Effects of cow's milk beta-casein variants on symptoms of milk intolerance in Chinese adults: A multicentre, randomised controlled study

Abstract

Abstract Background A major protein component of cow’s milk is β-casein. The most frequent variants in dairy herds are A1 and A2. Recent studies showed that milk containing A1 β-casein promoted intestinal inflammation and exacerbated gastrointestinal symptoms. However, the acute gastrointestinal effects of A1 β-casein have not been investigated. This study compared the gastrointestinal effects of milk containing A1 and A2 β-casein versus A2 β-casein alone in Chinese adults with self-reported lactose intolerance. Methods In this randomised, crossover, double-blind trial, with a 3-day dairy washout period at baseline, subjects were randomised to consume 300 mL of milk containing A1 and A2 β-casein (ratio 58:42; conventional milk) or A2 β-casein alone; subjects consumed the alternative product after a 7-day washout period. Urine galactose was measured at baseline after a 15 g lactose load. Subjects completed 9-point visual analogue scales for gastrointestinal symptoms (borborygmus, flatulence, bloating, abdominal pain, stool frequency, and stool consistency) at baseline and at 1, 3, and 12 h after milk consumption. Results A total of 600 subjects were included. All six symptom scores at 1 and 3 h were significantly lower after consuming A2 β-casein versus conventional milk (all P

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R E S E A R C H Open Access
Effects of cow’s milk beta-casein variants
on symptoms of milk intolerance in
Chinese adults: a multicentre, randomised
controlled study
Mei He
1
, Jianqin Sun
2
, Zhuo Qin Jiang
3
and Yue Xin Yang
4,5*
Abstract
Background: A major protein component of cow’s milk is β-casein. The most frequent variants in dairy herds are
A1 and A2. Recent studies showed that milk containing A1 β-casein promoted intestinal inflammation and
exacerbated gastrointestinal symptoms. However, the acute gastrointestinal effects of A1 β-casein have not been
investigated. This study compared the gastrointestinal effects of milk containing A1 and A2 β-casein versus A2 β-
casein alone in Chinese adults with self-reported lactose intolerance.
Methods: In this randomised, crossover, double-blind trial, with a 3-day dairy washout period at baseline, subjects
were randomised to consume 300 mL of milk containing A1 and A2 β-casein (ratio 58:42; conventional milk) or A2
β-casein alone; subjects consumed the alternative product after a 7-day washout period. Urine galactose was
measured at baseline after a 15 g lactose load. Subjects completed 9-point visual analogue scales for
gastrointestinal symptoms (borborygmus, flatulence, bloating, abdominal pain, stool frequency, and stool
consistency) at baseline and at 1, 3, and 12 h after milk consumption.
Results: A total of 600 subjects were included. All six symptom scores at 1 and 3 h were significantly lower after
consuming A2 β-casein versus conventional milk (all P<0.0001). At 12 h, significant differences remained for
bloating, abdominal pain, stool frequency, and stool consistency (all P<0.0001). Symptom scores were consistently
lower with A2 β-casein in both lactose absorbers (urinary galactose ≥0.27 mmol/L) and lactose malabsorbers
(urinary galactose <0.27 mmol/L).
Conclusion: Milk containing A2 β-casein attenuated acute gastrointestinal symptoms of milk intolerance, while
conventional milk containing A1 β-casein reduced lactase activity and increased gastrointestinal symptoms
compared with milk containing A2 β-casein. Thus, milk-related gastrointestinal symptoms may result from the
ingestion of A1 β-casein rather than lactose in some individuals.
Trial registration: NCT02878876, registered August 16, 2016. Retrospectively registered.
Keywords: Beta-casein, Lactase, Lactose, Intolerance
* Correspondence: yxyang@263.net
4
Department of Food Nutrition, National Institute for Nutrition and Health,
Chinese Center for Disease Control and Prevention, Beijing, China
5
Chinese Nutrition Society, 6# Guang An Men Nei. Street, Fenghua Square,
Building A, Unit 5, Room 1601/1602, Xuanwu District, Beijing 100053,
People’s Republic of China
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
He et al. Nutrition Journal (2017) 16:72
DOI 10.1186/s12937-017-0275-0
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Background
β-casein is a major protein component of cow’s milk,
and numerous variants have been described, including
the A1 and A2 types. The A1 and A2 types differ in
terms of the amino acid at position 67, being histidine in
the A1 type and proline in A2 β-casein. The presence of
histidine in the A1 type increases the protein’s suscepti-
bility to cleavage of the preceding seven amino acids,
yielding β-casomorphin-7 (BCM-7), an exorphin with
moderate agonistic activity on μ-receptors [1–3]. The
knowledge that A1 β-casein may yield a functionally ac-
tive exorphin has propelled research to understand
whether A1 β-casein and BCM-7 affect gastrointestinal
function or cause/exacerbate gastrointestinal inflamma-
tion in cell/animal models [4–9] and in people with milk
or lactose intolerance [10, 11].
Thus far, however, very few studies have compared the
effects of A1 and A2 β-casein on the gastrointestinal sys-
tem in humans. In one recent study by Jianqin et al., 45
Chinese adults with self-reported intolerance to milk
were randomised to receive milk containing either A2 β-
casein or milk containing A1 and A2 β-casein (ratio
40:60; conventional milk) in a double-blind, 2×2 cross-
over manner with a washout period of 2 weeks before
the study and between each phase [11, 12]. In that study,
consumption of conventional milk was associated with
increases in symptoms of post-dairy digestive discomfort
and longer gastrointestinal transit times compared with
milk containing only A2 β-casein. Consumption of con-
ventional milk was also associated with increased small
intestine inflammation and increased serum inflamma-
tory biomarkers, as well as increased serum BCM-7 con-
centrations. In addition, when the subjects were divided
according to their lactase activity based on urinary gal-
actose tests, subjects with lactase deficiency reported
greater gastrointestinal disturbances after consuming
conventional milk compared with subjects with normal
lactase activity, and that neither subgroup reported
worsening of symptoms when they consumed milk con-
taining A2 β-casein. Lactase deficiency was confirmed
based on the subject’s reduced ability to metabolise
lactose.
The results of this study prompted the hypothesis that
the acute symptoms of milk intolerance (including self-
reported lactose intolerance) in some people might be
related to the presence of A1 β-casein in cow’s milk and
that eliminating A1 β-casein could avoid these symp-
toms. Accordingly, the present study was designed to
further evaluate the results of the study by Jianqin et al.
[11, 12] by enrolling a much larger sample of subjects
with self-symptoms of milk intolerance. Our primary ob-
jective was to compare the acute effects of milk contain-
ing either A2 β-casein or both A1 and A2 β-casein
(conventional milk) on gastrointestinal symptoms in
subjects with self-reported lactose intolerance and
gastrointestinal discomfort. Our secondary objective was
to examine whether these symptoms were correlated
with lactase activity or the ability of these subjects to
metabolise lactose.
Methods
Ethics
This three-centre, two-way parallel-group, crossover,
randomised, double-blind controlled study was con-
ducted in accordance with the Declaration of Helsinki
(1996), Good Clinical Practice, and applicable regulatory
requirements. The protocol was approved by the central
institutional review board of the Shanghai Nutrition
Society. All subjects provided written informed consent
before study entry. The trial was registered on Clinical-
Trials.gov with the ID NCT02878876.
Subjects
Subjects were enrolled by the Chinese National Nutri-
tion Society across three sites (Beijing, Guangzhou, and
Shanghai). Males or females aged 20–50 years with self-
reported lactose intolerance and digestive discomfort
after consuming traditional milk were eligible for this
study. Subjects were excluded if they had any of the fol-
lowing: any eating disorder, metabolic and/or chronic
disease that was deemed likely to interfere with the
study outcome measures, other than lactose intolerance;
acute infection or gastroenteritis at time of enrolment;
gastrointestinal disease likely to interfere with the study
outcome measures, such as gastroesophageal reflux dis-
ease, irritable bowel disease, or Crohn’s disease; known
allergy to cow’s milk products; doctor-diagnosed
immunodeficiency and any severe disease. Because the
incidence of lactose intolerance may vary with age, we
planned to enrol approximately similar numbers of sub-
jects in two age groups: 20–35 and 36–50 years.
Study products
Milk containing either A1 and A2 β-casein (conventional
milk) or only A2 β-casein was provided by The a2 Milk
Company (Shanghai, China). Both products were con-
firmed to be identical (Table 1), except for the β-casein
content. The ratio of A1 β-casein to A2 β-casein in con-
ventional milk was 58:42. A1 and A2 β-casein levels in
the study milk were measured by an independent labora-
tory (Analytica Laboratories, Ruakura, New Zealand)
using ultra-high-performance liquid chromatography–
diode array detector–tandem mass spectrometry.
Study design
Figure 1 shows the design of the study. After a screening
period, eligible subjects underwent urinary galactose
tests and completed a visual analogue scale (VAS) to
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assess their baseline gastrointestinal symptoms [13].
Subjects were then randomized to one of two treatment
sequences in which they received conventional milk on
Day 1 and milk containing A2 β-casein on Day 8, or vice
versa.
Randomisation was performed using a computer-
generated list prepared on site. Subjects were stratified
by age group (20–35 years/36–50 years) to either
sequence 1 (A1/A2➔A2) or sequence 2 (A2➔A1/A2) ac-
cording to the allocation numbers prepared in sealed
envelopes. The allocation list was generated using SAS
statistical software (SAS Institute, Cary, NC, USA).
Subjects returned to the study site on Day 1 after a 3-
day washout of dairy products and a 12-h overnight fast.
The subjects were instructed to avoid dairy products
during the washout, but were not provided any non-
dairy milk products during this time. At the study site,
the subjects provided a urine sample and consumed 300
mL of the allocated milk type in accordance with the
allocation schedule.
All milk products were provided in a double-blind
manner, and subjects used a food diary to record milk
intake and adherence to each intervention. In Beijing,
the study products were prepared and repackaged by
technicians assigned by the principal investigator. At the
other sites, the study products were prepared by the
sponsor. All products were identical in packaging and
were only labelled with the subject’s identification
number.
At 1 h after consuming the milk product, the subjects
ate breakfast and completed the VAS for gastrointestinal
symptoms. The breakfast was congee and a steamed bun
in Beijing, and comprised fried chicken, congee and
bread in Shanghai and Guangzhou. At 3 h, subjects
Table 1 Nutritional composition of the two types of milk
Nutrient (per 100 mL) A2 milk A1 milk
Energy (kJ) 278 270
Protein (g) 3.3 3.3
Fat (g) 3.7 3.5
Saturated fat (g) 2.4 2.1
Carbohydrate (g) 5 4.8
Sodium (mg) 37 45
Calcium (mg) 117 120
Lactose 5 4.8
A1 conventional milk containing A1 and A2 β-casein, A2 milk containing
A2 β-casein
Fig. 1 Study design. VAS, visual analogue scale
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provided a urine sample and completed the VAS for
gastrointestinal symptoms. After completing these
assessments, the subjects could leave the study site. At
12 h, subjects in Beijing and Guangzhou completed the
VAS for gastrointestinal symptoms. This VAS was com-
pleted via a telephone call. The 12-h follow up assess-
ment was not possible at Shanghai owing to limited
resources and logistical constraints.
The subjects continued a dairy-free diet for 7 days
and, on Day 8, they returned to the study site to repeat
the study procedures, and this time they consumed the
opposite milk product. The subjects were also asked to
complete food frequency questionnaires to assess dietary
adherence.
Objectives
The primary objective was to compare the effects of
consuming milk containing solely the A2 type of β-ca-
sein with conventional milk containing both A1 and A2
types of β-casein on acute self-recorded lactose intoler-
ance and gastrointestinal discomfort occurring within
several hours of consuming milk. Therefore, the primary
endpoint was gastrointestinal symptom scores assessed
by VAS. Secondary objectives included the following: (1)
to compare the effects of both milk products on lactase
activity, which was assessed in terms of urinary galactose
after an oral lactose load; (2) to compare/contrast the
shifts in lactase activity induced by the type of β-casein
with self-reported symptoms of lactose intolerance; and
(3) to determine whether age was correlated with the
symptoms of milk intolerance or urinary galactose, the
subjects were also divided into two age groups (20–35
years and 36–50 years). Consequently, urinary galactose,
as a surrogate marker of lactase activity, was included as
a secondary endpoint.
Gastrointestinal symptom scores
Baseline gastrointestinal symptoms were evaluated
before product intervention by asking the subjects to
report their symptoms at the last time they consumed
milk. The following gastrointestinal symptoms were
assessed by the VAS: borborygmus, flatulence, bloating,
abdominal pain, stool frequency, and stool consistency.
Each symptom was assessed using a 9-point scale, where
0 = not at all and 9 = very serious. Improvements in
gastrointestinal symptoms after consumption of milk
containing A2 β-casein relative to conventional milk
were then classified as follows: no symptoms; significant
improvement in symptoms (reduction in score of ≥4 for
that symptom); slight improvement in symptoms (reduc-
tion in score of 1 to ≤3 for that symptom); or no differ-
ence or worsening in symptoms (no change in score or
an increase in the score).
Food frequency questionnaire
Subjects were asked to complete food frequency question-
naires (Additional file 1: Table S1) to document adherence
to the dairy-free diet during the 3-day and 7-day washout
periods. The questionnaires were conducted by the inves-
tigators on days 1 and 8 at each study site. The question-
naires recorded the food intake in the previous 24 h. The
subjects also confirmed that no dairy product was con-
sumed during the washout period.
Urinary galactose
Urinary galactose was measured as an indicator of lac-
tase deficiency using a fluorescence spectrum analysis
method. To assess the effects of lactose malabsorption
on gastrointestinal symptoms, the subjects were divided
into two subgroups according to their change in urinary
galactose at 3 h after ingestion of 15 g of lactose at base-
line (300 mL of conventional milk). Individuals with a
urinary galactose concentration <0.9 mmol/L after a 50-
g oral lactose intake are considered lactose malabsorbers
[14, 15]. Therefore, we used 0.9*(15/50)=0.27 mmol/L as
the threshold to distinguish lactose absorbers from
malabsorbers.
Adverse events
Adverse events occurring any time between enrolment
and up to 30 days last assessment were to be recorded.
The subjects were asked to report adverse events via
telephone or at each visit. The adverse events were clas-
sified in terms of their seriousness and relationship with
the study product by the subject’s investigator.
Statistics
Sample size
Based on our clinical experience together with data
published in previous studies, it was determined to enrol
approximately 630 subjects (210 at each participating
site) to achieve a total of 600 subjects after accounting
for a dropout rate of 5%. The sample size was estimated
based on prior studies [11] reporting a confidence of p<
0.05 for upper gastrointestinal inflammation and the
greater sample size of our study was determined to be
sufficient to examine gastrointestinal symptoms puta-
tively linked to milk consumption. Enrolment was
stopped once the planned sample size had been enrolled.
Data analysis
Baseline characteristics and adverse events are sum-
marised descriptively in terms of the mean and standard
deviation, and number (%) of subjects. Gastrointestinal
symptoms were analysed using generalised estimating
equations (GEE) for ordinal repeated measures, with
fixed effects of study product (A1 or A2), study visit (1
or 2), and age group (20–35 or 36–50 years old), a
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random subject effect nested within the sequence of
study treatment (A1➔A2 or A2➔A1), and adjusted for
baseline symptom scores. Contrast tests were generated
to compare means for each product. Urinary galactose
concentrations were analysed using mixed effects ana-
lysis of variance with fixed effects of study product (A1
or A2), study visit (1 or 2) and age group (20–35 or 36–
50 years old), a random subject effect nested within the
sequence of study treatment (A1➔A2 or A2➔A1), and
adjusted for baseline symptom scores. Type III tests of
fixed effects were used to test the effect of study prod-
ucts. Contrast tests were generated to compare means
for each product. Differences in gastrointestinal symp-
toms and urinary galactose concentrations were analysed
using the Kruskal–Wallis test and analysis of variance,
respectively.
Considering the geographical (environmental and life-
style) differences between the three sites, and because
the VAS for gastrointestinal symptoms was not assessed
at 12 h in Shanghai, it was planned to analyse the data
for all three sites individually and then by all three sites
combined.
The residual plots for all VAS variables showed no vio-
lations of normality for data at 1 and 3 h, although some
skewness was apparent at 12 h. Accordingly, all VAS
scores are presented as the median (range).
Results
Subjects
The first subject was enrolled on January 18, 2016
(Shanghai), and the last subject completed the study on
April 19, 2016 (Beijing). A total of 1200 subjects were
initially screened and 642 started the study, 230 in
Beijing, 210 in Guangzhou, and 202 in Shanghai. In
Beijing, 13 subjects discontinued the study and question-
naires were not completed by 17 subjects. In addition,
10 subjects in Guangzhou and 2 in Shanghai discontin-
ued the study. Therefore, data were available for 600
subjects (200 per site). The characteristics of participat-
ing subjects are summarized in Table 2. All of the sub-
jects were non-regular milk drinkers and had not
consumed milk for at least 1 month before enrolment.
However, some subjects reported that they consumed
yoghurt up to twice a week (120 mL per time). The re-
sults from the food frequency questionnaire revealed
that all subjects that completed the study showed adher-
ence to a dairy-free diet during the washout periods.
Table 2 Subject characteristics
Site Beijing Guangzhou Shanghai
Sequence A1➔A2 (n=100) A2➔A1 (n=100) A1➔A2 (n=100) A2➔A1 (n=100) A1➔A2 (n=100) A2➔A1 (n=100)
n/mean %/SD n/mean %/SD n/mean %/SD n/mean %/SD n/mean %/SD n/mean %/SD
Males 56 56% 48 48% 39 39% 41 41% 52 52% 43 43%
Age (years) 37.2 8.3 35.5 8.5 36 7.6 36.3 7.6 35 8.9 35 9.3
20–35 years 50 50% 50 50% 50 50% 50 50% 50 50% 50 50%
30 4.6 28.3 4.2 39.8 4.4 30.1 4.3 27.3 5 26.9 4.1
36–50 years 50 50% 50 50% 50 50% 50 50% 50 50% 50 50%
44.3 3.7 42.7 4.8 42.2 4.2 42.5 4.4 42.6 3.8 43.2 4.6
Weight (kg) 69.7 11.5 68.7 11.3 60.1 10.4 60.4 11.2 62.7 12.1 63.5 11.3
Height (cm) 171.1 10 168.7 9.6 164.5 7.0 163.6 7.4 166.9 7.9 164.2 12
Site All sites
Sequence A1➔A2 (n=300) A2➔A1 (n=300)
n/mean %/SD n/mean %/SD
Males 147 49% 132 44%
Age (years) 36 8.3 35.6 8.5
20–35 years 150 50% 150 50%
29 4.8 28.4 4.4
36–50 years 150 50% 150 50%
43 4 42.8 4.6
Weight (kg) 64.2 12 64.2 11.7
Height (cm) 167.5 8.8 165.5 10.1
A1 conventional milk containing A1 and A2 β-casein, A2 milk containing A2 β-casein
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Gastrointestinal symptoms
There were no significant effects of visit or age group on
symptom scores (Additional file 1: Table S2). Therefore,
data are grouped according to the intervention received.
The gastrointestinal symptom VAS scores recorded are
summarised according to the study intervention in Table
3. As indicated in this table, the symptom scores for all
six symptoms were consistently lower with milk contain-
ing A2 β-casein than with conventional milk at both 1
and 3 h after consumption (all P<0.0001). The differ-
ences in symptom scores between the two products were
also observed at all three sites separately (Additional file
1: Table S3). Differences in symptom scores were still
apparent at 12 h for bloating, abdominal pain, stool fre-
quency, and stool consistency in Beijing and for abdom-
inal pain, stool frequency, and stool consistency in
Guangzhou.
Significant between-group differences in gastrointes-
tinal symptoms were also observed at 1 and 3 h between
the two groups when data for all three sites were com-
bined (all P<0.0001) (Table 3). At 12 h, significant differ-
ences remained for bloating, abdominal pain, stool
frequency, and stool consistency (all P<0.0001), but not
flatulence (P=0.059) or borborygmus (P=0.458). These
findings indicate that milk containing A2 β-casein was
associated with less severe gastrointestinal symptoms
within 1–12 h after consumption compared with con-
ventional milk.
We also classified gastrointestinal symptom scores ac-
cording to the direction of change at 1, 3, and 12 h rela-
tive to the baseline scores in terms of no symptoms,
significant improvement, slight improvement, and no
difference. As indicated in Additional file 1: Table S4
(subjects at each study site separately) and Table 4 (all
subjects combined), there was a greater trend towards
slight improvements in the gastrointestinal symptoms
when the subjects consumed milk containing A2 β-
casein as compared with their consumption of conven-
tional milk.
Effects of age on gastrointestinal symptoms
The effect of age on gastrointestinal symptoms was ex-
amined. As indicated in Additional file 1: Tables S2 and
S5, age group did not have a significant impact on
gastrointestinal symptoms when evaluated using GEE
analysis or Kruskal–Wallis test.
Urinary galactose concentrations
Table 5 shows the urinary galactose concentrations at
baseline and at 3 h after the consumption of milk con-
taining A2 β-casein or conventional milk. As would be
expected after the consumption of milk, the urinary
Table 3 Gastrointestinal symptom scores (all study sites combined)
Variable Time Visit 1 (baseline) Visit 2 GEE analysis
A1 (n=300)
a
A2 (n=300)
a
A1 (n=300)
a
A2 (n=300)
a
Product (A2 vs. A1)
Median Range Median Range Median Range Median Range Estimate SE P
Borborygmus 1 h 3 0–62 0–63 0–62 0–6 1.327 0.097 <0.0001
3h 3 0–62 0–63 0–62 0–5 1.912 0.103 <0.0001
12 h 0 0–20 0–20 0–20 0–2 0.134 0.180 0.458
Flatulence 1 h 2 0–62 0–53 0–61 0–5 1.516 0.098 <0.0001
3h 3 0–62 0–53 0–62 0–4 1.869 0.101 <0.0001
12 h 0 0–30 0–30 0–30 0–2 0.245 0.130 0.059
Bloating 1 h 2 0–61 0–63 0–61 0–5 1.474 0.098 <0.0001
3h 3 0–62 0–53 1–62 0–4 1.922 0.096 <0.0001
12 h 2 0–51 0–42 0–41 0–3 0.883 0.126 <0.0001
Abdominal Pain 1 h 0 0–50 0–51 0–50 0–3 0.714 0.082 <0.0001
3h 2 0–51 0–42 0–60 0–3 1.903 0.101 <0.0001
12 h 2 0–41 0–32 0–51 0–3 1.887 0.124 <0.0001
Stool Frequency 1 h 0 0–50 0–40 0–50 0–3 0.749 0.088 <0.0001
3h 2 0–50 0–32 0–50 0–3 2.276 0.110 <0.0001
12 h 1 0–20 0–21 0–30 0–2 1.778 0.156 <0.0001
Stool Consistency 1 h 0 0–50 0–40 0–50 0–2 0.799 0.092 <0.0001
3h 2 0–50 0–42 0–50 0–2 2.375 0.115 <0.0001
12 h 1 0–30 0–21 0–30 0–2 2.051 0.149 <0.0001
A1 conventional milk containing A1 and A2 β-casein, A2 milk containing A2 β-casein, GEE generalized estimating equation, SE standard error
a
n=400 at 12 h (the VAS was not assessed at 12 h in Shanghai)
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galactose concentrations increased between baseline and
3 h in both groups. However, the magnitude of the in-
crease was significantly greater when the subjects con-
sumed milk containing A2 β-casein than when they
consumed conventional milk, with consistent changes in
each site and in all subjects combined. Age group was
not associated with differences in urinary galactose con-
centrations based on the GEE analysis (Additional file 1:
Table S6)
Relationship between malabsorption and gastrointestinal
symptoms
Overall, 170 (28.3%) and 430 (71.7%) of subjects at all
three sites combined were classified as lactose absorbers
and lactose malabsorbers, respectively. The proportions
of lactose absorbers and lactose malabsorbers were simi-
lar at all three sites separately (Beijing: 48 [24.0%] vs 152
[76.0%]; Guangzhou: 69 [34.5%] vs 131 [65.5%]; Shang-
hai: 53 [26.5%] vs 147 [73.5%]). For subjects in all sites
combined, the gastrointestinal symptom scores were sig-
nificantly lower for milk containing A2 β-casein as com-
pared with conventional milk at both 1 and 3 h in
lactose absorbers and lactose malabsorbers (all
P≤0.0002) (Table 6). The symptom scores for bloating,
abdominal pain, stool frequency, and stool frequency at
12 h after consumption were also significantly lower for
milk containing A2 β-casein compared with conven-
tional milk in both lactose absorbers and lactose malab-
sorbers (all P<0.0001). In addition, there were trends
towards slight improvements in gastrointestinal symp-
toms after consuming milk containing A2 β-casein com-
pared with conventional milk (Additional file 1: Table
S7). Similar results were observed when the data were
analysed for each site separately (data not shown).
Table 4 Proportions of subjects with improvements in gastrointestinal symptoms after consuming milk containing A2 β-casein
relative to milk containing A1 β-casein (all study sites combined)
Measurement Improvement Borborygmus Flatulence Bloating Abdominal Pain
n% n % n % n %
1h(n=600) No symptom 50 8.3% 61 10.2% 112 18.7% 119 19.8%
Significant improvement 9 1.5% 9 1.5% 6 1.0% 1 0.2%
Slight improvement 310 51.7% 314 52.3% 279 46.5% 88 14.7%
No difference 231 38.5% 216 36.0% 203 33.8% 392 65.3%
3h(n=600) No symptom 33 5.5% 29 4.8% 62 10.3% 240 40.0%
Significant improvement 8 1.3% 9 1.5% 5 0.8% 6 1.0%
Slight improvement 362 60.3% 341 56.8% 338 56.3% 131 21.8%
No difference 197 32.8% 221 36.8% 195 32.5% 223 37.2%
12 h (n=400)
a
No symptom 51 12.8% 102 25.5% 83 20.8% 121 30.3%
Significant improvement 0 0.0% 0 0.0% 1 0.3% 0 0.0%
Slight improvement 4 1.0% 24 6.0% 105 26.3% 140 35.0%
No difference 345 86.3% 274 68.5% 211 52.8% 139 34.8%
Measurement Improvement Stool Frequency Stool Consistency All Symptoms
n% n % n %
1h(n=600) No symptom 120 20.0% 128 21.3% 16 2.7%
Significant improvement 1 0.2% 1 0.2% 282 47.0%
Slight improvement 65 10.8% 61 10.2% 236 39.3%
No difference 414 69.0% 410 68.3% 66 11.0%
3h(n=600) No symptom 304 50.7% 310 51.7% 4 0.7%
Significant improvement 0 0.0% 1 0.2% 480 80.0%
Slight improvement 114 19.0% 87 14.5% 100 16.7%
No difference 182 30.3% 202 33.7% 16 2.7%
12 h (n=400)
a
No symptom 199 49.8% 202 50.5% 21 5.3%
Significant improvement 0 0.0% 0 0.0% 127 31.8%
Slight improvement 25 6.3% 22 5.5% 186 46.5%
No difference 176 44.0% 176 44.0% 66 16.5%
a
The VAS was not assessed at 12 h in Shanghai
He et al. Nutrition Journal (2017) 16:72 Page 7 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Adverse events
Overall, 16 adverse events were reported during the
study. Adverse events reported by subjects in Beijing
were flu (2, 1.0%), croup (2, 1.0%), upper respiratory
infection (3, 1.5%), anal fissure (1, 0.5%), urinary tract in-
fection (1, 0.5%), and atopic dermatitis (1, 0.5%). Four
subjects in Guangzhou reported adverse events, which
were cough without other related symptoms (2, 1.0%),
pneumonia (1, 0.5%), and bronchitis (1, 0.5%). Pneumo-
nia (1, 0.5%) and torticollis (1, 0.5%) were reported in
Shanghai. None of the adverse events were considered
related to the study products, and none of the adverse
events were classified as serious.
Discussion
Ho et al. [10] and Jianqin et al. [11] performed prelimin-
ary studies to compare the effects of conventional milk
and milk containing only A2 β-casein on gastrointestinal
symptoms in humans. Ho et al. [10] revealed that milk
containing A1 β-casein was associated with significantly
softer stool showing higher consistency scores, as deter-
mined using the Bristol Stool Scale, compared with milk
containing A2 β-casein. In addition, consumption of A1
β-casein milk was associated with increased faecal cal-
protectin, a marker of intestinal inflammation [16].
Meanwhile, Jianqin et al. [11] revealed that consumption
of conventional milk was associated with greater symp-
toms of post-dairy digestive discomfort in subjects with
self-reported lactose intolerance. The worsening of
gastrointestinal symptoms was apparent in lactose
tolerant and lactose intolerant subjects. A subsequent
analysis [17] of the study by Jianqin et al. revealed in-
creased concentrations of inflammatory biomarkers and
BCM-7 after consumption of milk containing both β-
casein types compared with consumption of milk
containing only A2 β-casein. However, the studies by Ho
et al. [10] and Jianqin et al. [11] were relatively small, in-
volving 40 and 45 subjects, and warranted confirmation
in larger-scale studies. Nevertheless, the results
highlighted a link between A1 β-casein, gastrointestinal
inflammation, and symptoms of milk intolerance. Not-
ably, subjects confirmed to be lactose malabsorbers tol-
erate milk containing only A2 β-casein, even though the
lactose level was similar to that of conventional milk,
suggesting that the type of β-casein may contribute to
the symptoms of lactose intolerance in some people.
Accordingly, the objectives of the present were to
compare the effects of consuming milk containing either
A2 β-casein or conventional milk containing both A1
and A2 β-casein on acute self-recorded lactose intoler-
ance and gastrointestinal discomfort occurring within
several hours of consuming milk. In addition, we sought
to examine the effects of both milk products on lactase
activity to determine if changes in lactase activity are
linked to the changes in self-reported symptoms of milk
intolerance. We also examined whether age was corre-
lated with a shift in lactase activity and the symptoms of
milk intolerance.
This cross-over study of 600 Chinese subjects with self-
reported milk intolerance revealed significant differences
Table 5 Urinary galactose concentrations after consumption of the study products
Site Time Visit 1 (baseline) Visit 2 LSM difference
A1 (n=300)
a
A2 (n=300)
a
A1 (n=300)
a
A2 (n=300)
a
Product (A2 vs. A1)
Mean (mmol/L) SD Mean (mmol/L) SD Mean (mmol/L) SD Mean (mmol/L) SD Estimate SE P
Beijing Baseline 0.72 0.30 0.71 0.30 0.70 0.31 0.71 0.28
3 h 0.84 0.33 1.06 0.39 0.81 0.31 1.07 0.42 0.243 0.034 <0.0001
Change from
baseline
0.12 0.27 0.35 0.43 0.11 0.31 0.36 0.47 0.244 0.038 <0.0001
Guangzhou Baseline 0.72 0.32 0.73 0.33 0.73 0.38 0.70 0.38
3 h 0.85 0.49 1.01 0.39 0.96 0.58 1.07 0.69 0.142 0.044 0.002
Change from
baseline
0.13 0.40 0.28 0.32 0.23 0.46 0.37 0.59 0.144 0.045 0.002
Shanghai Baseline 0.71 0.26 0.71 0.25 0.72 0.28 0.72 0.21
3 h 0.85 0.36 0.99 0.39 0.83 0.37 1.02 0.42 0.165 0.034 <0.0001
Change from
baseline
0.14 0.37 0.28 0.41 0.12 0.31 0.30 0.38 0.165 0.036 <0.0001
All sites Baseline 0.72 0.29 0.72 0.30 0.72 0.32 0.71 0.30
3 h 0.85 0.40 1.02 0.39 0.87 0.44 1.05 0.53 0.183 0.022 <0.0001
Change from
baseline
0.13 0.35 0.30 0.39 0.15 0.37 0.34 0.49 0.184 0.023 <0.0001
A1 conventional milk containing A1 and A2 β-casein, A2 milk containing A2 β-casein, LSM least-squares mean, SD standard deviation, SE standard error
a
n=400 at 12 h (the VAS was not assessed at 12 h in Shanghai)
He et al. Nutrition Journal (2017) 16:72 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

in gastrointestinal symptoms after the consumption of
milk containing A2 β-casein or conventional milk. Of
note, the gastrointestinal symptom scores were signifi-
cantly lower at 1, 3 and 12 h after consumption of milk
containing A2 β-casein relative to the consumption of
conventional milk. These results suggest that elimination
of A1 β-casein from the diet was associated with reduced
severity of acute gastrointestinal symptoms after milk in-
take in this population.
It is important to note that the baseline symptoms
were evaluated before consumption of either milk prod-
uct by asking the subjects to report their symptoms at
the last time they consumed milk. Accordingly, the sub-
jects possibly recalled their worst experience. To avoid
this potential source of bias, the analyses of gastrointes-
tinal symptoms were adjusted for baseline scores to ac-
count for individual differences.
The exact mechanism by which acute exposure to A1
β-casein augments gastrointestinal symptoms relative to
exposure to A2 β-casein is unclear, but we speculate that
inflammation might be a contributing factor. This is
supported by the studies by Ho et al. [10], Deth et al.
[17], and Trivedi et al. [18] who noted increases in the
concentrations of inflammatory biomarkers following
exposure to A1 β-casein. However, these studies
involved longer durations of exposure than our study, in
which symptoms were assessed up to 12 h after expos-
ure. To our knowledge, no studies have examined the
acute effects of A1 β-casein exposure on gastrointestinal
inflammation in humans.
Although no studies have examined the acute ef-
fects of A1 β-casein, some studies have investigated
the acute effects of other dietary proteins on inflam-
matory biomarkers.
Table 6 Effects of lactose malabsorption on gastrointestinal symptoms
1 h Absorbers (n=170) Malabsorbers (n=430)
A1 A2 GEE estimate A1 A2 GEE estimate
Median Range Median Range Estimate SE P Median Range Median Range Estimate SE P
Borborygmus 3 0–62 0–6 1.562 0.218 <0.0001 3 0–62 0–5 1.244 0.116 <0.0001
Flatulence 3 0–62 0–5 1.826 0.212 <0.0001 2 0–61 0–5 1.395 0.119 <0.0001
Bloating 2 0–61 0–5 1.698 0.201 <0.0001 2 0–61 0–6 1.406 0.12 <0.0001
Abdominal Pain 1 0–50 0–4 1.552 0.206 <0.0001 0 0–50 0–5 0.396 0.107 0.0002
Stool Frequency 1 0–50 0–4 1.328 0.212 <0.0001 0 0–50 0–3 0.518 0.121 <0.0001
Stool Consistency 1 0–50 0–4 1.339 0.207 <0.0001 0 0–50 0–4 0.566 0.128 <0.0001
3 h Absorbers (n=170) Malabsorbers (n=430)
A1 A2 GEE estimate A1 A2 GEE estimate
Median Range Median Range Estimate SE P Median Range Median Range Estimate SE P
Borborygmus 3 0–52 0–5 1.701 0.206 <0.0001 3 0–62 0–6 2.022 0.124 <0.0001
Flatulence 3 0–52 0–5 1.424 0.199 <0.0001 3 0–62 0–5 2.079 0.133 <0.0001
Bloating 3 0–52 0–5 1.617 0.196 <0.0001 3 0–62 0–4 2.066 0.125 <0.0001
Abdominal Pain 2 0–60 0–4 1.375 0.204 <0.0001 2 0–60 0–4 2.149 0.134 <0.0001
Stool Frequency 1 0–50 0–3 1.866 0.222 <0.0001 2 0–50 0–3 2.454 0.136 <0.0001
Stool Consistency 1 0–50 0–4 1.904 0.216 <0.0001 2 0–50 0–3 2.586 0.146 <0.0001
12 h
a
Absorbers (n=117) Malabsorbers (n=283)
A1 A2 GEE estimate A1 A2 GEE estimate
Median Range Median Range Estimate SE P Median Range Median Range Estimate SE P
Borborygmus 0 0–20 0–2–0.479 0.375 0.202 0 0–20 0–2 0.317 0.211 0.133
Flatulence 1 0–30 0–3 0.165 0.251 0.509 0 0–30 0–2 0.297 0.159 0.062
Bloating 2 0–41 0–4 1.074 0.242 <0.0001 2 0–51 0–4 0.812 0.15 <0.0001
Abdominal Pain 2 0–31 0–3 2.239 0.253 <0.0001 2 0–51 0–3 1.793 0.156 <0.0001
Stool Frequency 1 0–30 0–2 1.618 0.282 <0.0001 1 0–20 0–2 1.854 0.192 <0.0001
Stool Consistency 1 0–20 0–1 1.988 0.291 <0.0001 1 0–30 0–2 2.077 0.183 <0.0001
A1 conventional milk containing A1 and A2 β-casein, A2 milk containing A2 β-casein
a
The VAS was not assessed at 12 h in Shanghai
He et al. Nutrition Journal (2017) 16:72 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

For example, Kristjánsson et al. [19] investigated mu-
cosal inflammatory reactivity to cow’s milk protein and
wheat gluten in 20 patients with coeliac disease and 15
healthy controls. The mucosal reactions to these
proteins were assessed 15 h after exposure. Of note, the
gluten challenge induced neutrophil activation and nitric
oxide synthesis. Ten patients showed strong inflamma-
tory reactions to cow’s milk protein. Six patients sensi-
tive to cow’s milk were also challenged with casein and
α-lactalbumin. In this experiment, casein induced an in-
flammatory response similar to that elicited by cow’s
milk. These findings suggest that casein elicits an inflam-
matory response similar to that elicited by gluten in pa-
tients with coeliac disease. These results are consistent
with the study by Trivedi et al. [18] who reported that
A1 β-casein-derived BCM-7 and gluten-derived exor-
phin share a mechanistic pathway for inducing oxidative
stress in cultured human gut epithelial cells and neur-
onal cells.
Holmer-Jensen et al. [20] conducted a randomized
crossover study in which 11 obese non-diabetic subjects
consumed a fat-rich mixed meal containing cod protein,
whey isolate, gluten, or casein. They observed some dif-
ferences in the acute effects of dietary protein on post-
prandial inflammatory biomarkers. Intriguingly, all four
proteins were associated with reductions in monocyte
chemoattractant protein-1 and increases in CCL5/
RANTES. The whey protein meal was associated with
the smallest reduction in monocyte chemoattractant
protein-1 and the largest increase in CCL5/RANTES
compared with the other meals.
Pal and Ellis [21] compared the effects (within 6 h) of
whey protein, caseinate, and glucose on blood pressure,
vascular function, and inflammatory markers in 20 over-
weight and obese postmenopausal women. Although
systolic blood pressure, diastolic blood pressure, and
augmentation index decreased initially after each meal,
there were no significant differences in these variables
between the glucose, casein, or whey groups. Moreover,
they found no differences in plasma inflammatory
markers.
Finally, Nestel et al. [22] found no changes in systemic
inflammatory and atherogenic biomarkers after ingestion
of a variety of dairy products (low-fat milk, or 45 g fat
from butter, cream, yoghurt, or cheese) in 12 overweight
subjects after a single meal. Moreover, in a 4-week study
of 12 subjects who consumed 50 g dairy fat daily as either
butter, cream and ice cream (non-fermented) or cheese
plus yoghurt (fermented) dairy foods, there were no ap-
parent differences in fasting biomarker concentrations be-
tween the non-fermented and fermented dairy products.
Unfortunately, none of these studies assessed gastrointes-
tinal symptoms and changes in plasma inflammatory
markers might not be correlated with local inflammation.
Nevertheless, the results of these studies suggest that
dietary proteins might have differential effects on gastro-
intestinal inflammation, and further studies might be ne-
cessary to examine whether changes in localised
gastrointestinal inflammation are correlated with gastro-
intestinal symptoms.
It is also important to consider that lactose might con-
tribute to the gastrointestinal symptoms in this cohort
of subjects with self-reported lactose intolerant. Indeed,
an increase in gastrointestinal symptoms was observed
when the subjects consumed conventional milk. How-
ever, the symptoms were reduced when the subjects
consumed milk containing only A2 β-casein, indicating
that A1 β-casein-induced inflammation may be linked to
the symptoms of lactose intolerance.
To examine the impact of lactose malabsorption on
gastrointestinal symptoms, we divided the subjects as
lactose absorbers and lactose malabsorbers, based on the
results of the urinary galactose test. Of note, the gastro-
intestinal symptoms after consumption of milk contain-
ing A2 β-casein were comparable between the lactose
absorbers and lactose malabsorbers.
Based on these findings, we propose the hypothesis
that the gastrointestinal symptoms in some subjects with
self-reported lactose intolerance might be related to A1
β-casein rather than lactose itself. This seems feasible
considering that the lactose concentrations were com-
parable in both milk products.
We also explored the possibility that age had an im-
pact on gastrointestinal symptoms or the correlation be-
tween lactose malabsorption and gastrointestinal
symptoms. As indicated in Additional file 1: Tables S2,
S5 and S6, age was not significantly associated with
gastrointestinal symptoms. However, because the upper
age range was limited to 50 years, it is possible that older
subjects might experience more severe gastrointestinal
symptoms after dairy intake.
The results of this study should be interpreted with
care, considering the limitations of this study, especially
in terms of the mechanistic link between the observed
compromise in lactose digestion and the type of β-
casein. In addition, we used an indirect method to assess
lactase activity. Last, the consumption of other non-
dairy foods and drinks by subjects is a potential con-
founder; however, rather than deny intake of food to
participants, we ensured that any foods and drinks con-
sumed were dairy-free and that there was consistency in
food intake type for both interventions. Further studies
are warranted to examine the putative role of A1 β-
casein in gastrointestinal inflammation, the effects of in-
flammation on the expression and/or activity of lactase
enzyme, and the proportion of people with lactose in-
tolerance who would benefit from excluding A1 β-casein
from their diet. Additionally the effects of long-term
He et al. Nutrition Journal (2017) 16:72 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

exposure to milk in terms of the changes in gastrointes-
tinal health need to be examined in future trials, and
whether chronic exposure conditions desensitise the
gastrointestinal tract to A1 β-casein consumption.
Conclusions
In conclusion, this study showed that consumption of
milk containing A2 β-casein attenuated the acute gastro-
intestinal symptoms following milk intake relative to
conventional milk containing A1 and A2 β-casein in
Chinese subjects with self-reported lactose intolerance.
Gastrointestinal symptoms after consuming milk con-
taining A2 β-casein were consistently reduced in both
lactose absorbers and lactose malabsorbers. These find-
ings suggest that, in some individuals with self-reported
lactose intolerance, the adverse gastrointestinal symp-
toms following milk intake might be related to the
presence of A1 β-caseininmilkratherthanlactose
itself.
Additional file
Additional file 1: Table S1. Food frequency questionnaire. Table S2.
Gastrointestinal symptom scores at each study site: GEE analysis of visit and
age group. Table 3. Gastrointestinal symptom scores in individual study
sites.Table4.Proportions of subjects with improvements in gastrointestinal
symptoms at each study site. Table 5. Differences in gastrointestinal
symptoms between the two age groups by study product (all study sites
combine). Table 6. Urinary galactose concentrations after consumption of
the study products: LSM difference for visit and age group. Table 7.
Proportions of subjects with improvements in gastrointestinal symptoms
according to lactose absorption/malabsorption. (XLSX 37 kb)
Abbreviations
BCM-7: Beta-casomorphin 7; GEE: Generalised estimating equations;
VAS: Visual analogue scale
Acknowledgements
This study was funded by The a2 Milk Company Limited. The authors thank
the Chinese National Nutrition Society for help with enrolling subjects and
Nicholas D. Smith, PhD (Edanz Group), for providing medical writing support,
which was funded by The a2 Milk Company Limited. The authors would also
like to acknowledge the clinical research organization S.P.R.I.M. China
(Shanghai) Consulting Co., Ltd. for conducting the clinical trial.
Funding
This study was funded by The a2 Milk Company Limited.
Availability of data and materials
The datasets supporting the conclusions of this article are included within
the article and its additional files.
Authors’contributions
YXY was the lead investigator and helped design the study; JS helped
design the study. All authors contributed to the conception and design of
the study, selected variables of interest, and contributed to the manuscript.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
The protocol was approved by the central institutional review board of the
Shanghai Nutrition Society. All subjects provided written informed consent
before study entry.
Consent for publication
Not applicable.
Competing interests
All authors report having received honoraria from The a2 Milk Company
Limited.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Beijing Research Institute for Nutritional Resources, Beijing, China.
2
Clinical
Nutrition Center, Huadong Hospital Affiliated to Fudan University, Shanghai,
China.
3
Department of Nutrition, School of Public Health, Sun Yat-Sen
University, Guangzhou, China.
4
Department of Food Nutrition, National
Institute for Nutrition and Health, Chinese Center for Disease Control and
Prevention, Beijing, China.
5
Chinese Nutrition Society, 6# Guang An Men Nei.
Street, Fenghua Square, Building A, Unit 5, Room 1601/1602, Xuanwu District,
Beijing 100053, People’s Republic of China.
Received: 11 May 2017 Accepted: 24 August 2017
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