Gastrointestinal response to A1 vs A2 milk
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In case you're not up to speed with A1 and A2 milk, well, it all boils down to type of cow and type of milk produced. Anyone with a handle on autism research history will have probably heard about the opioid-excess hypothesis . The long-and-short-of-it is that casein, the protein found in milk and dairy products, is eventually metabolised into it's constituent amino acids. Along the way, short chains of amino acids called peptides are formed. Some of these peptides look (chemically) similar to compounds like morphine and are hence referred to as the casomorphins (casein derived morphine-like). The opioid-excess hypothesis suggested that these exogenously derived peptides mimic some of our own naturally occurring morphine-like compounds that we all have, and disrupt typical functioning in this area to such an extent that it may correlate with some of the signs and symptoms called autism. I talked about something similar quite recently.
Granted, such a model looks a little simplistic these days knowing what we think we know about the very plural autisms and the ESSENCE of cormorbidity. Still, such a hypothesis did seem to fit in well with the suggested effectiveness of a casein-free (and gluten-free) diet for some on the autism spectrum, and also some work looking at the opioid receptor blocker that is naltrexone (see here) and autism. It is with the structure of those peptides in mind that we come to the differences suggested for A1 and A2 milk. Y'see not every cow or other mammal produces the same kind of casein protein in their milk and hence peptide formulations can vary also. For A2 casein, the idea is that beta-casomorphin fragment 1-7 (BC 1-7), a peptide formed during digestion is not the same as the BC1-7 from A1 milk (see here) particularly when it comes to a single amino acid change (proline over histidine) .
After such a long-winded explanation, we come back to the Ho paper and some interesting findings...
- First things first, this was a double-blind, randomised cross-over study looking at "gastrointestinal effects" in adults under conditions of either A1 or A2 milk consumption. Two weeks of either A1 or A2 milk consumption (with an appropriate washout period in between) were completed.
- The very informative Bristol Stool Chart was used to grade poop (stool) consistency alongside other more physiological measures such as faecal calprotectin.
- Results: "The A1 beta-casein milk led to significantly higher stool consistency values". That and a correlation between stool consistency and reports of abdominal pain for participants when on the A1 milk compared with when on A2 milk. Ergo, it didn't seem that A2 milk did anything over and above A1 milk, rather that consumption didn't seem to be linked to the symptoms noted when drinking A1 milk.
Appreciating the authors' call for further study in this area, I was intrigued by these results. Not so many moons ago, I came across the paper by Barnett and colleagues  talking about greater gastrointestinal (GI) transit time in rats fed A1 milk over A2 milk (see here for some additional commentary from one of the study authors). One might very well overlap those rodent reports with the more recent Ho results in terms of how longer transit time from A1 milk might mean greater discomfort bearing in mind some of the literature on longer transit time and "pain and distension"  in certain conditions. Interestingly, the authors ask that research not only focus on confirmation of their results but: "confirmation in a larger study of participants with perceived intolerance to ordinary A1 beta-casein-containing milk" which begs the question: who and what ailments are being reported?
That all being said, not all the literature on A2 milk is so directional. Take for example the paper by Crowley and colleagues  (open-access) looking at the question of milk consumption correlating with the functional bowel issue constipation. They concluded that: "that removal of CMP [cow's milk protein] from the diet of children with CFC [chronic functional constipation] significantly increased the number of bowel motions and improved constipation". Their results however did not show any significant effect based on casein type when looking at A1 and A2 milk. Constipation, by the way, is also something talked about with some autism in mind (see here) and particularly the findings from Afzal and colleagues  which concluded: "Multivariate regression analysis showed consumption of milk to be the strongest predictor of constipation in the autistic group".
I am quite interested in this whole area of different milks from different animals potentially possessing different qualities which might impact on physiology particularly if eventually applied to conditions like autism, or at least some comorbidity. I think back to the post I did on milk derived opioid peptides and methylation status (see here) as also being important, as might be the work on something like the use of camel milk (see here) bearing in mind the adverse publicity our humped friends have received recently. As per my previous caveat, I don't think we are in a position yet to advocate changes in milk drinking practices for specific groups based on the available literature, but there might be quite a bit more research to do in this important area...
To close, I know this might sound a little odd but am I the only father with young children who know Barry Scott on sight?
 Ho S. et al. Comparative effects of A1 versus A2 beta-casein on gastrointestinal measures: a blinded randomised cross-over pilot study. Eur J Clin Nutr. 2014 Jul 2.
 Shattock P. & Whiteley P. Biochemical aspects in autism spectrum disorders: updating the opioid-excess theory and presenting new opportunities for biomedical intervention. Expert Opin Ther Targets. 2002 Apr;6(2):175-83.
 Truswell AS. The A2 milk case: a critical review. Eur J Clin Nutr. 2005 May;59(5):623-31.
 Barnett MP. et al. Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats. Int J Food Sci Nutr. 2014 Mar 20.
 Cann PA. et al. Irritable bowel syndrome: relationship of disorders in the transit of a single solid meal to symptom patterns. Gut. May 1983; 24(5): 405–411.
 Crowley ET. et al. Does Milk Cause Constipation? A Crossover Dietary Trial. Nutrients 2013; 5: 253-266
 Afzal N. et al. Constipation with acquired megarectum in children with autism. Pediatrics. 2003 Oct;112(4):939-42.
Ho, S., Woodford, K., Kukuljan, S., & Pal, S. (2014). Comparative effects of A1 versus A2 beta-casein on gastrointestinal measures: a blinded randomised cross-over pilot study European Journal of Clinical Nutrition DOI: 10.1038/ejcn.2014.127
This is the first systematic review, to our knowledge, of published studies investigating the gastrointestinal effects of A1-type bovine β-casein (A1) compared with A2-type bovine β-casein (A2). The review is relevant to nutrition practice given the increasing availability and promotion in a range of countries of dairy products free of A1 for both infant and adult nutrition. In vitro and in vivo studies (all species) were included. In vivo studies were limited to oral consumption. Inclusion criteria encompassed all English-language primary research studies, but not reviews, involving milk, fresh-milk products, β-casein, and β-casomorphins published through 12 April 2017. Studies involving cheese and fermented milk products were excluded. Only studies with a specific gastrointestinal focus were included. However, inclusion was not delimited by specific gastrointestinal outcome nor by a specific mechanism. Inclusion criteria were satisfied by 39 studies. In vivo consumption of A1 relative to A2 delays intestinal transit in rodents via an opioid-mediated mechanism. Rodent models also link consumption of A1 to the initiation of inflammatory response markers plus enhanced Toll-like receptor expression relative to both A2 and nonmilk controls. Although most rodent responses are confirmed as opioid-mediated, there is evidence that dipeptidyl peptidase 4 stimulation in the jejunum of rodents is via a nonopioid mechanism. In humans, there is evidence from a limited number of studies that A1 consumption is also associated with delayed intestinal transit (1 clinical study) and looser stool consistency (2 clinical studies). In addition, digestive discomfort is correlated with inflammatory markers in humans for A1 but not A2. Further research is required in humans to investigate the digestive function effects of A1 relative to A2 in different populations and dietary settings.
β-casein, β-casomorphin, gastrointestinal tract, humans, in vitro, in vivo, inflammation, milk
In this review, we address the scientific evidence for A1-type bovine β-casein (A1) compared with A2-type bovine β-casein (A2) being associated with gastrointestinal issues. We do this within a systematic framework in accordance with section 6 of the Australia New Zealand Food Standards Code (1) by using defined criteria and including in vivo and in vitro studies (all species) published through 12 April 2017. Articles that do not have an explicit relevance to gastrointestinal issues were excluded.
As background, β-casein makes up ∼30% of the total protein contained in bovine milk and may present as 1 of 2 major genetic types: A1 and A2 (2). The distinguishing structure between these 2 forms of β-casein is the presence of either histidine (His67) in A1 or proline (Pro67) in A2 at position 67 of this 209–amino acid protein, with A1 being consequential to a point mutation from Pro67 to His67 occurring in ancestors to modern European-type cattle (2). Consequently, the milk produced commercially in many countries contains a mixture of A1 and A2 (2). The His67 mutation is absent in purebred Asian and African cattle. Similarly, the presence of a histidine mutation at the equivalent position in other mammalian species, including humans, is either absent or extremely rare (3, 4).
Within modern European-type cattle, there are additional derivative β-casein proteins through mutations at other points of the protein chain, which can be grouped within the A1 and A2 types. The most important of these is type B β-casein, which, like A1, contains His67. Other A1 and A2 caseins can be considered minor. Most studies are not explicit as to the presence or absence of these minor variants and refer only to either “A1” or “A2.” Accordingly, throughout this review we use the same terminology but recognize that other unrecorded minor subvariants of these types may also have been present.
Although His67 within A1 is susceptible to proteolytic cleavage, Pro67 within A2 is not. Thus, A1s have the potential to release short β-casomorphin (BCM) opioid peptides, including BCM-7, during gastrointestinal digestion (Table 1). The avoidance of A1 is feasible within dairy-based diets through the consumption of goat, sheep, and buffalo milk or through the consumption of bovine milk from the native Asian and African bovine breeds, or through the consumption of milk from genetically selected herds of European-type cattle that are certified free of the His67 mutation. Such herds are being developed in many countries.
Structure of BCMs released from bovine milk1
|Peptide||Corresponding β-casein location||Structure|
|Peptide||Corresponding β-casein location||Structure|
Bovine milk that is free of A1 is now available commercially in a range of countries, including Australia, the United Kingdom, the United States, New Zealand, and The Netherlands, and is widely promoted as beneficial for people who suffer from milk intolerances. Infant formula containing casein but free of A1 is now marketed widely in China and Australia and is promoted commercially as being more gentle on the infant digestive system.
To our knowledge, this is the first scientific review to focus specifically on intolerances and gastrointestinal effects. An earlier review by Truswell (5) was not systematic, did not focus specifically on gastrointestinal effects, and has been criticized for ignoring relevant studies (6, 7). A review by the European Food Safety Authority (EFSA) undertaken in 2008 (2) noted that BCM-7 was an opioid peptide released by A1 but not A2, but did not find convincing evidence for physiologic effects in humans. The EFSA study did not investigate intolerances and gastrointestinal effects. In our study, we focus on the available science both before and subsequent to these studies. Although the underlying science of casomorphins has been known for >30 y, the scientific evidence specific to gastrointestinal effects has largely emerged since 2009.
Criteria for inclusion
Both in vitro and in vivo animal studies and human clinical trials that reported outcomes relevant to a comparison between the digestion and potential health impacts of A1 and A2 in the gastrointestinal system were included in the review. Studies involving milk, milk products, β-casein, and BCM peptides of various lengths were considered relevant. For in vivo animal and human clinical studies, inclusion was limited to those studies in which the test material was administered orally. Relevant outcome measures were as follows: release of BCM in actual or simulated gastrointestinal digestion of milk, fresh-milk products, or casein; opioid agonist activity after digestion of milk, milk products, or casein; and variations in biomarkers of bloating and abdominal pain or discomfort after the consumption of milk, fresh-milk products, or casein. However, the selection of studies was not delimited by any specific mechanism or outcome as long as it had a gastrointestinal relevance. Studies involving fermented and aged dairy products, such as cheese, were not included. The authors considered that the presence of cultures in fermented and aged products raises 2 separate questions—1) how much BCM is released during the cheese-making process and 2) how much BCM is released during in vitro and in vivo digestion—which are sufficiently complex to warrant a separate review.
English-language literature searches were undertaken by using PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), Scopus (https://www.elsevier.com/solutions/scopus), and ScienceDirect (http://www.sciencedirect.com/) (filtered for Agricultural and Biological Sciences; Biochemistry, Genetics and Molecular Biology; and Health Sciences), initially in March 2016, but updated through 12 April 2017 with the use of the following search terms: A2 AND Milk; beta-casein AND A1 OR A2; casomorphin; beta-casomorphin; beta-casomorphin-7; beta-casomorphine; beta-casomorphine-7; A1_betacasein OR A2_betacasein; and b-cm OR 7 bcm7 OR bcm-7. In addition, 1 relevant study (8) was identified from the EFSA DATEX Working Group report (3), 1 (9) was identified from a citation in a selected study (10), and 1 (11) was provided by one of the authors.
Data were extracted manually and independently for each search database. Studies that measured outcomes relevant to potential direct effects of A1 compared with A2 in the gastrointestinal tract were selected. Studies that did not contain relevant outcome measures or that were only available as abstracts of conference presentations were excluded. Studies were assessed manually for bias on the basis of information provided in each publication (see Supplemental Tables 1–6).
A total of 3287 unique studies were identified (Figure 1). The overwhelming majority of studies were excluded on the basis that the reported variables did not include oral exposure to the test materials for in vivo studies; did not include markers relevant to gastrointestinal bloating, abdominal pain or discomfort, or both; were not original results; or were reviews. Additional exclusion criteria included that the original references could not be sourced, were not available in English, or the references were conference papers or abstracts that contained data published elsewhere in full. Thirty-five studies were identified for review directly from the search results, and 4 were identified from other sources as described. Of these, 11 described the digestion of bovine milk or fresh-milk products within in vitro studies and 5 within in vivo studies, 11 reported bovine BCM activity in the gastrointestinal tract within in vitro studies, and 15 reported results from milk or bovine BCM within in vivo studies. Some studies were allocated to more than one category during review depending on the relevant content. Although mention of BCM activity was not a required condition for study selection, it was found that β-casein studies of gastrointestinal effects consistently referred to either BCM-related analyses or BCM-related explanatory hypotheses.
PRISMA flowchart. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.
PRISMA flowchart. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.
Release of BCMs from β-casein
Eleven in vitro studies that reported enzymatic digestion of milk and milk products or casein, and 5 in vivo digestion studies (including those in humans), met the inclusion criteria for review (Supplemental Tables 1 and 2). One study reported both in vitro and in vivo data.
In vitro studies
The release of bioactive peptides from milk and milk products with opioid properties was first discovered in an in vitro system by Brantl and Teschemacher (12) in 1979 while searching for pronase-resistant opioids in bovine milk and milk products that were similar to those reported previously in plasma. Chloroform-methanol extracts of enzyme-digested commercial cow milk, casein digests, and milk-based baby food were found to inhibit contraction of isolated guinea pig ileum. Furthermore, this activity was blocked by the opioid antagonist (−)naloxone, suggesting that milk peptides in the extracts had μ-opioid receptor agonist activity. The fresh-milk samples were collected monthly over a 7-mo period from a local farm. Opioid activity was found in casein extracts prepared from milk collected in May, June, and October but not in July, August, September, or November. An explanation for this differential finding may reflect random sampling, herd composition, and health status. In a follow-up study, similar chloroform-methanol extracts of digested casein were shown to have μ-opioid receptor activity in isolated guinea pig ileum (13), and the active peptide was isolated and identified by using amino acid sequencing as BCM-7 (14, 15). The sequence identified was cross-mapped to bovine β-casein corresponding to the amino acid sequence 60–66 (16).
Subsequent studies have shown that longer BCM peptides, but not BCM-7 itself, can be released by simulated gastric digestion with pepsin, but that a multistage digestion system as is present in the human stomach and small intestine is necessary for the release of BCM-7 and other smaller BCMs. Svedberg et al. (17) reported that large amounts of BCM-7 immunoreactive material was released after simulated gastrointestinal digestion of β-casein (unspecified variant or variants) in a multienzyme system comprising the following: 1) pepsin; 2) lipase, amylases, proteases, and bile acids; and finally, 3) intestinal mucosal peptidase. Only small amounts of BCM-7 immunoreactive material were found after pepsin digestion alone. This finding was confirmed in a small follow-up study in 4 healthy adults fitted with intestinal tubes who consumed the same β-casein (17). The BCM-7 immunoreactive material identified in these studies was not further identified as BCM-7 itself and potentially indicates the release of a longer-chain peptide that includes the BCM-7 amino acid sequence. In another more recent study, pepsin digestion of commercial bovine β-casein released both His9 and Pro9 BCM-11 but not BCM-7 (18). These data indicate that pepsin alone is insufficient and that the release of BCM-7 occurs beyond the stomach.
It has been shown that pancreatin, a mixture of several digestive enzymes produced by the pancreas and only found in the small intestine (1), releases BCM-7 from His67 β-caseins (i.e., A1 and B β-casein types) but not Pro67 (i.e., A2 type) variants (16). Elastase was identified as the essential component enzyme in the pancreatin responsible for cleaving the Ile66–His67 bond to release BCM-7 from longer BCM peptides (19). More recently, De Noni (20) and Ul Haq et al. (21) both confirmed the release of BCM-7 after simulated gastrointestinal digestion of milk from A1/A1 and A1/A2 but not from A2/A2 cows. Ul Haq et al. (21) also verified that elutes containing BCM-7 showed opioid activity in an isolated rat ileum assay.
One research group has consistently reported small amounts of BCM-7 in fresh milk and in hydrolyzed milk from both A1/A1 and A2/A2 cows after an intensive 24-h acidic (pH 2.0) pepsin digestion (22, 23). The presence of BCM-7 in fresh milk is not confirmed elsewhere in the literature, and it has alternatively been proposed that this finding may be explained by proteolysis of caseins by enzymes associated with somatic cells in the milk, which are normally associated with clinical or subclinical mastitis (2, 24). However, it could be more likely that small amounts of BCM-7 detected by this group after pepsin digestion were the result of extended acidic hydrolysis rather than enzymatic action and are not reflective of human multienzyme gastrointestinal digestion in which gastric digestion may occur for <1 h and is unlikely to exceed 6 h (25). From the same research group, and by using an alternative pepsin-trypsin-elastase sequential digestion system, Cieślińska et al. (23) reported that the concentrations of BCM-7 were highest in milk hydrolysates from A1/A1 cows and 25- to 27-fold higher than that reported for pepsin digestion alone. In hydrolysates of milk from A1/A2 cows, the concentrations of BCM-7 measured were ∼50–60% of those from the A1/A1 milk. BCM-7 was also detected at low concentrations in milk hydrolysates from A2/A2 cows in this study, although the concentrations were consistently <10% of those measured from A1/A1 milk. Although this study was undertaken by using simulated gastric and intestinal digestion, the detection of BCM-7 from A2/A2 milk stands in contrast to other studies that have shown no release of BCM-7 from the milk of A2/A2 cows (19–21).
In vivo studies
BCMs were first identified in the gut contents after the consumption of 100 g of a commercial, acid-precipitated bovine casein in a small study with 2 mini-pigs fitted with a duodenal cannula (26). The duodenal extract contained 23 tyrosine-containing peptides from which electrophoresis produced a main band that was subsequently resolved by HPLC into 14 peptides, predominantly BCM-5, but also minor bands of BCM-7 and a BCM-4 amide.
A similar result was reported from a small study in 4 healthy men fitted with a gastric tube positioned in the proximal small intestine after the consumption of 1 L raw milk (β-casein variants not reported) (17). Analysis of the BCM-7 immunoreactive peptides recovered indicated that, although they contained the BCM-7 sequence, much of the material was suspected to be a longer-peptide sequence, possibly indicative of A2s. In a separate study, BCM-7 immunoreactive material, with chain lengths of 12–13 amino acids, was also found in the plasma of young dogs (aged 2 and 4 wk), but not in adult dogs, after oral administration of cow milk (β-casein variants not reported) and canine milk (27). All of these studies indicate the release of BCM peptides but do not directly show the release of BCM-7 itself from β-casein. In addition, because the β-casein variants were not identified, it is unclear from these studies alone whether the longer BCM peptides detected, including the [Pro8]BCM-11 identified by Meisel (26), came from A1 or A2. The formation of a number of β-casein peptides was also reported in samples collected from the stomach and duodenum from a small study in 6 adult humans after the consumption of milk and yogurt; however, no peptides that contained sequences corresponding to β-casein positions 60–70 were reported (7).
In a current single-blind parallel study, 16 healthy adults were randomly assigned to 2 groups of 8 participants (28). Each group consumed daily, for 9 d, a protein controlled meal (1.4 g · kg−1 · d−1), including a protein shake containing 30 g of either milk casein (equivalent to ∼1 L milk) or whey protein. The β-casein status of the source milk was not reported. On day 9, samples were withdrawn by nasojejunal tube, positioned to collect contents from the proximal jejunum, at multiple times postconsumption. Analysis of peptides was undertaken by using tandem MS. β-Casein was reported to be the predominant precursor of peptides recovered, accounting for 61.2% of total recovered casein peptides. There were 20 peptides identified that had amino acid sequences that included all or part of the β-casein 60–66 sequence (i.e., BCM-7) (Table 2). Six peptides cleaved before β-casein position 67, including BCM-7, were identified with collection frequencies between 0.73 and 0.14. The cumulative quantity of 4 of these peptides [β-casein57–66, β-casein58–66, β-casein59–66, and β-casein60–66 (the last being BCM-7)] was highest at 30 min postconsumption, at 3.60 ± 0.35 mg. An average total of 4 mg BCM-7 was recovered from the jejunal effluent ≤2 h postconsumption, corresponding to a concentration of 17 μmol/L in 304 mL (mean volume) of the jejunal effluent collected. Only 2 long peptides containing intact His67 were identified, β-casein59–72 and β-casein57–72, both with very low frequencies (0.06 and 0.02, respectively). A total of 14 peptides containing an intact Pro67 bond (i.e., peptides of the A2 type) were identified. The most frequently found was β-casein59–68 (i.e., Pro67–10 amino acid peptide) at a frequency of 0.65. Three other Pro67 peptides—β-casein57–68 (i.e., Pro67–12 amino acid peptide), β-casein58–68 (i.e., Pro67–11 amino acid peptide), and β-casein59–67(i.e., Pro67–9 amino acid peptide)—were identified with frequencies between 0.31 and 0.35. β-Casein60–68 (i.e., Pro67–BCM-9) was identified at a frequency of 0.15. These data support the in vitro data that His67 (i.e., A1 and B) β-casein is readily cleaved at position 67.
Frequency of detection of jejunal BCM peptides after consumption of bovine casein by healthy adults