Safety of Bifidobacterium Breve PS1 for Infants
Condition: Infant Development; Safety Issues
Study type: Interventional

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.
    

Detailed 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
    

Intervention name(s): Infant formula supplemented with Bifidobacterium breve PS1; Infant formula NOT supplemented with Bifidobacterium breve PS1
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): Feeding with an infant formula containing 7 log10 cfu of the strain per gram of formula.; Feeding with the same infant formula but without supplementation with the B. breve strain
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Infants were classified into 2 groups. All infants in the probiotic group consumed an infant formula containing a freeze-dried probiotic (7 log10 CFU of B. breve PS1 per gram of formula). All infants in the control group consumed the same formula but without probiotic supplementation.
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Basic Science