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ORIGINAL ARTICLE
Comparative effects of A1 versus A2 beta-casein
on gastrointestinal measures: a blinded randomised
cross-over pilot study
SHo
1
, K Woodford
2
, S Kukuljan
3
and S Pal
1
BACKGROUND/OBJECTIVES: At present, there is debate about the gastrointestinal effects of A1-type beta-casein protein in cows’
milk compared with the progenitor A2 type. In vitro and animal studies suggest that digestion of A1 but not A2 beta-casein affects
gastrointestinal motility and inflammation through the release of beta-casomorphin-7. We aimed to evaluate differences in
gastrointestinal effects in a human adult population between milk containing A1 versus A2 beta-casein.
SUBJECTS/METHODS: Forty-one females and males were recruited into this double-blinded, randomised 8-week cross-over study.
Participants underwent a 2-week dairy washout (rice milk replaced dairy), followed by 2 weeks of milk (750 ml/day) that contained beta-
casein of either A1 or A2 type before undergoing a second washout followed by a final2weeksofthealternativeA1orA2typemilk.
RESULTS: The A1 beta-casein milk led to significantly higher stool consistency values (Bristol Stool Scale) compared with the A2
beta-casein milk. There was also a significant positive association between abdominal pain and stool consistency on the A1 diet
(r= 0.520, P= 0.001), but not the A2 diet (r=−0.13, P= 0.43). The difference between these two correlations (0.52 versus −0.13) was
highly significant (Po0.001). Furthermore, some individuals may be susceptible to A1 beta-casein, as evidenced by higher faecal
calprotectin values and associated intolerance measures.
CONCLUSIONS: These preliminary results suggest differences in gastrointestinal responses in some adult humans consuming milk
containing beta-casein of either the A1 or the A2 beta-casein type, but require confirmation in a larger study of participants with
perceived intolerance to ordinary A1 beta-casein-containing milk.
European Journal of Clinical Nutrition (2014) 68, 994–1000; doi:10.1038/ejcn.2014.127; published online 2 July 2014
INTRODUCTION
Cows’milk contains ~ 32 g of protein per litre, of which ~ 80% is
casein protein and ~ 20% is whey.
1
Beta-casein is the second most
abundant casein type in cows’milk and comprises ~ 30% of total
milk protein.
2
There are two families of beta-casein proteins,
known as A1 and A2 beta-casein ‘types’.
3
The A1 type variant
arose in European herds from the original A2 type ~ 5000–10 000
years ago from a Proline
67
to Histidine
67
point mutation.
3
In
countries that have dairy cows of northern European ancestry, the
relative proportions of the co-dominant A1 to A2 beta-casein
alleles are typically 1:1 in cows, which then produce the same ratio
of A1 to A2 beta-casein in milk. This tends to be lower in breeds
from Southern Europe. However, this ratio depends on the specific
breeding history of the dominant breeds.
4
Once milk or milk
products are consumed, the action of digestive enzymes in the
gut on A1 beta-casein releases the bioactive opioid peptide beta-
casomorphin-7 (BCM-7);
4–8
in contrast, A2 beta-casein releases
much less and probably minimal amounts of BCM-7 under normal
gut conditions.
7–10
BCM-7 is a mu-opioid receptor ligand,
8,11
and
mu-opioid receptors are expressed widely throughout human
physiology, including the gastrointestinal tract.
12
Two animal studies have investigated the effects of A1 versus
A2 beta-casein on gastrointestinal effects directly.
13,14
Barnett
et al.
14
showed that feeding rodents milk containing A1
beta-casein resulted in significantly delayed gastrointestinal transit
time compared with milk containing A2 beta-casein.
14
This delay
could be eliminated by administration of the opioid blocker
naloxone, which suggests that the gastrointestinal transit delay with
A1 feeding is an opioid-mediated effect. They also demonstrated a
significant 40% upregulation of dipeptidyl peptidase-4 in the
jejunum of A1- relative to A2-fed rodents.
14
Dipeptidyl peptidase-4
not only breaks down BCM-7 quickly
15
but it also degrades the gut
incretin hormones rapidly;
16
in humans, the incretin hormones
modulate insulin and glucose metabolism,
17
gastric emptying
18
and
antroduodenal motility.
19,20
Interestingly, Barnett et al.
14
also
showed that A1 feeding relative to A2 feeding significantly
increased the colonic activity of the inflammatory marker
myeloperoxidase by ~65%, an effect also negated by the opioid
blocker naloxone. Similarly, Haq et al.
13
showed in mice fed a
milk-free basal diet supplemented with A1 relative to A2 beta-
casein that MPO levels were increased significantly by 204%,
whereas A2 beta-casein had no effect relative to controls.
13
Further,
they showed significant increases in intestinal interleukin-4,
immunoglobulin E and leukocyte infiltration with A1 compared
with A2 feeding.
13
Intestinal inflammation disturbs colonic micro-
biota composition and enhances pathogen growth, which can
affect stool composition and output.
21
BCM-7 has also been reported to alter human intestinal
lymphocyte proliferation.
22,23
In vitro, BCM-7's effects on human
colon goblet-like cells (HT29-MTX cells) include increasing mRNA
1
School of Public Health, Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia;
2
Agricultural Management Group, Lincoln University, Christchurch,
New Zealand and
3
A2 Dairy Products Australia Pty Ltd., Melbourne, Victoria, Australia. Correspondence: Professor S Pal, School of Public Health, Curtin Health Innovation Research
Institute, Curtin University, GPO Box U1987, Perth, WA 6845, Australia.
E-mail: s.pal@curtin.edu.au
Received 14 March 2014; revised 9 May 2014; accepted 24 May 2014; published online 2 July 2014
European Journal of Clinical Nutrition (2014) 68, 994–1000
© 2014 Macmillan Publishers Limited All rights reserved 0954-3007/14
www.nature.com/ejcn
concentration of the mucin MUC5AC, depending on mu-opioid
receptor activation.
24
BCM-7 also induces rapid secretion of
intestinal mucus through the activation of the enteric nervous
system and opioid receptors.
25
More recently, bovine BCM-7 has
been detected in the jejunal effluents in humans fed 30 g of casein
in amounts compatible with a biological action,
5
which confirms
the identification ~30 years earlier of immunoreactive BCM-7
materials in the aspirated small intestinal contents of healthy male
adults following milk intake.
26
Bovine immunoreactive BCM-7 has
also been detected in the blood of human infants fed cows’milk-
based infant formula;
27,28
Kost et al.
27
showed with chromato-
graphic characterisation that a material with the same molecular
mass and polarity as BCM-7 was contained in the immunoreactive
BCM-7 of those infants who were fed formula.
27
As A1 beta-casein can result in the production of the opioid
BCM-7
6–9
and because Barnett et al.
14
have shown opioid-related
gastrointestinal effects with A1 but not with A2 beta-casein
feeding (by comparing saline to naloxone), a physiologically
plausible mechanism exists by which milk containing A1 beta-
casein may be responsible for a range of gastrointestinal effects
described above. However, no studies have assessed whether A1
relative to A2 beta-casein-containing milk imparts different
gastrointestinal effects in human adults. The aim of this study
was to compare the gastrointestinal effects of dietary A1 versus A2
beta-casein-containing milk in adults using subjective and
objective measures of gastrointestinal performance.
MATERIALS AND METHODS
Study design and participants
This 8-week cross-over study saw 12 men and 29 women (19–68 years)
from Perth, Western Australia, randomised to one of two groups for
2 weeks, following a 2-week dairy washout in which rice milk substituted
dairy milk: (1) milk containing beta-casein of A1 type (n= 21); or (2) milk
containing beta-casein of A2 type (n= 20) (Figure 1). Participants under-
went a second 2-week dairy washout before crossing to the alternative
milk intervention for another 2 weeks. Of the randomised participants at
study entry, a subgroup (n= 10) had self-reported intolerance to
commercial milk, containing a mix of A1 and A2 beta-casein. Exclusion
criteria were as follows: (1) milk allergy; (2) diagnosed lactose intolerance;
(3) pregnancy/ lactation; (4) cardiovascular events in the last 6 months; (5)
opioid consumption; (6) antibiotic treatment in the previous 8 weeks; and
(7) immunosuppressive medication or anti-inflammatory drugs in the
4 weeks before screening. Study recruitment and intervention was
conducted from November 2011 to October 2012. Participants were
randomised in the order of recruitment using a simple sequence
generated from www.randomization.com by the researcher (SH). This
study was approved by the Curtin University Human Research Ethics
A1 Milk
(n=21)
Telephone Screening (n=79)
Dairy Washout 1: 14 days (n=41)
Randomisation
Crossover 1: 14 days (n=41)
A2 Milk
(n=20)
Provided consent (n=42)
Withdrew
(n=3)
Withdrew
(n=1)
At 4 weeks
(n=18)
A1 Milk
(n=19)
Dairy Washout 2: 14 days (n=37)
Crossover 2: 14 days (n=36)
A2 Milk
(n=17)
Excluded (n=37)
Did not meet criteria (n=10)
Declined to participate (n=27 )
Declined to participate (n=1)
End of Trial (n=36)
At 4 weeks
(n=19)
Withdrew from A1
group (n=1)
Figure 1. Participant flowchart.
Intestinal effects of A1 vs A2 beta-casein
SHoet al
995
© 2014 Macmillan Publishers Limited European Journal of Clinical Nutrition (2014) 994 –1000
Committee (HR 102/2011) and written informed consent was obtained
from all participants.
Interventions
Washout rice milk. Participants replaced all dairy milk with supplied rice
milk (So Natural Rice Milk, Freedom Foods, Taren Point, NSW, Australia) for
both 2-week washouts and were instructed to avoid all other dairy. A dairy-
free alternative list and information relating to hidden dairy sources was
provided.
A1 and A2 beta-casein diets. During the 2-week A1 and A2 beta-casein
interventions, participants were instructed to consume 750 ml/day of their
allocated milk (containing ~ 7.5 g of either A1 or A2 beta-casein) over the
day and to avoid all other dairy products. Both milk products were
produced in November 2011, at Leppington Pastoral Company, NSW,
Australia, by cows genotyped as homozygous for A1 beta-casein (A
1
A
1
)or
A2 beta-casein (A
2
A
2
) based on genotyping tail hair follicle material, which
was performed at Genomnz (AgResearch Invermay Agricultural Centre,
Mosgiel, New Zealand). Milk was processed and packed in identical 1-l UHT
plain packages (blinding participants and the investigator to each milk
intervention) by Pactum Australia Pty Limited, Taren Point, NSW, Australia.
The A1 and A2 milk were both standardised to the following nutrition
profile per 100 ml: energy 189 kJ, total protein 3.1 g, total fat 2.5 g and
lactose 5.2 g; no other known differences existed. Nano-liquid chromato-
graphy electrospray ionisation mass spectrometry analysis (Australian
Proteome Analysis Facility, Macquarie University, Sydney, NSW, Australia)
of the A1 and A2 milk showed that the A1-type beta-casein proportion of
total beta-casein was 499% in the A1 milk and ⩽0.5% in the A2 milk.
Participants recorded their daily milk intake on compliance calendars.
Assessments
Participants attended four clinical visits, including baseline visits after both
dairy washouts and assessment visits after consuming the A1 and A2 diets.
Anthropometry, diet and physical activity measurements. At all visits,
anthropometric measurements were collected in the School of Public
Health Research Clinic at Curtin University. Height was measured without
shoes to the nearest 0.5 cm using a stadiometer. Weight was measured
using a digital scale (Omron, Kyoto, Japan). BMI was calculated as kg/m
2
.
During the first washout, at the start and during the milk interventions,
participants kept a 3-day household measures food diary on two weekdays
and one weekend day to monitor dietary intake. Data were analysed with
Foodworks Professional 2007, Xyris Software, Kenmore Hills, QLD, Australia
based on data from the AUSNUT database. At each visit, participants
completed the IPAQ (International Physical Activity Questionnaire)
29
to
monitor physical activity.
Gut inflammation. Faecal calprotectin is a non-invasive marker of
gastrointestinal inflammation.
30,31
Participants collected faecal samples at
home on the morning of each of the two assessment visits using kits
provided. Several heterogeneous stool portions were collected from the
day’sfirst stool passed onto the provided collection tray. Specimens were
stored at Curtin University at −80 °C before being sent to Dorevitch
Laboratories (Heidelberg, Victoria, Australia) for assessment. Faecal
calprotectin was measured by a single-step enzyme-linked immunosor-
bent asssay using antibodies against six epitopes found on the calprotectin
molecule.
Gastrointestinal symptom recording. Participants recorded symptoms of
bloating, abdominal pain, flatus and difficulty in voiding as they occurred
in a Symptom Report Diary according to a severity scale (0 = none; 1 = mild;
2 = moderate; 3 = severe) on all days during both interventions and during
dairy washouts. The validated Bristol Stool Scale (BSS) participant-
recording system
32
was used to assess bowel frequency (number of
bowel motions/day) and stool consistency (1 = separate hard lumps like
nuts; 2 = sausage-shaped but lumpy; 3 = like a sausage or snake but with
cracks on its surface; 4 = like a sausage or snake, smooth and soft; 5 = soft
blobs with clear-cut edges; 6 = fluffy pieces with ragged edges, a mushy
stool; 7 = watery, no solid pieces).
Sample size. There are no data available on the effects of A1 relative to A2
beta-casein-containing milk on gastrointestinal symptoms in humans, and
as such this study must be considered a pilot study so that powering of
future studies can be performed.
Statistical analysis
Statistical analyses were conducted using IBM SPSS Statistics Version 20
(IBM Corp., Chicago, IL, USA). Output data were first tested for normality
(Kolmogorov–Smirnov test), and depending on outcomes they were
analysed using either parametric paired t-tests (physical activity and mean
2-week Bristol Stool analyses) or non-parametric Wilcoxon signed-rank test
(faecal calprotectin, bloating, abdominal pain, flatus and voiding difficulty).
Parametric analyses are presented as means ±s.e.m., whereas non-parametric
analyses are presented as means for descriptive purposes together with
non-parametric statistics as appropriate to the specific comparison. Linear
associations between measures are reported as Pearson’sr.
RESULTS
Baseline characteristics
Baseline data following the first washout are presented as
means ± s.d. and range (Table 1). There were no between-
treatment group differences before the study commencement
or at the start of intervention 1.
Study attrition, adherence and changes in milk, calcium, energy
and fibre intake
Four (9.8%) participants withdrew from the study (one from the A2
and three from the A1 diets) and one failed to provide a symptom
diary (Figure 1). Two withdrawals were from the self-identified
milk-intolerant subgroup. Mean compliance with the A1 and A2
diets was 96.2% (±5.3) and 96.4% (±6.6), respectively. Greater than
100% compliance stems from some participants consuming extra
study milk in tea/coffee/food. There were no significant between-
group differences for milk, energy, fibre or calcium intakes during
the intervention.
Stool consistency and bowel frequency
Stool consistency was assessed using the BSS (Table 2). BSS was
analysed as 2-week mean values for each participant on the A1
and A2 diets. Stool consistency values on the BSS were
significantly higher on the A1 diet compared with the A2 diet
when all participants were assessed, and this result was retained
when self-identified milk tolerants were considered alone
(Table 2). This result was stronger (both size effect and
significance) for women alone (Table 2). There were no significant
treatment order effects (data not shown). There were no
significant differences between the A1 and A2 diets for bowel
frequency, although a notable feature was considerable within-
group variation, ranging from 0.43 to 3.6 under A1 and from 0.36
to 4.5 under A2 (data not shown).
Subjective measures of intolerance symptoms
Bloating, abdominal pain, flatus and voiding difficulty, as reported
by all participants, were analysed as measures of digestive
discomfort. Although all mean values were numerically higher
on the A1 diet, none were statistically significant. For those who
self-identified as milk intolerant (n= 8), the mean A1 values were
considerably higher than A2 values for bloating (61% higher),
abdominal pain (38% higher) and voiding difficulty (83% higher).
However, given the small participant numbers in the self-
identified milk-intolerant group, it was not possible to demon-
strate statistically significant differences. In relation to these
subjective measures, there was evidence of an order of treatment
effect. For cases where the A1 diet was consumed first, bloating
and flatus were both significantly higher on the A1 than on the A2
diet (P= 0.05 and 0.048, respectively). For participants who
Intestinal effects of A1 vs A2 beta-casein
SHoet al
996
European Journal of Clinical Nutrition (2014) 994 –1000 © 2014 Macmillan Publishers Limited
consumed the A1 diet second, there were no significant
differences between the diets in any of these measures.
Cross-correlations by treatment
There were strong cross-correlations between the four subjective
intolerance measures on both diets (Table 3). The flatus with
bloating correlation on the A1 diet was significantly higher than
the correlation on the A2 diet (r= 0.63 versus r= 0.25, P= 0.02).
There was also a significant positive association between
abdominal pain and stool consistency on the A1 diet (r= 0.520,
P= 0.001), providing evidence that greater pain on the A1 diet is
associated with softer stool. In contrast, there was no relationship
between these two measures on the A2 diet (r=−0.13, P= 0.43).
The difference between these two correlations (0.52 versus −0.13)
was highly significant (Po0.001).
Faecal calprotectin
There were no overall differences in faecal calprotectin (FC)
between the A1 and A2 diets (mean values of 41.6 versus 35.8 μg/g
and median values of 15 versus 14 μg/g). Most cases fell within
the normal cutoff (o50 μg/g). However, eight cases stood out
from the others (Table 4). Five of these standout cases had FC
values of ⩾50 μg/g for both the A1 and A2 diets, and all of these
had the A1 diet first. Another three standout cases had FC values
4100 μg/g on the A1 diet, but o50 μg/g on A2 (Table 4). The five
cases with high FC values on both diets also had a general
tendency to have high values for the four subjective intolerance
measures relative to median values on both diets (Table 4).
Interestingly, those with high FC values on the A1 diet but not on
the A2 diet tended to have high subjective intolerance measures
for the A1 diet but not the A2 diet.
There were strong and statistically significant correlations
between FC and subjective intolerance measures when partici-
pants were on the A1 diet (Table 5). There was also a particularly
strong association with a composite index comprising these four
measures summed. When participants were on the A2 diet, these
relationships were absent in relation to bloating and abdominal
pain and considerably weaker on the composite measure, but still
present in relation to flatus and voiding difficulty. The difference in
the correlation measures between the A1 and A2 diets was
significant for abdominal pain (0.46 vs 0.03; P= 0.02) and bloating
(0.36 vs −0.02; P= 0.05).
DISCUSSION
In this study, the BSS measure of stool consistency was
significantly higher on the A1 versus A2 beta-casein diet, and
this finding was retained when self-identified milk intolerants
were excluded. The appropriate interpretation to be placed on
these BSS results requires careful consideration.
It has been shown that extremes in stool formation may reflect
gastrointestinal transit time,
33,34
where softer stools reflect faster
transit time. However, Davies et al.
35
have shown that BSS may not
always reflect the speed of gut transit excursions.
35
Importantly,
Barnett et al.
14
have shown clearly that A1 beta-casein feeding
delays gut transit through an opioid pathway in rats
14
and
confirmed earlier rodent study results
13
that A1 compared with A2
beta-casein feeding increases gut inflammation significantly,
as evidenced by myeloperoxidase levels. Together, these studies
are suggestive of the fact that the significantly higher BSS values
Table 2. Bristol Stool Scale analyses of stool consistency (mean ±s.e.m.)
Group A1 A2 Difference A1 −A2 P-value for paired t-test
All participants (n=36) 3.87 (0.11) 3.56 (0.15) 0.31 (0.14) 0.04
Women only (n=25) 3.93 (0.15) 3.50 (0.16) 0.43 (0.16) 0.01
Men only (n=11) 3.72 (0.15) 3.70 (0.31) 0.02 (0.28) 0.95
Self-described as milk tolerant (n=28) 3.82 (0.12) 3.47 (0.16) 0.35 (0.17) 0.04
Self-described as milk intolerant (n=8) 4.02 (0.28) 3.87 (0.34) 0.16 (0.29) 0.63
Table 1. Baseline characteristics of all participants (mean ±s.d. (range))
Characteristic All Self-described as milk
tolerant (n= 27) or
not described (n=1)
Self-described
as milk intolerant
(n=8)
Starting anthropometric characteristics
Age (years) 45.5 ±15.7 (19–68) 44.1 ±15 (21–68) 50.2 ±18.1 (19–66)
Height (cm) 165.8 ±7 (149–182) 166.4 ±(149–182) 163.6 ±6.7 (154–175.5)
Weight (kg) 69.2 ±14.8 (47.2–110.8) 68.2 ±14.4 (47.2–110.8) 72.6 ±16.8 (51.2–98.7)
BMI (kg/m
2
) 25.2 ±5.2 (16.5–43) 24.7 ±5.3 (16.5–43) 26.9 ±5 (20.6–36.5)
Systolic BP (mm Hg) 117.3 ±16.9 (82–157) 117.6 ±15.5 (93–152) 116.2 ±22.4 (82–157)
Diastolic BP (mm Hg) 73.5 ±9.6 (53–95) 74.2 ±8.8 (63–95) 71.1 ±12.5 (53–93)
Usual dietary characteristics
Energy (kJ/day) 7982 ±2482 (2901–13 842) 8278 ±2425 (2901–13842) 7058 ±2757 (3460–12 564)
Fibre (g/day) 24 ±11 (7–55) 24 ±9(7–49) 26 ±16 (9–55)
Milk (ml/day) 144 ±176 (0–633) 170 ±190 (0–633) 57 ±78 (0–185)
a
Calcium (mg/day) 842 ±401 (218–1639) 853 ±380 (218–1474) 808±492 (347–1639)
Usual physical activity
Physical activity (Met-min/week) 2330 ±3025 (0–13608) 2100 ±2461 (0–11 304) 3137 ±4631 (132–13 608)
Abbreviations: BMI, body mass index; BP, blood pressure. Anthropometric characteristics n=36 (11 male, 25 female); dietary characteristics n=35
(11 male, 24 female).
a
P=0.019 between tolerant and intolerant.
Intestinal effects of A1 vs A2 beta-casein
SHoet al
997
© 2014 Macmillan Publishers Limited European Journal of Clinical Nutrition (2014) 994 –1000
on A1 compared with A2 beta-casein diets are caused by
proinflammatory factors. This is reinforced by prior evidence that
intestinal inflammation is associated with malabsorption of fluids,
nutrients and electrolytes.
36,37
This explanation is also consistent
with the significant and positive association between abdominal
pain and stool consistency on the A1 diet.
Cows’milk is cited commonly as a cause of symptoms such as
bloating, abdominal distension, flatulence and disturbed voiding
(that is, digestive discomfort), and in the majority of cases lactose
may not be the mediator.
38–40
Given prior evidence that A1
beta-casein feeding can delay intestinal transit,
14
an alternative
explanation is that A1 beta-casein could create greater opportu-
nities for food fermentation and hence digestive discomfort within
the gastrointestinal system. Although the differences in digestive
discomfort measures between the two diets were not statistically
significant for this predominantly milk-drinking cohort of people,
the effect sizes suggest that this may be possible. However, a
much larger study of susceptible people is needed to either
confirm or refute this hypothesis.
The current pilot study shows three cases with abnormally high
FC values following 14 days of exposure to the A1 but not A2 beta-
casein diet. These case study FC results are consistent with prior
research regarding the pro-inflammatory characteristics of A1
beta-casein.
13,14
However, in themselves, these cases are insuffi-
cient to provide any conclusion. As with intolerance symptoms,
the present study protocol could have mitigated against high FC
rates as a consequence of susceptible people being either
unwilling to enrol or predisposed to study non-completion.
Considering all cases, it is apparent that there is overall
evidence for cross-correlation between subjective measures of
intolerance. There is also evidence for correlation between FC
values and subjective intolerance measures and also between
these measures of digestive discomfort and stool consistency. This
provides support for a finding that perceived symptoms of
digestive discomfort have a physiological basis. It is both notable
and intriguing that there is overall suggestion for these relation-
ships being stronger on the A1 diet.
CONCLUSION
Our pilot study demonstrated that consuming the A1 beta-casein
milk led to significantly higher BSS stool consistency values
compared with the A2 beta-casein milk among a normal milk-
drinking population. This finding may be linked to the known
digestive release of BCM-7 from milk containing A1 beta-casein.
FC values correlated highly with subjective measures of digestive
discomfort on the A1 diet but less so on the A2 diet. We also
showed for the A1 diet that greater abdominal pain is associated
with softer stool. Furthermore, some individuals may be
susceptible to A1 beta-casein as evidenced by higher FC values
and associated intolerance measures. These intolerance and
Table 4. Faecal calprotectin outlier cases and associated gastrointestinal measures
Characteristic Three cases with high FC on A1 but not A2
a
Five cases with high FC on both A1 and A2
a
Median for all
cases (n= 36)
Faecal calprotectin
A1 diet 427 102 130 103 81 51 131 61 14
A2 diet 32 38 30 367 171 50 129 53 14
Bloating
A1 diet 1.5 0.0 0.3 0.0 1.0 0.6 0.0 0.6 0.0
A2 diet 0.0 0.0 0.1 0.0 1.0 0.6 0.0 0.0 0.0
Abdominal pain
A1 diet 1.4 0.0 0.0 0.0 0.8 0.4 0.4 0.0 0.0
A2 diet 0.0 0.0 0.0 0.0 1.1 0.8 0.0 0.0 0.0
Flatus
A1 diet 2.0 0.2 1.3 1.8 1.5 1.2 1.1 1.9 0.5
A2 diet 2.0 0.0 1.4 1.4 1.5 1.0 1.2 1.3 0.6
Voiding difficulty
A1 diet 1.0 0.2 1.1 0.1 0.8 1.0 0.0 0.0 0.0
A2 diet 0.0 0.0 0.0 1.3 0.8 1.2 0.0 0.0 0.0
Stool consistency
A1 diet 5.2 3.7 3.1 3.0 3.5 4.2 3.6 3.7 4.0
A2 diet 4.0 1.4 3.8 3.3 3.1 3.8 3.5 3.8 3.8
Bowel frequency
A1 diet 2.1 0.9 0.6 0.9 0.9 1.1 2.4 2.0 1.3
A2 diet 1.5 0.7 0.6 1.0 1.3 1.2 2.1 2.0 1.3
Abbreviation: FC, faecal calprotectin.
a
Of the three cases with high FC on A1 but not A2, two cases had the A2 diet first. All other cases had the A1 diet first.
Table 3. Correlations (Pearson’sr) for subjective measures of
intolerance on the A1 and A2 diets
Characteristic Bloating Abdominal pain Flatus
A1 diet
Bloating 1.00
Abdominal pain 0.61*** 1.00
Flatus 0.63*** 0.44
**
1.00
Voiding difficulty 0.51
**
0.15 0.27
‡
A2 diet
Bloating 1.00
Abdominal pain 0.61*** 1.00
Flatus 0.25 0.14 1.00
Voiding difficulty 0.39* 0.24 0.29
†
*Po0.05; **Po0.01; ***Po0.001;
†
P=0.08;
‡
P=0.11.
Intestinal effects of A1 vs A2 beta-casein
SHoet al
998
European Journal of Clinical Nutrition (2014) 994 –1000 © 2014 Macmillan Publishers Limited
abnormally high FC results require confirmation with a larger
study of participants with perceived intolerance to ordinary A1
beta-casein-containing milk.
CONFLICT OF INTEREST
Dr Sonja Kukuljan is a salaried employee of A2 Dairy Products Australia. Professor
Keith Woodford consults to A2 Corporation as an independent scientific adviser. The
remaining authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This study was supported by a grant from A2 Dairy Products Australia, who also
supplied the milk. A2 Dairy Products Australia had no role in the data analysis of
this study.
AUTHOR CONTRIBUTIONS
All authors contributed to the research design (project conception, develop-
ment of overall research plan and study oversight). SH and SP conducted the
research (hands-on conduct of the experiments and data collection). KW
analysed the data and performed statistical analyses. SK, KW, SH and SP wrote
the paper. All authors had primary responsibility for the final content.
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Table 5. Correlations (Pearson’sr) between faecal calprotectin and
measures of intolerance on each of the A1 and A2 beta-casein diets
Characteristic FC on A1 diet
(n=36)
P-value FC on A2 diet
(n= 36)
P-value
Bloating 0.36 0.030 −0.02 0.930
Abdominal pain 0.46 0.005 0.03 0.880
Flatus 0.39 0.020 0.32 0.060
Voiding difficulty 0.35 0.040 0.56 0.001
Composite index of four subjective
intolerance measures
a
0.50 0.002 0.32 0.060
Stool consistency 0.14 0.420 −0.11 0.510
Bowel frequency 0.01 0.970 −0.11 0.510
Abbreviation: FC, faecal calprotectin.
a
The composite index comprises the
sum of bloating, abdominal pain, flatus and voiding difficulty.
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