Nutrition 91
92 (2021) 111472

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Nutrition
journal homepage: www.nutritionjrnl.com

Applied nutritional investigation

A partially hydrolyzed formula with synbiotics supports adequate

growth and is well tolerated in healthy, Chinese term infants: A
double-blind, randomized controlled trial
Ying Wang M.D., Ph.D. a,*, Zailing Li M.D., Ph.D. b, Jie-ling Wu M.D. c, Lili Zhang M.D., Ph.D. d,
Min Liu M.D., Ph.D. e, Meizhen Tan M.D. f, Akke Botma Ph.D. g, Mengjin Liu Ph.D. h, Kelly A. Mulder Ph.D. g,
Marieke Abrahamse-Berkeveld Ph.D. g, Wei Cai M.D., Ph.D. a
a
Division of Pediatric Gastroenterology and Nutrition, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
b
Peking University Third Hospital, Department of Pediatrics, Peking, China
c
Guangdong Women and Children Hospital, Department of Children Health Care, Guangzhou, China
d
Wuxi Children’s Hospital, Department of Children Health Care, Wuxi, China
e
Shanghai Public Health Clinical Center, Obstetrics Department, Shanghai, China
f
Guangzhou Women and Children’s Medical Centre, Obstetrics Department, Guangzhou, China
g
Danone Nutricia Research, Utrecht, The Netherlands
h
Danone Open Science Research Centre, Shanghai, China

A R T I C L E I N F O A B S T R A C T

Article History: Objectives: The aim of this study was to evaluate growth and gastrointestinal tolerance in infants fed a par-
Received 27 May 2021 tially hydrolyzed protein formula (pHF) with a synbiotic mixture of short-chain galacto-oligosaccharides and
Received in revised form 20 August 2021 long-chain fructooligosaccharides (scGOS/lcFOS; 9:1) and Bifidobacterium breve M-16V (test formula) com-
Accepted 24 August 2021
pared with an intact protein infant formula (IF) with scGOS/lcFOS (control formula).
Methods: This randomized, double-blind, controlled, multicenter trial enrolled healthy, fully formula-fed Chinese
Keywords:
infants (
44 d) who received either the test (n = 112) or control formula (n = 112) until 17 wk of age. Fully
Tolerance
breastfed infants served as a reference (n = 60). Anthropometrics, gastrointestinal symptoms, and adverse events
Prebiotic
Probiotic
were assessed monthly. Primary outcome was weight gain in grams per day from baseline to 17 wk of age.
Infant feeding Results: Equivalence in daily weight gain (primary outcome) was demonstrated between the test and control
Safety groups (estimated mean difference [SE]:
0.36 [0.93] g/d, 90% confidence interval [CI],
1.90 to 1.18) as well
as between each IF group and the breastfed reference group (test: 0.02 [1.05] g/d, 90% CI,
1.71 to 1.75; con-
trol: 0.36 [1.04] g/d, 90% CI,
1.35 to 2.08). There were no clinically relevant differences in gastrointestinal
tolerance or adverse events between the formula groups.
Conclusion: A pHF with synbiotics supports adequate growth and is well tolerated in healthy, term-born Chi-
nese infants. Additionally, infant growth and gastrointestinal tolerance measures of both IF groups were
comparable to the breastfed group and can be considered suitable and well tolerated for use.
© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/)

Introduction

Danone Open Science Research Center Shanghai provided the funding to conduct
the study. YW, ZL, LZ, ML, MT, and WC were responsible for the conceptualization
of the study and the investigation. AB, ML, KM, and MAB were involved in the study
methodology. KM, AB, and MAB were involved in the writing of the original draft.
All authors were involved in the writing, review, and editing of the final manuscript.
AB, KM, and MAB are employees of Danone Nutricia Research, Utrecht, The Nether-
lands. ML is an employee at Danone Open Science Research Centre, Shanghai, China.
The remaining authors have no conflicts of interest to declare.
*Corresponding author: Fax: 86-21-65791316.
E-mail address: 13611884226@126.com (Y. Wang).
Human milk (HM) provides the best nutrition for infants, sup-
porting them through critical periods of early life. Notably, HM
contains bioactive and immunologic components that may modu-
late the infant immune system and have benefits to growth, brain,
and gastrointestinal (GI) development [1
3]. For infants not
receiving HM, specifically designed infant formulas (IFs) are
required, and continuous efforts are made to improve the composi-
tion of IF to bring functionality closer to HM.

https://doi.org/10.1016/j.nut.2021.111472
0899-9007/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
2 Y. Wang et al. / Nutrition 91
92 (2021) 111472

Cow’s milk protein is the most common source of protein used
in IF and may be present as intact protein or as partially or exten-
sively hydrolyzed protein, specifically designed for infants with
cow’s milk allergy [4
6]. Initially, partially hydrolyzed protein for-
mulas (pHFs) were developed to reduce the allergenicity of and
risk for allergic sensitization by cow’s milk protein-based formulas
[7,8]. More recently, pHF has been suggested to have potential ben-
efits in functional GI disorders [7,9
11]. Due to limited data evalu-
ating growth adequacy and GI tolerance of pHFs in healthy IF-fed
infants, expert groups have called for studies evaluating both the
short- and long-term health outcomes [7,9].
Increasing evidence suggests that GI development including the
gut microbiota has a role in supporting optimal infant develop-
ment [12,13]. Environmental factors, including diet, influence
microbiota in early life, with well-characterized differences
between HM-fed and standard IF-fed infants [14
17]. Inclusion of
prebiotics, probiotics, or a combination of both (termed synbiotics)
in IF has resulted in stool microbiota closer to that of HM-fed
infants and may provide a benefit to nutrient digestion and absorp-
tion, as well as the development of the immune system [16
20].
To our knowledge, this prospective, randomized, double-blind,
controlled trial was the first aimed to demonstrate suitability of a
whey protein-based pHF with a specific synbiotic composition
comprised of the prebiotic mixture of short-chain galacto-oligosac-
charides and long-chain fructooligosaccharides (scGOS/lcFOS, 9:1)
and Bifidobacterium breve M-16V in healthy, term-born infants.
The primary objective of the present study was to investigate
growth adequacy, defined as equivalence in daily weight gain, of
infants who received the pHF compared with those fed a standard
IF containing intact protein and scGOS/lcFOS. Secondary outcomes
included other anthropometrics, parent-reported GI tolerance,
stool characteristics, and adverse events (AEs). As a reference, a
group of breastfed infants was included.
Participants and methods
Participants
This multicenter study included infants recruited from six study centers in
four cities in China; Shanghai (Xinhua Hospital, School of Medicine, Shanghai Jiao
Tong University; Shanghai Public Health Clinical Center), Beijing (Peking Univer-
sity Third Hospital), Guangzhou (Guangdong Province Women and Children Hos-
pital; Guangzhou Women and Children's Medical Centre), and Wuxi (Wuxi
Children's Hospital).
Healthy, term-born infants,
44 d of age whose parent(s) autonomously
decided to fully formula feed were invited to participate. Inclusion criteria was as
follows:
 Chinese ethnicity,
 Birth weight within the 10th to 90th percentile of INTERGROWTH-21st Inter-
national Standards [21],
 Head circumference at enrollment within normal range for age and sex (within
§2 SD World Health Organization [WHO] Child Growth Standards) [22],
 No current or previous illness/condition, and
 No participation in any other study involving investigational/marketed prod-
ucts.
sequences, which were then coded onto the IF tins. The unique code corresponded
to either the test or control IFs, which were stored at the study site. Upon study
enrollment, the investigator accessed a central interactive web-response system
to obtain the randomly assigned code for each infant. For twin or sibling infants
both participating in the study, one infant was randomized and the other was
assigned to receive the same IF. The tins with the corresponding code were dis-
pensed to the parent of the infant during study visits with the instruction to feed
only the assigned study IF ad libitum during the intervention period. Parents were
offered the opportunity to continue with the study product up to 6 mo of age on a
voluntary basis, at which point they would switch to a follow-on formula of their
choice.
All parents and study staff were blinded to IF assignment. The study was con-
ducted in compliance with the principles of the Declaration of Helsinki and all pro-
cedures were approved by Ethical Review Boards of participating hospitals. Before
screening, written informed consent was obtained from parent(s)/legal guardians
(parents) 18 y of age for each infant.
Study products
The test and control IFs were compliant with the China guideline: National
Food Safety standard General Guideline for Formula for Special Medical Purposes
Intended for infants (GB 25596-2010; test) and National Food Safety Standard
Infant Formula (GB10765-2010; control) and manufactured according to divisional
quality standards (ISO 22000; Table 1). Both IFs were provided in identical 800-g
tins.
The test pHF was a partially hydrolyzed non-ultrafiltrated cow’s milk protein-
based IF containing a synbiotic mixture of 0.8 g/100 mL scGOS/lcFOS (9:1 ratio)
and B. breve M-16V (3 £ 107 CFU/g). The control was a commercially available
standard IF (Aptamil Pronutra) and is an intact cow’s milk protein-based IF con-
taining a prebiotic mixture of 0.8 g/100 mL scGOS/lcFOS (9:1 ratio). Although
energy content was very similar in both products, it should be noted that the test
pHF contained a higher concentration of carbohydrates (7.2 versus 6.7 g/100 mL)
compensated by a lower level of fat content (3.4 versus 3.5 g/100 mL) compared
with the control IF.
Measurements
The primary objective of this study was to evaluate equivalence of daily
weight gain (g/d) from baseline to 17 wk of age between the test and control
groups, with a secondary objective to compare each IF group with the breastfed
group. Additional secondary objectives included equivalence of daily length and
head circumference gain, anthropometric measure z-scores, GI tolerance, and
occurrence of AEs.
Infants were assessed during study visits (V) at baseline (V1), 4 (V2), 8 (V3), 13
(V4), and 17 (V5) wk of age. For infants randomized at 28 to 33 d of age, V2 coin-
cided with V1, and for infants randomized >33 d of age, V2 was not applicable.
After written informed consent and checking of eligibility, the baseline character-
istics, including relevant medical history, were collected by interview at V1. Fully
formula-fed infants were randomized to one of the two study products and fully
breastfed infants enrolled in the reference group. Anthropometric measurements
were taken at each visit. Infants were weighed naked to the nearest gram on a cali-
brated electronic weighing scale (Seca 276, Seca, Germany). Recumbent length of
the infants was measured to the nearest 0.1 cm with a length board (Seca 416,
Seca). A non-stretchable measuring tape (Seca 201, Seca) was used to measure
head circumference. Anthropometric measurements were taken twice, with a
third taken if a substantial difference between measurements was recorded
(>50 g for weight and >5 mm for length and head circumference). The two closest
measurements were averaged for the statistical analysis.
Table 1
Composition of intervention infant formulas.

Unit Test Control

Fully breastfed infants were enrolled in the breastfed reference group if they
met these inclusion criteria and additionally their mothers had the intention to Energy kcal/100 mL 66 65
fully breastfeed until 17 wk of age and were not participating in any other clinical Fat g/100 mL 3.4 3.5
study involving other products during pregnancy/lactation. Saturates g/100 mL 1.6 1.6
Monounsaturates g/100 mL 1.2 1.3
Polyunsaturates g/100 mL 0.5 0.7
Study design
Protein g/100 mL 1.5 1.4
Whey g/100 mL 1.5 0.9
This was a double-blind, randomized controlled, two-arm parallel group
Casein g/100 mL
0.5
growth equivalence trial. This trial was registered in ClinicalTrials.gov (as
Carbohydrates g/100 mL 7.2 6.7
NCT03520764). Fully IF-fed infants were block randomized (block size of four) on
scGOS/lcFOS (9:1) g/100 mL 0.8 0.8
a 1:1 basis stratified by sex, to receive the test or control formula until 17 wk of
Bifidobacterium breve M-16V CFU/g 3 £ 107

age. The PLAN procedure of SAS statistical software was used to create the ran-
domization sequences. A study-independent statistical programmer generated the scGOS/lcFOS, short-chain galacto-oligosaccharides/long-chain fructooligosaccharides
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92 (2021) 111472
After V1, parents completed a 7-d paper diary before each visit, which col-
lected information on IF intake, GI symptoms, any illness or symptoms, and use of
medication or supplements. For GI symptoms, stool consistency and frequency as
well as regurgitation and vomiting were recorded. During the 7-d period, stool
consistency was scored for each defecation by the parents according to the
Amsterdam Stool Scale (watery, soft, formed, hard) [23] and frequency was the
total daily number of defecations. Number of occurrences was recorded for both
regurgitation (return of the milk into the mouth without force) and vomiting
(return of milk into the mouth with force). The investigator reviewed the diary
and documented all AEs and any concomitant medications at each visit. A postin-
tervention follow-up visit was planned at 12 mo of age, which is beyond the scope
of the present study and will be presented in a subsequent publication.
Statistical analysis
The a priori assumptions of the primary objective included a margin of equiva-
lence of §3 g/d [24], an equal within-group SD of 6 g/d in weight gain in both
intervention groups, and a difference between the intervention groups of 0.5 g/d.
A required sample size of 224 (112 per group) was calculated with the two one-
sided tests (a = 0.05; power of 0.80; dropout/non-compliance rate of 30%). An
interim analysis to review AEs and confirm the sample size estimation was con-
ducted after 109 randomized infants completed the visit at 17 wk of age and was
evaluated by an independent data monitoring committee. It was recommended
that the study continue without modification.
The equivalence analyses for daily weight gain (primary outcome) was per-
formed with the parametric growth curve mixed-effects model with a quadratic
function of time and included sex, sex £ time, sex £ time2, group, group £ time,
group £ time2, and birth weight as fixed effects and each infant’s intercept, time
and time2 as random effects. The following sensitivity analyses were performed to
evaluate the robustness of the results:
 Excluding second twin/sibling;
 Excluding outliers or influential participants;
 Adding imbalanced covariates to the model (e.g., mode of delivery, pooled
site).
Equivalence analyses for growth outcomes used the per protocol (PP) as the pri-
mary analysis data set. Eligibility of data for the PP population was assessed on
blinded data before analyses and defined on a per visit level (where applicable)
with major protocol deviations including not meeting inclusion criteria, a duration
of >7 d post-baseline to transition fully to the study IF, missing all post-baseline
anthropometric measurements, and >3 d of non-study IF (or any IF for the breastfed
group) or complementary food use. In the case of infant withdrawal from the study,
all available data was considered, and no data imputation was performed.
A comparison of the IF groups with the breastfed reference group was also done
as a secondary objective using the above primary parameter analysis model includ-
ing all three groups. For comparison with the WHO growth standard, anthropomet-
ric z-scores were calculated using a macro provided by the WHO [22].
The all subjects treated population (infants who received some study IF) was
used for all analyses of parent-reported GI tolerance parameters (stool characteris-
tics, regurgitation, vomiting), and AEs. Study product intake was derived as the
Fig. 1. Participant flow through the study from enrollment to study completion for the ASE, ASR, completed, and PP populations. One IF-fed infant withdrew immediately
after enrollment before randomization. An AE was the reason for early termination for three infants in the test group, all of which were cows’ milk protein allergy or intoler-
ance, and two infants in the control group, one for lactose intolerance and one for eczema and rotavirus gastroenteritis. AE, adverse event; ASE, all subjects enrolled; ASR, all
subjects randomized; AST, all subjects treated; BF, breastfed; IF, infant formula; PP, per protocol.
3
volume prepared minus the volume leftover and the mean daily IF intake per day
(mL/d) and body weight (mL/kg) were calculated for each visit. For both study IF
intake and parent-reported GI tolerance parameters, three of seven completed
diary days with no missing data was required. Stool characteristics included mean
stool frequency (number of stools per day) and stool consistency (percentage of
stools with certain consistency over all diary days within the visit week). The
occurrence of frequent watery stools (passage of three or more watery stools in a
day) and the occurrence of infrequent hard stools (two or less defecations per
week and [if any] with a hard consistency) were also calculated from the stool con-
sistency and frequency data for each visit. Regurgitation and vomiting were ana-
lyzed as the occurrence of regurgitation or vomiting at least once on 1 d and the
occurrence of frequent regurgitation or vomiting (2 to 3 d with three or more
regurgitation or vomiting episodes).
Unless otherwise specified, parameters were summarized with descriptive
statistics and for comparison between groups; two sample t tests or Mann
Whit-
ney U tests were used for continuous data. Dichotomous outcome parameters
were compared with the Miettinen
Nurminen Score test. For the AE analyses,
estimates of the risk differences between IF groups were also provided along with
the corresponding Miettinen
Nurminen 95% confidence intervals (CIs) for all
events that occurred in at least four participants in either IF group. For all second-
ary parameters, P-values are unadjusted. All statistical analyses were conducted
according to a predefined statistical analysis plan finalized before data unblinding
using SAS version 9.4 (SAS, Cary NC, USA).
Results
Participant characteristics
From April 2018 to November 2019, we enrolled 285 healthy,
term-born, Chinese infants (IF fed: 224, breastfed: 60; one infant
withdrew before randomization; Fig. 1). In the IF groups, 190
infants completed V5 with 175 PP completers. For the breastfed
reference group, 53 infants completed V5 with 51 PP completers.
Baseline characteristics among the randomized groups were rela-
tively well balanced with a few exceptions (Supplementary Table
1, PP). Compared with the control group, the test group had more
girls, more twins, a lower median baseline age, more Cesarean
deliveries, and a lower incidence of familial allergy. Infant expo-
sure to cigarette smoke was only reported for 5 IF-fed infants and
1 breastfed infant.
Due to the severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) pandemic, some parents were not allowed or not
willing to attend the study site. Ten infants, of which 9 were part
of the PP population (6 in the test and 3 in the control group) had
anthropometric measurements taken by their parents using house-
hold measuring tools after providing clear instructions and
4 Y. Wang et al. / Nutrition 91
92 (2021) 111472

Table 2
Daily parent-reported infant formula intake per day and per kilogram body weight in the per-protocol population*

mL/d mL¢kg¢d
1

Test Control Test Control

n (missingy) Mean (SD) n (missing) Mean (SD) n (missing) Mean (SD) n (missing) Mean (SD)

All subjects randomized population

Visit 2 83 (7) 783 (221) 69 (6) 804 (175) 83 (7) 182 (43.9) 69 (6) 185 (39.2)
Visit 3 93 (0) 927 (210) 96 (2) 894 (189) 93 (0) 172 (33.4) 96 (2) 162 (30.5)
Visit 4 93 (1) 941 (246) 96 (0) 916 (205) 93 (1) 147 (34.6) 96 (0) 141 (30.4)
Visit 5 94 (0) 970 (251) 96 (0) 974 (197) 94 (0) 138 (31.4) 96 (0) 137 (27.2)
Per-protocol population
Visit 2 73 (6) 809 (215) 68 (3) 803 (176) 73 (6) 187 (41.8) 68 (3) 185 (39.4)
Visit 3 86 (0) 922 (212) 94 (1) 894 (188) 86 (0) 171 (34.1) 94 (1) 162 (30.3)
Visit 4 86 (0) 927 (243) 93 (0) 914 (206) 86 (0) 146 (34.8) 93 (0) 141 (30.6)
Visit 5 85 (0) 961 (239) 90 (0) 975 (194) 85 (0) 137 (30.6) 90 (0) 137 (25.8)
*Average daily consumed volume per visit was calculated if at least three diary days were filled out completely, including volume of water and number of scoops used and the
volume of the left over reported for each day.
y
Missing includes those who were participating in the study but did not provide 3 d of completed study product intake data in the diary. Infants who withdrew from the
study are not included as missing.

guidance documents how to measure their infant as accurate as
possible. During only 11 of the 873 observations in these random-
ized groups, anthropometric measurements were collected by
parents (1.3%). The blinded measurements were discussed by the
study team using the longitudinal data plotted in growth charts
before statistical analysis and considered accurate and reliable and,
therefore, included in the analyses. It is of note that none of the
outliers or influential participants identified during statistical anal-
ysis had any home measurements taken. However, acknowledging
the potential higher inaccuracy of home measurements and the
importance of standardization of measurements, an additional
(post hoc) analysis was performed excluding these home measure-
ments for confirmation.
Study product intake
The average daily IF intake was similar among IF-fed groups
over the intervention period (Table 2). The mean (SD) daily IF
intake at V2 was 809 (215) mL/d and 803 (176) mL/d for the test
and control groups, respectively, increasing to 961 (239) mL/d in
the test group and 975 (194) mL/d in the control group at V5 (PP).
Growth outcomes
Growth outcomes were highly similar among both IF groups
and the breastfed reference group (Table 3). An equivalent daily
weight gain (g/d) between the test and control groups from base-
line to 17 wk was demonstrated. The difference in estimated
means (SE) of daily weight gain between the IF groups was
0.36
(0.93) g/d (90% CI,
1.90 to 1.18 g/d) for the PP population. Also
after exclusion of home measurements, equivalence was demon-
strated for the PP population with estimated differences in means
(SE) of daily weight gain between the IF groups of
0.28 (0.94) g/d
(90% CI,
1.83 to 1.28). The equivalence was confirmed with the
sensitivity analyses and the all subjects randomized population.
Equivalence in daily weight gain was also achieved between the
breastfed reference and both the test and control groups. The esti-
mated mean difference (SE) between the test and breastfed refer-
ence groups was 0.02 (1.05) g/d (90% CI,
1.71 to 1.75 g/d) and
between the control and breastfed reference groups was 0.36
(1.04) g/d (90% CI,
1.35 to 2.08 g/d).
Equivalence in daily length gain (mm/d) was achieved between
the test and control groups, but not between either IF groups or
the breastfed reference group. The difference in estimated means
(SE) between the test and control group was 0.01 (0.02) mm/d
(90% CI,
0.05 to 0.03 mm/d). After exclusion of home measure-
ments, equivalence was confirmed with a difference of estimated
means (SE) between both formula groups of 0.001 (0.02) mm/d
(90% CI,
0.04 to 0.04 mm/d). Between the test and breastfed refer-
ence groups the difference in estimated means (SE) was 0.08 (0.03)
mm/d (90% CI, 0.04
0.13 mm/d) and between the control and
breastfed reference groups it was 0.09 (0.03) mm/d (90% CI,
0.04
0.14 mm/d). For daily head circumference gain, equivalence
was achieved between the test and control groups, also when
home measurements were excluded, as well as between each IF
group and the breastfed reference group (data not shown).
Compared with the WHO growth standards, the mean z-scores
for weight-for-age, length-for-age, head-circumference-for-age,

Table 3
Gain in weight, length, and head circumference from baseline to visit 5 (17 wk of age) in the per-protocol population*

Test (n = 89) Control (n = 97) Breastfed (n = 56)

Mean Estimate (SE) 95% CI Mean Estimate (SE) 95% CI Mean Estimate (SE) 95% CI

Weight gain
g 3242 (67.9) 3108
3376 3279 (64.9) 3150
3407 3209 (83.5) 3044
3373
g/d 32 (0.67) 30.7
33.3 32.4 (0.64) 31.1
33.6 31.9 (0.83) 30.2
33.5
Length gain
mm 119.4 (1.76) 115.9
122.9 120 (1.74) 116.6
123.5 109.9 (2.27) 105.4
114.4
mm/d 1.18 (0.02) 1.14
1.21 1.19 (0.02) 1.15
1.22 1.09 (0.02) 1.05
1.14
Head circumference gain
mm 55.3 (1.09) 53.1
57.4 54.9 (1.09) 52.7
57 54.1 (1.40) 51.3
56.8
mm/d 0.55 (0.01) 0.52
0.57 0.54 (0.01) 0.52
0.56 0.54 (0.01) 0.51
0.56
*Data were modeled with a parametric growth curve mixed model with a quadratic function of time.
 Y. Wang et al. / Nutrition 91
92 (2021) 111472
Fig. 2. Estimated mean (§95% CI) WHO Growth Standard z-scores weight-for-age (A), length-for-age (B), BMI-for-age (C), head circumference-for-age (D), mid-upper arm cir-
cumference-for-age (E), per age category for the test, control, and the breastfed reference per-protocol population. BMI, body mass index; WHO, World Health Organization.
and body mass index-for-age were all within or very close to the §
0.5 SD bandwidth for the test, control, and breastfed reference
groups, suggesting adequate infant growth (Fig. 2). Highly similar
growth z-score patterns were observed after exclusion of home
measurements (data not shown). The mid-upper arm circumfer-
ence was measured at 13 and 17 wk of age and the corresponding
median z-score was between the +0.5 and +1 SD bandwidth at 13
wk and close to +1 SD at 17 wk.
Parent-reported gastrointestinal tolerance
Average stool frequency decreased slightly over the study
period from a median (Q1, Q3) of 1.9 (1.1
3.3) stools per day at V2
to 1 (0.7
1.4) at V5 among the IF groups (Fig. 3). There was only
one minor statistically significant difference between the IF
groups; the median (Q1, Q3) of 1 (0.7
1.7) stool per day in the test
group was slightly higher than in the control group of 0.9 (0.6
1.3;
P = 0.049) at V5. In the breastfed reference group, the median (Q1,
Q3) stool frequency was 4.3 (2.8
6) stools per day at V2 and 1.6
(0.9
2.3) at V5.
Most stools were scored as soft, with no statistically significant
differences between IF groups at any time point (Fig. 3). The per-
centage of watery stools among IF-fed infants was very low (Sup-
plementary Table 2). Some watery stools were reported in the
breastfed reference group with a median (Q3) of 0% (23.1%) and 0%
(66.7%) of stools at V2 and V5, respectively. The occurrence of fre-
quent watery stools was also low among IF-fed infants from V2
(test: 8.4%, control: 7.2%, P = 0.788) to V5 (test: 2.1%; control: 4.2%,
P = 0.423). For the breastfed reference group, frequent watery
stools were 34.6% and 18.9% of infants at V2 and V5, respectively.
Hard stools were rarely reported during the intervention period
among all infants (Supplementary Table 2). The occurrence of
infrequent hard stools was also rare (<1%) among all of the infants
with no statistically significant differences between the IF groups
at any time point.
There were no statistically significant differences in the occur-
rence of parent-reported regurgitation or vomiting between the IF
groups at any time point. The occurrence of any or frequent regur-
gitation or vomiting were similar between the IF infants and
5
breastfed reference groups at all timepoints (Supplementary Table
3). In general, the occurrence of both regurgitation and vomiting
decreased over the intervention period from V2 to V5 for all
infants.
Adverse events
The occurrence of at least one AE was reported for 100 (47.8%)
IF-fed infants (test: 52.9%; control: 43.0%, P = 0.151) and 16 (26.7%)
breastfed infants. There were no statistically significant nor clini-
cally relevant differences between the number or type of AEs or
serious AE (SAEs) between the IF groups. The most common AEs
were skin and subcutaneous tissue disorders (test: 22 [21.6%]; con-
trol: 20 [18.7%]; breastfed: 6 [10%]), with eczema being the most
reported for 18 (17.6%), 17 (15.9%), and 5 (8.3%) of infants in the
test, control, and breastfed groups, respectively. Infections and
infestations (test: 16 [15.7%]; control: 20 [18.7%]; breastfed group:
5 [8.3%]) and GI disorders (test: 16 [15.7%]; control: 13 [12.1%];
breastfed: 6 [10%]) were also commonly reported AEs. The number
of AEs related to the study product was comparable between IF
groups (test: 24 [23.5%]; control: 26 [24.3%]), with skin and subcu-
taneous tissue disorders being the most reported as related to the
study product (test: 14 [13.7%]; control: 16 [15.9%]), followed by GI
disorders (test: 9 [8.8%]; control: 9 [8.4%]). None of the AEs
reported as related to the study product were classified as severe.
The occurrence of at least one SAE was reported for 8 (3.8%) infants
(test: 5 [4.9%]; control: 3 [2.8%], P = 0.430; breastfed: 0). All SAEs
were classified as an infection and infestation and none were
reported as related to the study products.
Discussion
To our knowledge, this prospective, randomized, double-blind,
controlled trial was the first to evaluate the suitability of a whey
protein-based pHF with a specific synbiotic composition comprised
of the prebiotic mixture scGOS/lcFOS (9:1) and B. breve M-16V
compared with a standard intact cow’s milk protein-based for-
mula. Equivalence in daily weight gain was demonstrated (primary
outcome), and both IFs were well tolerated without any
6 Y. Wang et al. / Nutrition 91
92 (2021) 111472

Fig. 3. Average stool frequency (n/d) (A) and mean percentage of stool consistency (B) for the all subjects treated population and the breastfed reference group. For stool fre-
quency, only visit 5 was statistically significantly different between the test and control groups (P = 0.049), by Mann
Whitney test. No statistically significant differences in
stool consistency between IF groups at any time point. Percentage of stool consistency was calculated by patient per visit, defined per consistency category as the number of
stools with certain consistency over all diary days within that visit divided by the total number of stools (of any consistency) over all diary days within that visit.

statistically significant or clinically relevant differences in number
or type of AEs. Additionally, infant growth and GI tolerance meas-
ures of both IF groups were comparable to the breastfed group and
can be considered as suitable for use in healthy term infants.
The growth of Chinese infants in this study was comparable to
infant growth observed in studies evaluating IFs in both China
and Europe [25
28]. We demonstrated equivalence in daily
weight gain between both the test and control groups with the
breastfed reference group. In contrast, we observed a slightly
higher length gain in both IF groups compared with the breastfed
reference group. This is consistent with other IF studies in China
[29,30]. One study showed that compared with IF-fed infants,
infants receiving HM had a slightly lower length between 6 and
12 mo of age, and that infants in the Chinese study population
had slightly higher weight-for-age and length-for-age z-scores
compared with WHO standards from 2 to 12 mo of age [31].
Although WHO growth standards included breastfed infants
from six countries, China was not represented and studies show
that Chinese infants and/or children may have a higher median
weight, length, and head circumference than WHO reference val-
ues, although not all infants in those studies were exclusively
breastfed [31
33]. In the present study, the weight of infants
was only slightly higher than the WHO growth standards, with
mean weight-for-age z-scores around 0.5 SD among all infants by
 Y. Wang et al. / Nutrition 91
92 (2021) 111472
17 wk of age. The length-for-age z-score for the IF-fed infants
was also around 0.5 SD; whereas for the breastfed infants it was
closer to 0. Together, the equivalence in daily weight gain
between the IF-fed and breastfed infants in the present study as
well as the comparison with the WHO growth standards con-
firms the growth adequacy in infants of both the pHF (test) and
the commercial IF (control).
Several previous studies have shown that the addition of
scGOS/lcFOS and/or B. breve M-16 V was well tolerated [25,34
36]
and this was confirmed in the present study combining the synbi-
otic mixture with a pHF. At 17 wk of age, the median reported stool
frequency was slightly higher for the test participants than for the
control group (1 versus 0.9 stools per day, P = 0.049, unadjusted).
The clinical relevance of this observation in healthy infants
remains to be elucidated. Most notably, the median percentage of
stool consistency was reported as soft for 100% of stools at V3 to
V5 for infants in both the IF and the breastfed reference groups.
Consistent with this was the rare occurrence of infrequent hard
stools (<1%) among all infants. It has long been documented that
breastfed infants have softer stools than IF-fed infants [37
39]. In
the present study, both the test and control products contained
scGOS/lcFOS (0.8 g/100 mL), and the test pHF additionally con-
tained B. breve M-16V. The stool softening effect of both scGOS/
lcFOS and B. breve M-16V has previously been attributed to the
bifidogenic effect of these pre- and probiotics [35,40,41]. Although
a similar effect might have occurred in the present study, explain-
ing the soft stools observed, this requires further investigation to
confirm. In a study comparing pHF with an intact protein IF, both
containing scGOS/lcFOS, »80% of infants had a stool consistency
reported by parents as soft, and hard stools were nearly absent
[25]. A previous epidemiologic survey in China showed functional
constipation prevalence in 6.2% in infants 0 to 3 mo of age [42].
Additionally, there appeared to be no difference in intake between
the test and control groups, indicating a similar acceptability of
both products by infants and their parents. Overall, GI tolerance
was comparable between infants in both the test and control
groups with the breastfed reference group, particularly with
respect to stool consistency and regurgitation.
In the present study, number and type and relatedness of AEs
were not statistically significantly nor clinically relevantly different
between IF groups. The nature of AEs were those typically
expected in young infants [42,43]. Eczema was the most commonly
reported AE in the present study (test: 17.6%; control: 15.9%, P >
0.05), and was higher than what has been reported by other
growth and safety trials of pHF and IF in European infants
[25,26,44]. However, in the present study, eczema was also
reported as an AE for 8.3% of the breastfed infants, which might
indicate a population-specific increased sensitivity to such AEs.
Infant cohort studies in other countries have shown that several
factors are associated with eczema development in infants, includ-
ing maternal history of allergy or eczema, cesarean delivery, antibi-
otic use during pregnancy, and East Asian ethnicity [45
47]; the
present study population did have a number of these risk factors.
Eczema development is clearly multifactorial and given the simi-
larity in prevalence between intervention groups and prevalence
in the breastfed reference group, the occurrence in our study is not
considered a safety concern.
This study had several key strengths, including its randomized,
double-blind, controlled design, the confirmed powered sample
size, and the rigorous, strict high-quality standards applied during
conduct and evaluation. The authors also had several limitations to
address. Despite the randomized design, there were some notable
differences in baseline characteristics between the IF groups. The
potential effect of these differences was investigated in the
7
sensitivity analyses and did not influence the conclusions of our
growth analyses (primary outcome). The study was conducted in
part during the SARS-CoV-2 pandemic and as a result, several
infant anthropometrics were being taken by the parents at home,
potentially affecting the accuracy of these measurements.
Although the number was limited (1.3% of all measurements), and
visual assessment of the unblinded measurements did not reveal
any signs for unreliability of the collected data (and therefore
remained part of the prespecified statistical analysis), additional
analyses were performed excluding these home measurements,
which did not reveal any significant effect on the outcomes. Addi-
tionally, it is possible that due to the pandemic, the usual environ-
ment of infants was affected, for example with reduced exposure
to external environments due to a lockdown situation. However, it
is anticipated that this has not substantially affected the key out-
comes of this study.
Conclusion
The present study demonstrated that a pHF with a synbiotic
mixture of scGOS, lcFOS, and B. breve M-16 V supports adequate
growth, is well tolerated, and is suitable for use in healthy, term-
born infants. A follow-up of these infants at 12 mo of age aims to
evaluate the intervention effects on longer-term outcomes.
Author Roles and Responsibilities
Conceptualisation, Y.W., Z.L., L.Z., M.L., M.T., W.C.; Investigation,
Y.W., Z.L., L.Z., M.L., M.T., W.C.; Methodology, A.B., M.L., K.M., and
M.A-B.; Writing
original draft, K.M., A.B., and M.A-B.; Writing

review & editing, Y.W., Z.L., L.Z., M.L., M.T., A.B., M.L., K.M., M.A-B.,
W.C.
Declaration of interests
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
The authors acknowledge the families who participated in this
study. They also acknowledge the research staff at the study sites
for their hard work and dedication. The contribution of the Dragon
study team, especially Stephanie Song from Danone Open Science
Research Center (Shanghai), Jasmine Ho from Danone Research
(Singapore), and Maya Marintcheva-Petrova, Ramona Grigorescu,
Meint-Jan Elzinga, Berdine Draijer, Marion Kaspers, and Sonakshi
Shankar from Danone Nutricia Research (Utrecht) is also acknowl-
edged.
Supplementary materials
Supplementary material associated with this article can be
found in the online version at doi:10.1016/j.nut.2021.111472.
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