus A2 beta-casein proteins on gastrointestinal effects directly (29, 30). Feeding rodents milk containing A1 beta-casein resulted in significant delays in GI transit time and increased colonic activity of the inflammatory marker myeloperoxidase (MPO), compared to milk containing A2 beta-casein (29).

Similarly, feeding mice a milk free basal diet supplemented with A1 relative to A2 beta-casein also resulted in significant MPO level increases (by 204%), whereas A2 beta-casein had no effect relative to controls (30).

Cows’ milk protein (31, 32) and more specifically the casein milk protein (33, 34) have been shown previously to be associated with various effects on the gastrointestinal tract, including the inhibition of motility (34, 35), with evidence implicating exogenous opioids like BCM-7 as a mediator (36-39).

In dogs, a comparison of casein and soy protein on various GI motility measures (e.g. force and contraction frequency) showed that casein reduced these parameters significantly and that pretreatment with naloxone (an opioid antagonist) blocked this effect (35), suggesting a role for exogenous opioids like BCM-7. Such BCM-7 effects on GI motility are physiologically plausible, since BCM-7 is a mu-receptor ligand and mu-receptor activation is known to affect the mechanics of intestinal propulsion (40).

A2 milk may assist some people with milk mediated digestion symptoms

A human randomised crossover study comparing the effects of A1 versus A2 beta-casein milk proteins (A1 vs A2 milk) on gastrointestinal (GI) outcomes has shown significant differences in stool consistency, with stools on A1 being overall looser (7). For people with looser stools on the A1 milk, there was very strong evidence that this was associated with more abdominal pain (P=0.001). This relationship was absent when the same people consumed A2. The difference between these two correlations was highly significant (Figure 4). As the A1 and A2 milk both contained lactose, the GI effects of the A1 milk can be attributed to the A1 beta-casein protein rather than lactose, so A2 milk may assist some with digestive wellbeing.


Figure 4: Significant correlation between stool consistency and abdominal pain with A1 beta-casein milk (*P<0.001)

Forty-one people were recruited into this double-blinded, randomised crossover study. Most participants at study entry considered themselves not to have problems digesting ordinary milk. Participants underwent a 2-week dairy washout (rice milk replaced all dairy), followed by two weeks of 750mL/milk/day containing beta-casein of either the A1 or A2 type, before undergoing a second washout followed by a final two weeks of the alternative A1 or A2 type milk.

In addition to the above results, this study identified that while on the A1 milk, higher gut inflammation (faecal calprotectin) correlated with higher abdominal pain (r=0.46, P=0.005) and higher bloating (r=0.36, P=0.03) scores but that on the A2 milk and in the same people, these relationships were absent. Again, the difference in the correlation measures was significant for: 1) gut inflammation and abdominal pain (A1, 0.46 vs A2, 0.03; P=0.02); and 2) gut inflammation and bloating (A1, 0.36 vs A2, 0.02; P=0.05).

A sub-group analysis of study participants with self-reported intolerance to ordinary A1 containing milk (n=8) showed further that the A1 milk resulted in more GI symptoms than the A2 milk (Figure 5), and while it was not possible to demonstrate statistically significant differences, the magnitude of these differences between A1 versus A2 milk may be clinically significant. Future studies examining this in different population groups with gastrointestinal conditions, such as irritable bowel syndrome, are needed.


Figure 5: The mean A1 scores were considerably higher for abdominal pain (38% higher), bloating (61% higher) and voiding difficulty (83% higher) versus the mean A2 scores

Given the previous animal research that shows A1 beta-casein feeding delays gut transit via an opioid pathway (29) and that A1 compared with A2 beta-casein feeding significantly increases gut inflammation as evidenced by MPO levels (29, 30), these studies collectively suggest the softer stools observed by people consuming A1 compared with A2 beta-casein milk (7) are caused by proinflammatory factors coupled with GI transit time effects.

While further clinical trials are needed to confirm these preliminary results in humans relating to GI response differences between A1 and A2 beta-casein proteins in milk, the following has been established:

  • Digestion of A1 beta-casein yields BCM-7, while A2 beta-casein does not or if it does, at a very low rate (15);
  • BCM-7 is an exogenous opioid that can bind mu-opioid receptors expressed in cells throughout the body, including those found in digestive tissue (24, 41);
  • Opioid mediated regulation of gastrointestinal motility is well documented (25), with mu-receptor activation producing effects on the mechanics of intestinal propulsion (40);
  • BCM-7 has the potential to be produced, absorbed and circulated in humans, particularly infants (42, 43);
  • BCM-7 induces rapid secretion of intestinal mucus (in the first 30 min. of stimulation) in rodents via activation of the enteric nervous system and opioid receptors (44, 45).

Lactose intolerance information is available on the ASCIA website


  1. Boyce J.A, et al., (2010). Guidelines for the diagnosis and management of food allergy in the United States: report of the NIAID-sponsored expert panel. J Allergy Clin Immunol. 126(6 Suppl), S1-58. External link
  2. Campbell A.K, et al., (2005). The molecular basis of lactose intolerance. Science progress. 88(Pt 3), 157-202. External link
  3. Jussila J, et al., (1969). Lactase deficiency and a lactose-free diet in patients with “unspecific abdominal complaints”. Acta Med Scand. 186(3), 217-22. External link
  4. Carroccio A, et al., (1998). Lactose intolerance and self-reported milk intolerance: relationship with lactose maldigestion and nutrient intake. Lactase Deficiency Study Group. J Am Coll Nutr. 17(6), 631-6. External link
  5. Johnson A.O, et al., (1993). Correlation of lactose maldigestion, lactose intolerance, and milk intolerance. Am J Clin Nutr. 57(3), 399-401. External link
  6. Pelto L, et al., (1999). Milk hypersensitivity in young adults. Eur J Clin Nutr. 53(8), 620-4.External link
  7. Ho S, et al., (2014). Comparative effects of A1 versus A2 beta-casein on gastrointestinal measures: a blinded randomised cross-over pilot study. Eur J Clin Nutr. 68(9), 994-1000. External link
  8. Phelan M, et al., (2009). Casein-derived bioactive peptides: Biological effects, industrial uses, safety aspects and regulatory status. International Dairy Journal. 19(11), 643-54. External link
  9. Formaggioni P, et al., (1999). Milk protein polymorphism: Detection and diffusion of the genetic variants in Bos genus. Ann. Fac. Med. Vet. Univ. Parma. XIX: 127-165. External link
  10. Ng-Kwai-Hang K.F & Grosclaude F, (2002). Genetic polymorphism of milk proteins. In: Fox PFaM, P.L.H editor. Advanced Dairy Chemistry: Volume 1: Proteins, Parts A&B. New York: Kluwer Academic/Plenum Publishers. p. 739-816. External link
  11. Kaminski S, et al., (2007). Polymorphism of bovine beta-casein and its potential effect on human health. J Appl Genet. 48(3), 189-98. External link
  12. Lonnerdal B, et al., (1990). Cloning and sequencing of a cDNA encoding human milk beta-casein. FEBS Lett. 269(1), 153-6. External link
  13. Provot C, et al., (1989). Complete nucleotide sequence of ovine betacasein cDNA: inter-species comparison. Biochimie. 71(7), 827-32. External link
  14. accession e. Goat beta-casein [cited Dec. 2014]. External link
  15. Scientific Report of EFSA prepared by a DATEX Working Group on the potential health impact of ß-casomorphins and related peptides. EFSA Scientific Report (2009). 231, 1-107 [cited Dec. 2014]. External link
  16. De Noni I, (2008). Release of ß-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine ß-casein variants and milk-based infant formulas. Food Chemistry. 110(4), 897-903.External link
  17. Jinsmaa Y & Yoshikawa M, (1999) Enzymatic release of neocasomorphin and beta-casomorphin from bovine beta-casein. Peptides. 20(8), 957-62. External link
  18. Ul Haq M.R, et al., (2015). Release of beta-casomorphin-7/5 during simulated gastrointestinal digestion of milk beta-casein variants from Indian crossbred cattle (Karan Fries). Food chemistry. 168, 70-9. Epub 2014/08/31. External link
  19. Hartwig, A., et al., (1997). Influence of genetic polymorphisms in bovine milk on the occurrence of bioactive peptides. Seminar on milk protein polymorphism (pp. 459–460). IDF Special Issue no. 9702. Brussels: International Dairy Federation. No external link
  20. Woodford K, (2007). Devil in the Milk: Illness, Health and Politics: A1 and A2 Milk. Wellington New Zealand: Craig Potton Publishing. No external link
  21. Brantl V, et al., (1979). Novel opioid peptides derived from casein (beta-casomorphins). I. Isolation from bovine casein peptone. Hoppe Seylers Z Physiol Chem. 360(9), 1211-6. External link
  22. Henschen A, et al., (1979). Novel opioid peptides derived from casein (beta-casomorphins). II. Structure of active components from bovine casein peptone. Hoppe Seylers Z Physiol Chem. 360(9), 1217-24. External link
  23. Lottspeich F, et al., (1980). Novel opioid peptides derived from casein (beta-casomorphins). III. Synthetic peptides corresponding to components from bovine casein peptone. Hoppe Seylers Z Physiol Chem. 361(12), 1835-9. External link
  24. Brantl V, et al., (1981). Opioid activities of beta-casomorphins. Life Sci. 28(17), 1903-9. External link
  25. Pleuvry B.J, (1991). Opioid receptors and their ligands: natural and unnatural. Br J Anaesth. 66(3), 370-80. External link
  26. De Noni I & Cattaneo S, (2010). Occurrence of beta-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion. Food chemistry. 119(2), 560-6. External link
  27. Boutrou R, et al., (2013). Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans. Am J Clin Nutr. 97(6), 1314-23. External link
  28. Svedberg J, et al., (1985). Demonstration of beta-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans. Peptides. 6(5), 825-30. External link
  29. Barnett M.P, et al., (2014). Dietary A1 beta-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 beta-casein in Wistar rats. Int J Food Sci Nutr. 65(6), 720-7. External link
  30. Ul Haq M.R, et al., (2014). Comparative evaluation of cow ß-casein variants (A1/A2) consumption on Th2-mediated inflammatory response in mouse gut. Eur J Nutr. 53(4), 1039–1049. External link
  31. Andiran F, et al., (2003). Cows milk consumption in constipation and anal fissure in infants and young children. J Paediatr Child Health. 39(5), 329-31. External link
  32. Iacono G, et al., (1998). Intolerance of cow’s milk and chronic constipation in children. N Engl J Med. 339(16), 1100-4. External link
  33. Defilippi C & Gomez E, (1995). Effect of casein and casein hydrolysate on small bowel motility and D-xylose absorption in dogs. Neurogastroenterol Motil. 7(4), 229-34. External link
  34. Hara H, et al., (1992). Different effects of casein and soyabean protein on gastric emptying of protein and small intestinal transit after spontaneous feeding of diets in rats. Br J Nutr. 68(1), 59-66. External link
  35. Defilippi C, et al., (1995). Inhibition of small intestinal motility by casein: a role of beta casomorphins Nutrition. 11(6), 751-4. External link
  36. De Ponti F, et al., (1989). Effect of beta-casomorphins on intestinal propulsion in the guinea-pig colon. J Pharm Pharmacol. 41(5), 302-5. External link
  37. Daniel H, et al., (1990). Effect of casein and beta-casomorphins on gastrointestinal motility in rats. J Nutr. 120(3), 252-7. External link
  38. Schulte-Frohlinde E, et al., (1994). Effect of bovine beta-casomorphin-4-amide on gastrointestinal transit and pancreatic endocrine function in man In: Brantl V, Teschemacher H, eds. Beta-Casomorphins and related peptides: recent developments. New York VCH Weinheim; 155-60. No external link
  39. Kromer W, et al., (1980). Opioids modulate periodicity rather than efficacy of peristaltic waves in the guinea pig ileum in vitro. Life Sci. 26(22), 1857-65. External link
  40. Ward S.J & Takemori A.E, (1983). Relative involvement of receptor subtypes in opioid-induced inhibition of gastrointestinal transit in mice. J Pharmacol Exp Ther. 224(2), 359-63. External link
  41. Zoghbi S, et al. (2006). beta-Casomorphin-7 regulates the secretion and expression of gastrointestinal mucins through a mu-opioid pathway. Am J Physiol Gastrointest Liver Physiol. 290(6), G1105-13. External link
  42. Kost N.V, et al. (2009). Beta-casomorphins-7 in infants on different type of feeding and different levels of psychomotor development. Peptides. 30(10), 1854-60. External link
  43. Wasilewska J, et al., (2011). The exogenous opioid peptides and DPPIV serum activity in infants with apnoea expressed as apparent life threatening events (ALTE). Neuropeptides. 45(3), 189-95.External link
  44. Claustre J, et al. (2002). Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum. Am J Physiol Gastrointest Liver Physiol. 283(3), G521-8. External link
  45. Trompette A, et al., (2003). Milk bioactive peptides and betacasomorphins induce mucus release in rat jejunum. J Nutr. 133(11), 3499-503. External link
Back to Health Professionals
Back to top