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Journal of the American College of Nutrition, Vol. 25, No. 5, 415-419 (2006)
Published by the American College of Nutrition

Safety and Tolerance of a Probiotic Formula in Early Infancy Comparing Two Probiotic Agents: A Pilot Study

Zvi Weizman, MD, FACN and Ahmed Alsheikh, MD

Pediatric Gastroenterology and Nutrition Unit, Soroka Medical Center, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva, ISRAEL

Address reprint requests to: Zvi Weizman, MD, Head, Pediatric Gastroenterology and Nutrition Unit, Soroka Medical Center, P.O. Box 151, Beer-Sheva 84101, ISRAEL. E-mail: wzvi{at}bgumail.bgu.ac.il


   ABSTRACT
TOP
 FOOTNOTES
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: To compare the safety and tolerance of two formulas, supplemented with different probiotic agents, in early infancy.

Design: Prospective randomized placebo-controlled trial.

Setting: Clinics of a University Medical Center.

Subjects: Full-term healthy infants aged less than 4 months.

Intervention: Infants were randomly assigned for 4 weeks to a standard milk-based formula supplemented with either Bifidobacterium lactis (BB-12), Lactobacillus reuteri (ATCC 55730) or a probiotics-free formula.

Measures of Outcome: Growth parameters, daily characteristics of feeding, stooling and behavior, and side effects.

Results: Fifty-nine infants, aged 3–65 days, were included. Subjects in all three groups were similar at entry in terms of gestational age, birth weight, sex, growth parameters and breast feeding rate prior to the study. The supplemented formulas were well accepted and did not reveal any adverse effects. A comparison of growth parameters, and variables of feeding, stooling and crying and irritability did not reveal any significant differences between groups.

Conclusions: The use of formula supplemented with either Lactobacillus reuteri or Bifidobacterium lactis in early infancy, was safe, well tolerated and did not adversely affect growth, stooling habits or infant behavior.

Key words: infant formula, probiotics, safety, tolerance


   INTRODUCTION
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Probiotics are viable non-pathogenic bacteria that colonize the intestine and modify the intestinal microflora and their metabolic activities with beneficial effect for the host [1,2]. Breast-fed infants develop a probiotic-rich gut microflora with less pathogenic bacteria, compared to formula-fed individuals [3,4]. This effect has been considered one of the mechanisms for the lower rate of infectious diarrhea and allergic disorders in breast-fed infants [5,6].

Infant and follow-up formula supplemented with probiotics are currently marketed worldwide, aiming to mimic some of the beneficial effects of human milk. Probiotic bacteria are generally regarded as non-pathogenic, since they occur naturally in the intestine. These microorganisms have been extensively studied in adult humans with no observed adverse effects in normal use conditions [7,8]. However, in contrast to a wealth of information pertaining to the safety of these agents in adults, the pediatric literature is limited [9–11]. Therefore, several health authorities like the American Food and Drug Administration approved the use of a probiotic formula only for infants older than 4 months of age [12].

The present pilot study was conducted to evaluate the safety of a probiotic formula in early infancy, comparing two species: Bifidobacterium lactis (BB-12) and Lactobacillus reuteri. In addition we assessed the possible influence of these microorganisms on growth and also on feeding, stooling and behaviour characteristics.


   METHODS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects
Full-term healthy infants, aged 3–65 days, from similar socioeconomic areas, were recruited for this prospective, randomized, double-blind placebo-controlled trial, through meetings with parents. We excluded infants with prematurity (<36 weeks gestation), birth weight lower than 2500 grams, congenital anomalies, chronic disease, failure to thrive (weight loss of >2 percentiles), allergy or atopic disease and recent (less than four weeks) exposure to probiotics or antibiotics.

Intervention
All infants were not breast-fed due to parental decision prior to recruitment to the study. All participants were fed a humanized cow’s milk formula (Materna Premium, Stage I, Materna Laboratories, Maabarot, Israel). The formula composition is described in Table 1. Each infant was randomly assigned to be fed for 4 weeks by one of the following: the above formula, supplemented with either Bifidobacterium lactis (BB-12) (CHR Hansen, Denmark), or with Lactobacillus reuteri (ATCC 55730, also called SD 2112) (BioGaia AB, Sweden), or the same formula with no supplement of probiotics. The concentration of microorganisms in each supplemented formula was 1 x 107 CFU/gm of formula powder or 2.2 x 108 CFU/180 ml of prepared formula.


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  		Table 1. Composition of Study Formula

 
Follow-up Parameters
Each participant underwent a physical examination, including determination of growth parameters (weight, length and head circumference) at baseline and at 4 weeks. The parents filled a daily questionnaire for seven days at the first and fourth weeks of the trial. This questionnaire included the following feeding, behavior and stooling characteristics, as described in a previous study [13]. In all the following parameters, the meaning of a minimal score was "normal" and a maximal score meant "worst". Feeding parameters included number of meals per day, response to food (on a 1–5 scale), daily formula volume and daily number of regurgitation and vomiting episodes. Daily evaluation of behavior characteristics included number of severe crying episodes, crying nature (on a 1–4 scale), number of night awakenings and a restlessness score (on a 1–5 scale). Stooling parameters included daily number of bowel movements, stooling effort (on a 1–4 scale), stool consistency (on a 1–4 scale) and a daily gas score (on a 1–4 scale). The parents were instructed to report daily on every complaint or symptom. In addition, each family had a weekly telephone call from the research team to monitor possible adverse effects.

Outcome Measures
The primary outcome measures were clinical adverse effects including deviations of growth parameters. Secondary measures were undesired changes of feeding (regurgitation, vomiting), restlessness and stooling characteristics (diarrhea, constipation).

Ethics
The study protocol was approved by the local ethics committee of the Soroka University Medical Center and the Ben Gurion University. A written informed consent was obtained for each infant from both parents.

Statistical Analysis
Before the conduct of the study we estimated that for a two-sided test, at the 0,05 significance level, with a power of 85%, a sample size of 20 patients in each group would be sufficient to detect a difference of 20% between groups in terms of growth parameters or in the other scores described above, based on a previous study [13]. Randomization was performed by the random-digit method, on the basis of computer-generated numbers. For the analysis of the baseline parameters categorical data were compared using the chi-square test, and numerical data were compared with the use of ANOVA. For the comparison of the outcome measures we used ANOVA with the Tukey test. The data from all randomized patients were analyzed on an intention-to-treat basis. Differences were considered to be significant at the level of p < 0.05. All reported p values are two-sided. All other values are mean ± SD unless otherwise indicated. The analysis was performed with SPSS software (standard version 10).


   RESULTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
A total of 66 infants were assessed for eligibility, and 7 of them were excluded due to exclusion criteria. The remaining 59 infants constituted the intention-to-treat population. Ten infants were withdrawn due to poor compliance and protocol violation, 4 in the Bifidobacterium group and 3 in each of the two other groups. None of these withdrawals had formula-associated complaints or adverse effects. There were no significant differences between groups at randomization in terms of sex, gestational age, enrollment age, birth weight and percentiles of weight, length and head circumference at entry (Table 2).


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  		Table 2. Baseline Characteristics of All Participants

 
Outcome measures are presented in Table 3. The mean daily formula volume did not differ significantly between groups. Groups also did not differ in terms of food compliance, daily number of meals and daily number of regurgitation and vomiting episodes. A comparison of the other daily variables: stooling scores (effort, fecal consistency, gas); number of bowel movements; crying and restlessness scores; number of severe crying episodes and number of night awakenings—did not differ significantly between groups. One case of acute illness was noted in each group: otitis media in the control group, and upper respiratory infection in each of the two other groups. No adverse effects were noticed throughout the study in all subjects.


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  		Table 3. Outcome Measures in the Three Groups

 

   DISCUSSION
 
The present study is innovative in two respects. First, to the best of our knowledge, there are no other published studies assessing safety and tolerance of a probiotic formula in healthy infants less than three months of age. In addition, it is unique in comparing two different probiotic agents, and in showing that this supplementation did not adversely affect neither the health nor the daily habits of the infants. However, this pilot study is limited by a relatively small number of participants and a short follow up duration. These preliminary results should be validated in future long term, large-scale studies.

Because lactobacilli and bifidobacteria are the most commonly used and reported probiotics, we selected one representative of each group to be included in this study. The presence of these bacteria in the human intestine has been considered to be one of the most important aspects of a healthy intestinal microflora. Although these two bacteria may posses many similarities in their health promoting effects, they belong to different taxonomic groups with significant differences in many phenotypic and genotypic properties [12].

Although in the present study probiotic supplementation did not influence parameters of infant irritability and crying, a recent study performed in infants aged 3–24 months has demonstrated that a long term consumption of formulas supplemented with both Bifidobacterium lactis and Streptococcus thermophilus resulted in reduced reporting of colic and irritability [13].

In addition, this concept is supported by recent data demonstrating different colonization patterns of lactobacilli in colicky and non-colicky infants [14]. Many infant formulas worldwide have been recently supplemented with probiotic agents aiming to mimic some of the beneficial effects of human milk [15]. This represents a fascinating concept, and the currently available scientific knowledge justifies the careful use of some of these products. However, as of today the potential risks of such a formula in small infants are not very well documented. Despite recent important advances, many literature data are still preliminary. To be widely used, a probiotic must also be safe. Lactobacilli and bifidobacteria are among the predominant groups of the normal microflora of the human intestine. In many clinical trials these two agents appear to be safe for the general adult and pediatric populations [16,17].

In newborns, more than in adults, probiotic microorganisms are likely to colonize the intestine because they are able to induce changes in mucus composition and epithelial gene expression. Consequently, they influence the characteristics and the stability of the infant gut microflora [18].

Probiotic bacteria beneficially affect the host intestinal microbial balance and may improve immunity. They stimulate the immune system to produce secretory IgA, they enhance macrophage activity and they are also able to degrade food antigens. In addition they play a major role in the maturation of the intestinal barrier and in alleviating intestinal inflammation [19].

In the present study no fecal analysis of intestinal colonization was carried out. However, based on the average daily intake of formula, the mean daily ingested dose of probiotic microorganisms was 1.2 x 109 CFU/day, and according to a recent study, oral administration of lactobacilli at levels ranging from 108 to 1010 CFU/day has lead to transient colonization of the infant gastrointestinal tract [20].

Probiotics have been used with some success to treat diaper rash and constipation, to decrease the incidence and severity of acute diarrheal illnesses and respiratory infections, and to prevent atopic disease in high-risk infants [21–23]. However, there are almost no controlled clinical studies assessing various species or strains of probiotic microorganisms for a specific indication. The present study compares, in a controlled manner, the clinical efficacy of two different probiotic agents for one particular clinical application, and is unique in this respect. Different types of probiotic bacteria exert different effects based on specific capabilities and enzymatic activities, even within one species [24]. In vitro selection criteria for probiotic bacteria of human origin looking at correlation with in vivo findings are required [25]. When the great variety of species and strain characteristics are considered, it becomes clear that a proven probiotic effect of one strain or species can not be claimed to hold in another [26]. Therefore, more controlled clinical studies comparing different types of bacteria for a specific indication are warranted.


   ACKNOWLEDGMENTS
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors are thankful to Drora Leader and Chaim Zegerman of Materna Laboratories for their kind help and cooperation. They also thank Ilana Gelernter, from the Statistics Laboratory, Tel Aviv University, for the careful statistical analysis. In addition they are also thankful to the research team and the infants with their parents for making this study possible.


   FOOTNOTES
TOP
 FOOTNOTES
ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The study was supported by a research grant from Materna Laboratories, Maabarot, Israel.

Some of the study data were presented at the American Pediatric Societies Meeting, Seattle, Washington, May 2003, and at the European Society of Parenteral and Enteral Nutrition Annual Meeting, Cannes, France, September 2003. They appear in abstract form in Pediatr Res 53:174A, 2003, and in Clin Nutr 22:S69, 2003.

Received March 24, 2005. Accepted June 8, 2006.


   REFERENCES
TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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