Effectiveness of Growing-Up Milk Containing Only A2 β-Casein on Digestive Comfort in Toddlers: A Randomized Controlled Trial in China

Background: Emerging clinical evidence indicates the potential gastrointestinal (GI) benefits of milk containing only A2 β-casein, but data from randomized controlled trials is sparse among pediatric populations. We aimed to evaluate the effectiveness of growing-up milk (GUM) containing only A2 β-casein on GI tolerance in toddlers. Methods: A total of 387 toddlers aged 12–36 months were recruited in Beijing, China, and randomized in a 1:1:1 ratio to consume one of two commercially available A2 GUMs (combined in the analysis as A2 GUM) or continue their current feeding regimen of conventional milk for 14 days. The primary outcome was the total Gut Comfort Score (GCS) (range: 10–60; higher values indicate greater GI distress) derived from a 10-item (score range: 1–6 per item) parent-reported questionnaire, reflecting GI tolerance. Results: The GCS (mean ± SD) was comparable between the A2 GUM and conventional milk groups on day 7 (14.7 ± 5.0 vs. 15.0 ± 6.1, p = 0.54) and day 14 (14.0 ± 4.5 vs. 14.3 ± 5.5, p = 0.51). Parents reported less constipation in those consuming A2 GUM vs. conventional milk on day 14 (1.3 ± 0.6 vs. 1.4 ± 0.9, p = 0.020). Among 124 participants with minor GI distress at baseline (GCS ≥ 17, top tertile range 17–35), GCS was significantly lower in those consuming A2 GUM on day 7 (18.2 ± 5.1 vs. 21.2 ± 6.8, p = 0.004) and day 14 (17.1 ± 5.3 vs. 19.6 ± 6.3, p = 0.026), as were individual GI symptoms (all p < 0.05). In the toddlers without GI issues at baseline (GCS < 17), a low GCS was maintained throughout the study period after switching to A2 GUM (mean values range 10–13). Conclusions: Growing-up milk containing only A2 β-casein were well-tolerated and associated with lower parent-reported constipation scores after two weeks when compared to conventional milks. In healthy toddlers with minor GI distress, A2 GUM improved overall digestive comfort and GI-related symptoms within one week.


Introduction
Functional gastrointestinal disorders (FGIDs), a mixture of age-dependent, chronic or recurrent symptoms without evident structural or biochemical abnormalities [1], are common in infants and young children. The prevalence of FGIDs in infants was reported 30% worldwide in 2016 [2], 27% in the US in 2015 [3], and 34% in China in 2014 [4].

Study Setting and Design
This randomized, controlled, open-label study was conducted in a community healthcare center in Beijing, China, from September 2018 to January 2019. Healthy toddlers aged 12-36 months were recruited by trained physicians and randomized to receive one of two A2 GUM brands that were commercially available in China at the time of the study start (A2 GUM group A and A2 GUM group B) or to the conventional milk group for 14 consecutive days. The nutritional compositions of both A2 GUMs were comparable and similar; therefore, the two A2 GUM groups were combined as the A2 GUM group in the analysis below. This study was approved by the Peking University Institutional Review Board (IRB00001052-18063). All participants' parents or caregivers completed an informed consent form.

Participants
To be eligible for the study, toddlers (1) were born full-term (37-42 weeks gestation), (2) had a birth weight of 2.5 to 4.5 kg, and (3) were habitually consuming any cow's milk, conventional milks, and/or dairy products and (4) were 12-36 months of age. Exclusion criteria were (1) chronic infectious, metabolic, genetic, or other diseases potentially impacting GI function or feeding practices, (2) known cognitive and developmental disorders, or (3) currently using or had ever used therapeutic infant formulas (i.e., hypoallergenic, lactose-free, or anti-regurgitation formulas).

Randomization and Interventions
At enrollment, the physician received an instant WeChat Quick Response (QR) code from the randomization schedule, which was scanned by participants to receive their allocation information. A random block size of 9 was applied to guarantee balanced allocation. Allocation was masked from the participants and physicians until baseline information was completely collected. All participant data and allocation information were stored on a secure server hosted by the Peking University Maternal and Child Health WeChat platform, compliant with local data security requirements. After enrollment, participants were randomized to A2 GUM group A, A2 GUM group B, or the conventional milk group in a 1:1:1 ratio. Participants in A2 GUM group A received formula A (Askeaton, Ireland), and those in A2 GUM group B received formula B (Shanghai, China) in an openlabel manner. Both A2 formulas are GUMs designed for toddlers aged 12 months or older, and all β-casein proteins the formulas contained are of A2 origin. Based on the nutritional information provided on the product label, the nutritional composition of both A2 formulas, including total protein and lactose levels and the presence of prebiotics, are comparable. Toddlers in the A2 GUM groups were instructed to consume at least 300 mL of the study milks per day (two servings, 150 mL each) during the 14-day intervention period, and those in the conventional milk group maintained their habitual diet. Milk intake was assessed using a parent-reported milk intake diary at baseline, and on days 5-7 and 12-14 to record the type and volume of milk consumed over a 24 h period.

Study Outcomes
The primary outcome was the total GCS, evaluated using a parent-reported toddler gut comfort questionnaire. The questionnaire was administered at baseline, on three consecutive days from days 5-7, and on three consecutive days from days 12-14, to collect the prevalence and severity of gastrointestinal symptoms of toddlers. At baseline, parents completed the questionnaires under the guidance of physicians who had been unanimously trained on the content the questionnaire to minimize potential bias. Afterwards, parents completed the questionnaires by themselves. The questionnaire was modeled after a previously reported standardized infant gastrointestinal symptom questionnaire (IGSQ-13). It included ten questions related to GI symptoms (6 questions for stooling issues, constipation, diarrhea, gassiness, abdominal pain, and bloated) and related behaviors (4 questions for irritability, sleep problems, sleep duration during the day, and times of waking up during the night). The score for each individual question ranged from 1 to 6, assigned using a visual analog scale, leading to a total GCS ranging from 10 to 60. A higher score indicated a greater GI distress.
Secondary outcomes included stool characteristics, temperament, anthropometric parameters, and adverse events. Stool frequency and consistency were reported by parents prospectively over a 24 h period using a toddler stool diary at baseline, days 5-7, and days 12-14. Stool consistency was assessed using a validated 5-point scale (1 = watery, 2 = runny, 3 = mushy soft, 4 = formed, or 5 = hard) [19]. Temperament over the intervention period was assessed using a simplified 9-item Early Childhood Behavior Questionnaire [20] at day 14, which included attention focusing (1 question), activity level (1 question), sociability (2 questions), effortful control (1 question), irritability (2 questions), and soothability (2 questions). The score of each question ranged from 1 to 7 according to the frequency of behaviors observed by parents, with higher scores indicating higher frequency. Anthropometric parameters, including weight, length, and head circumference, were measured at clinic visits by experienced physicians at baseline and day 14 using standard methods. BMI (kg/m 2 ) was calculated as weight in kilograms divided by squared height in meters. The Z-score of each parameter was calculated according to the WHO child growth standards according to age and sex [21]. Adverse events (AEs), including incidence, severity, seriousness, and relation to study formula consumption, as well as concomitant medications and treatments, were reported by parents and checked by physicians throughout the study period. Predetermined GI AEs of interest included hard stool, constipation, difficulty with bowel movement, acute diarrhea, chronic diarrhea, and gastroesophageal reflux disease.

Sample Size
According to previous data collected using the Infant Gastrointestinal Symptom Questionnaire (IGSQ-13) [22][23][24], with a 2-sided significance level of p = 0.05 and 80% power, assuming a dropout rate of 10%, approximately 360 toddlers in total were needed to detect a mean difference of 1.25 points in the GCS (10% reduction from baseline mean GCS, and a standard deviation of 4.2) between the A2 GUM and conventional milk groups.

Statistical Analyses
Continuous variables were represented using the mean (standard deviation, SD) or median (interquartile ranges, IQR), and categorical variables were represented using frequency (percentage). Differences between milk groups were explored by t-test for means, Kruskal-Wallis test for medians, and chi-square test for frequencies. The average GCS and stool frequency and consistency scores at days 5-7 and days 12-14 were calculated to reflect digestive comfort or stool characteristics at day 7 and day 14, also for the following analyses. Multivariate-adjusted mean differences (95% CI) were calculated using analysis of covariance, adjusting for the baseline total GCS, intervention group, age at enrollment, sex, and whether or not they were ever breastfed. If a significant modification effect by baseline GCS was observed, subgroup analyses were then conducted within strata of baseline scores by tertiles, with participants in the top tertile defined as having minor GI distress. Independent t-tests were used to compare stool frequency and consistency, temperament, and anthropometric parameters (weight, length, head circumference, BMI, and Z-scores) between the two groups. A chi-square test was used to compare adverse events between groups. All analyses were based on the intention-to-treat principle using the full analysis set (FAS). Sensitivity analyses of the primary outcome were further conducted in the perprotocol set (PPS). All p values were 2-sided, and statistical significance was set at p < 0.05. All analyses were conducted using SAS/STAT software (version 9.4, SAS Institute, Inc., Cary, NC, USA).

Baseline Characteristics and Milk Intake
Of 387 enrolled toddlers, 359 completed the study. A total of 259 participants were allocated to the A2 GUM group, and 128 were allocated to the conventional milk group ( Figure 1). All baseline demographic characteristics of the participants were comparable between the two groups (Table 1).
On day 7 and day 14, the daily A2 GUM intake (mean ± SD) was 285 ± 144 mL and 285 ± 142 mL, respectively. The details of milk intake between the A2 GUM and conventional milk groups are presented in Table A1.

Figure 2.
Least-square means (LS means) and 95% confidence intervals (CI) for Gut Comfort Composite Score (GCS) by baseline GCS tertiles and group, day 7 and day 14. Models included adjustment for baseline GCS, age, sex, and breastfeeding. A negative value indicates that the GCS of the A2 GUM group was lower than that of the conventional milk group.

Temperament Question A2 GUM Conventional Milk p
Irritability: When having trouble completing a task (e.g., building, drawing, dressing), how often did your child get easily irritated? 5.2 ± 1.4 5.4 ± 1.6 0.66 Irritability: When s/he asked for something and you said "no", how often did your child have a temper tantrum? 4.2 ± 1.6 4.5 ± 1.6 0.37 Attention focusing: When engaged in play with his/her favorite toy, how often did your child play for more than 10 min? 4.8 ± 1.8 4.5 ± 1.9 0.061 Effortful control: During everyday activities, how often did your child pay attention to you right away when you called to him or her? 4.8 ± 1.7 4.5 ± 1.9 0.099 Sociability: When a familiar adult, such as a relative or friend, visited your home, how often did your child want to interact with the adult? 4.9 ± 1.7 4.7 ± 1.8 0.028 Sociability: When a familiar child came to your home, how often did your child seek out the company of the child? 4.5 ± 1.8 4.2 ± 1.9 0.047 Activity level: While playing indoors, how often did your child run through the house? 3.1 ± 1.7 3.4 ± 1.8 0.030 Soothability: When s/he was upset, how often did your child cry for more than 3 min, even when being comforted? 5.1 ± 1.6 5.2 ± 1.4 0.99 Soothability: When s/he was upset, how often did your child become easily soothed? 4.5 ± 1.6 4.2 ± 1.6 0.004 1-Never; 2-Very rarely; 3-Less than half the time; 4-About half the time; 5-More than half the time; 6-Almost always; 7-Always. p values < 0.05 were marked in bold.

Anthropometric Parameters
There were no significant differences in any anthropometric parameters on day 14 between the two groups (Table 4).

Adverse Events
The overall incidence of AEs was low and comparable between the groups (Table 5). In the A2 GUM group, a total of 19 events occurred in 10 (3.9%) participants, while a total of 8 events occurred in 5 (3.9%) participants in the conventional milk group. Of the predetermined GI AEs of interest, only constipation and diarrhea were observed, both with very low incidence and with no significant difference between the groups.

Discussion
In this randomized controlled study of healthy toddlers aged 12 to 36 months old, we found that cow's milk-based formulas containing only A2 β-casein were easy-to-digest and reduced parent-reported constipation compared to conventional formulas during the 14-day intervention period. Among toddlers with minor gastrointestinal distress, A2 GUM improved overall digestive comfort and gastrointestinal related symptoms, within one week of intervention.
In our population, low gut comfort scores at baseline indicated that the overall digestive tolerance was overall good at enrollment and was well maintained over the entire intervention period. We did not observe significant differences in overall digestive comfort or in most individual GI symptoms between the toddlers in the A2 GUM group and those in the conventional milk group, possibly due to the good digestive comfort of the study population at baseline and the relatively short intervention period. These findings suggested that the A2 GUM was as well tolerated as conventional milk in healthy toddlers with good digestive health. We found that parents tended to report less constipation of their toddlers in the A2 GUM group versus those in the conventional milk group on day 14. Constipation is most common during toddlerhood, with the prevalence as high as 30% [25][26][27]. Children with constipation reported greater impairment in quality of life than children with other gastrointestinal complaints [28,29], and might even have behavioral, social, and emotional problems [30]. A recent cross-over study among Chinese children aged 5-6 years with lactose intolerance demonstrated that those consuming A2 β-casein for five days were more likely to have significantly softer stools [19], similar to our findings. Another study conducted among Australian children aged 21-144 months showed a higher resolution of the proportion of constipation in A2 versus in A1 milk group (79% vs. 57%), but the proportions did not differ statistically possibly due to the limited sample size (n = 39) [17]. Hence, it is likely that the consumption of A2 GUM benefits toddlers with constipation. In addition, the number of waking-ups during the night was higher in A2 GUM group than conventional milk group, which was possibly due to the adaption of a new milk for toddlers in A2 GUM group [31].
It is worth noting that in this study, among toddlers in the top tertile of GCS who experienced minor gastrointestinal distress, there were greater improvements in overall digestive comfort and individual symptoms including constipation, diarrhea, gassiness, and abdominal pain, as soon as after 7 and 14 days of consumption. These findings were in line with previous studies which showed significant effects among participants with greater gastrointestinal distress [16,17,32]. Randomized controlled trials conducted among lactose-intolerant adults or children from the United States, Australia, and China, refs. [13][14][15]33], showed that milk containing only A2 β-casein improved GI symptoms and reduced digestive discomfort.
The digestive comfort of children might influence their temperament, including mood, behavior problems, and temper tantrums [34,35]. Previous findings also showed that physical discomfort was associated with negative emotionality and temperament [20,36,37]. Our data on temperament showed that toddlers consuming A2 GUM were more sociable and more easily soothed but less active than those consuming conventional milk.
One of the potential mechanisms underlying the effect of formulas containing only A2 versus those containing a mixture of A1 and A2 β-casein on digestive comfort is that the digestion of A1, but not A2, β-casein can produce BCM-7. In vivo studies in both humans and animals have shown that BCM-7 decreases GI motility and increases GI transit time [11,14,38], thus contributing to hard and dry stools, leading to GI symptoms such as constipation and abdominal pain. Additionally, BCM-7 was associated with changes in inflammatory responses and increased serum or luminal myeloperoxidase activity and concentrations of IL-4 and histamine, which can contribute to GI inflammation [11]. Previous studies have also demonstrated lower concentrations of BCM-7 or inflammatory biomarkers in adults who consumed milk containing only A2 β-casein compared to those who consumed milk containing both A1 and A2 β-casein [13,14,16].
Our findings among healthy toddlers may have public health and clinical relevance given the high prevalence of GUM consumption in pediatric populations. The prevalence is 75% in Chinese toddlers aged 12-23 months, and remains to be over 50% up to 35 months of age [39]. The strengths of this study include the prospective and randomized approach with a clear documentation of the amounts of milk consumed by each participant. Parent reports based on validated and published questionnaires on digestive comfort and temperament covering a variety of domains were utilized, and the relatively large sample size enabled us to perform subgroup analysis. Nevertheless, our study also had some limitations. First, we employed a relatively short feeding and follow-up period (2 weeks), which limited the examination of the potential long-term effects of A2 milk consumption. However, the GI benefits manifested after a short period of consumption of A2 GUM particularly in toddlers with minor GI distress at baseline in this study, within just one week. Second, the average A2 GUM intakes on day 7 and day 14 were slightly lower than recommended (285 mL vs. 300 mL), which may underestimate the effectiveness of A2 GUM. Third, data on complementary feeding other than milk products were not collected. Nevertheless, the RCT design was adopted to minimize potential bias introduced by complementary foods. Lastly, although participants were randomized to the study groups, the intervention was not blinded to them.

Conclusions
In conclusion, we found that cow's milk-based formulas containing only A2 β-casein were well-tolerated overall and reduced parent-reported constipation scores compared to conventional milks after two weeks. In healthy toddlers with minor GI distress at baseline, A2 GUM improved overall digestive comfort and GI-related symptoms, and improvements were seen within one week of the feeding intervention. The findings indicate that such formulas containing only A2 β-casein were as well tolerated as conventional milks for healthy toddlers in GI tolerance, but likely benefit to improve constipation or minor GI distress. Further studies with longer follow-up periods in diverse populations are warranted to confirm these findings and to investigate potential long-term beneficial effects of consuming milk products containing only A2 β-casein.