Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT05973812 |
| Other study ID # |
SAFEBIF1 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
December 12, 2012 |
| Est. completion date |
March 12, 2015 |
Study information
| Verified date |
July 2023 |
| Source |
Universidad Complutense de Madrid |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
The objective of this work was the characterization of the probiotic potential of
Bifidobacterium breve PS1, a strain originally isolated from human milk. Subsequently, its
safety and tolerance were evaluated in a trial including healthy, formula-fed 3-months-old
infants. A total of 187 infants were randomized into two groups: probiotic group (PG) and
control group (CG). Both groups received the same infant formula but, in the case of the PG,
it was supplemented with the strain. A total of 160 infants (80 per group) completed the
three months of intervention.
Description:
Introduction Some perinatal circumstances, such as the mode of birth, can alter the
composition of the infant gut microbiota. As an example, infants born by caesarean section
are prone to gut dysbiosis (including a depletion of bifidobacteria) in comparison with those
born by vaginal delivery, leading to an increased rate of gastrointestinal and respiratory
tract infections. Interestingly, breastfeeding can restore the Bifidobacterium population,
which in turn was associated with a decrease in the rate of various infections.
Despite the important functions that bifidobacteria may play in the infant gut, some studies
have shown that these bacteria may be present at a low concentration or even ab-sent in some
infants. Some factors, including lack of breastfeeding, early weaning, maternal/infant
antibiotic therapy or HMO composition may select against bifidobacteria in the mother-infant
dyad. Therefore, there is a need for strategies to develop functional foods, including infant
formula, to provide bifidobacterial strains naturally present in human milk to infants who
may be deprived of such microbes. In this context, the objectives of this work were, first,
to characterize of the potential probiotic properties of a bifidobacterial strain isolated
from human milk and, subsequently, to test its safety and tolerance in a population of
3-months-old formula-fed infants
Materials and Methods Study Design A randomized double-blinded controlled intervention study
with two study groups was carried out. Healthy, three month old infants, who due to their
mothers' choice were exclusively formula-fed from birth, were recruited into the study after
informed written consent was obtained from the parents. Investigators took special
precautions and care throughout their communication with the families in order to support and
protect, and not to discourage breastfeeding, and not to suggest bottle feeding to parents of
a breastfed baby. The exclusion criteria included history of mild or serious gastrointestinal
disorders (history of chronic diarrhea or constipation, gastroesophageal reflux),
gastrointestinal surgery, cow's milk protein allergy, metabolic disorders (diabetes, lactose
intolerance), immune deficiency, antibiotic prescription three-weeks prior to inclusion and
previous use of probiotic-containing formula. Exclusion criteria during the study were lack
of compliance with the study protocol, adverse events related to the consumption of the study
formula, not attending scheduled visits to the primary care center, and severe regurgitation
and/or colic that, according to pediatricians, needed prescription of a special formula.
The protocol was approved by the Ethics Committee of the Hospital Clínico (Madrid, Spain)
(Reference 10/017-E).
Sample size Sample size was estimated based on the primary outcome of average weight gain of
infants between baseline visit and visit 1 (180 ± 5 days of age). Based on previous
publications where growth was the primary outcome variable as part of a safety study, the
study was designed to have a power to detect a difference in weight gain equal to 0.5
standard deviations. Thus, at least 65 children would be needed in each formula group under
the assumption of non-inferiority (one-sided test), with a significance level of 2.5% and
power of 80%.
Study Formula A total of 187 infants (3-month-old) were enrolled and randomized into two
study groups (probiotic group [PG] and control group [CG]), according to a randomization
generated by a computer program. Infants received one of the following formula: (a) CG: a
standard powdered infant formula (HiPP 1, 600 g) with a nutritional composition in accordance
with current EU regulations, and (b) PG: the same formula supplemented with B. breve PS1 at a
concentration dose of 107 cfu/g of formula. B. breve PS1 was produced in the facilities of
Biopolis (Valencia, Spain). Both formulas were consumed by the infants until the age of 6
months (3 months' intervention period).
The concentration of the probiotic in the formula was analyzed and confirmed before and at
the end of the study. The original HiPP infant formula was purchased and prepared for the
appropriate use in a double-blind intervention trial. Therefore, the formula pow-der was
transferred to plain white undistinguishable tins and a subset was supplemented with the
strain PS1.
Preparation of both formulae were performed in a clean room provided of a aseptic pack-age
device in the facilities of the Dpt. of Galenic Pharmacy and Food Technology of the
Complutense University of Madrid. All study personnel (pediatricians and researchers) as well
as parents were kept blinded throughout the entire study period. The group assignment was
only revealed upon completion of the statistical analysis. The pediatricians prescribed the
amounts of formula per day to be administered to the infants following ESPGHAN guidelines.
Study outcomes Infants were scheduled to receive two clinical evaluations during the
intervention period: at baseline (3 months of age) and at the end of the study (6 months of
age). The primary outcome of the trial was average weight gain between baseline evaluation
and 6 months of age. Secondary outcomes included average length and head circumference gain,
ad-verse events associated with formula consumption, fecal bifidobacteria levels, and fecal
concentration of short-chain fatty acids (SCFA).
Fecal sample collection and analysis Fecal samples were collected from the diaper at both
study visits. Each fecal sample from each participant and sampling time was aliquoted in four
parts, preserved at -20 °C and processed within 1 week. Three aliquots were used to evaluate
the defined study parameters, and the remaining fourth aliquot was stored at -80 °C.
Bifidobacterial quantification in feces was performed by plating decimal dilutions of the
samples on MRS plates supplemented with L-cysteine (0.5 g/L) (MRS-Cys) agar plates, which
were incubated anaerobically (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in an anaerobic
workstation (MINI-MACS; DW Scientific, Shipley, United Kingdom) for 72 h at 37ºC. The
enumeration included the colonies that corresponded to Gram-positive catalase-negative and
F6PPK-positive. The detection and quantification of B. breve DNA in the fecal samples was
performed by real-time quantitative PCR as previously described.
For short chain fatty acids (SCFA) quantification, fecal samples were homogenized with 150 mM
NaHCO3 (pH 7.8) (1:5, wt/v) in an argon atmosphere. Samples were incubated for fermentation
during 24 h at 37°C and stored at -80 °C until the extraction. The extraction of SCFA was
performed by gas chromatography.
Statistical Analysis The quantitative data were expressed descriptively as the mean and
standard deviation (SD). When not normally distributed, the data were presented as the median
and inter-quartile range (IQR).
Anthropometric measurement data were summarized using the mean and standard deviation (SD),
while intestinal bacterial counts were summarized by median and inter-quartile range (IQR).
In addition, anthropometric measurements were converted to age and gender standardized
z-scores using the methodology recommended by the WHO and implemented in the R package anthro
version 1.0.0. Next, the student's t-test was used to compare means by treatment group of the
z scores for weight, length and head circumference at the 6-month time point. Intestinal
bacterial counts were compared using the Mann-Whitney-U non-parametric test. The tests were
performed at the two-sided 5% significance level and the 95% confidence intervals were
obtained for the estimates. For all the comparisons, differences were considered significant
at p< 0.05. The statistical analyses were conducted using R version 4.2.1
