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The Association of Bifidobacterium breve BR03 and B632 is Effective to Prevent Colics in Bottle-fed Infants

A Pilot, Controlled, Randomized, and Double-Blind Study

Giglione, Enza MD*; Prodam, Flavia PhD*; Bellone, Simonetta PhD*; Monticone, Sonia MD*; Beux, Sheila BS*; Marolda, Agostina MD*; Pagani, Alessia MD*; Di Gioia, Diana PhD; Del Piano, Mario MD; Mogna, Giovanni BS§; Bona, Gianni MD*

Author Information
Journal of Clinical Gastroenterology: November/December 2016 - Volume 50 - Issue - p S164-S167
doi: 10.1097/MCG.0000000000000693
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Infant colics represent a clinical condition in childhood, characterized by an uncontrollable crying that occurs without any apparent organic cause.1 They can also be associated with face redness, closed fists, thighs flexion, meteorism, and gas emission.

They are generally diagnosed according to Wessel’s “rule of three” (>3 h of crying a day, for >3 d a week, for >3 wk in a row).2 These crises tend to reach their maximum intensity at 6 weeks of age, mainly during late afternoon and night, and then to resolve spontaneously over time, in most cases.3

They represent a serious source of anxiety for the family, increasing hospital admissions (5.8% of infants),4 postpartum depression risk, and violence episodes, with higher stress levels for up to 3 years from these events.

The etiology is still unknown. Anyway, it is assumed that the following factors may be involved:

  • Lactose intolerance.
  • Food hypersensitivity.
  • Feeding difficulties.
  • Disorders of the enteric nervous system.
  • Alterations of pain transmission.
  • Gastroesophageal reflux.
  • Intestinal hormones.
  • Psychosocial factors.
  • Alteration of the intestinal microbiota.

In 1994, Lehtonen was the first to suggest that an altered intestinal microbiota composition in the very first months may induce intestinal colics in infants.

Human intestinal microbiota may be conceived as a mix of independent communities interacting with each other.

It is composed of about 1013 to 1014 microorganisms, mainly bacteria. The total number of microbiota genes is called “microbioma” and it is estimated to be 150 times the number of genes in the human genome.5

The microbiota acts as a real organ, whose activity can be influenced by diet, lifestyle, prebiotics, probiotics, and antibiotics.

Several studies revealed the predominance of bifidobacteria in breastfed infants, whereas bottle-fed infants show a mixed population where bifidobacteria are less represented.

Anyway, the differences in microbiota between breastfed and bottle-fed infants are temporary and become indistinguishable around 18 months of age; the intestinal microbiota composition in a 3-year-old child is already similar to that of an adult.6

Other factors conditioning the microbiota are gestational age and type of birth.

The microbiota would be involved, in addition to the proper metabolism of incoming substances to the gastrointestinal tract, even in a real immunomodulation and in endocrine and nervous transmission.

Colicky infants have a microbiota with a slow development and a lower stability over time.7 It also contains less lactobacilli and bifidobacteria, and a prevalence of gram-negative bacteria.

The stools of these children often show increased levels of calprotectin, an intestinal index of inflammation.



New epidemiological evidences suggest that exposure to cigarette smoke and to its metabolites may be related to colics; this might be connected to the increase of plasma and intestinal levels of motilin, due to cigarette smoke. In particular, maternal smoking during pregnancy seems to increase the risk of developing colics, more than postnatal exposition to smoke.8

Psychosocial Environment

Infant colics may be more frequent with an instable psychosocial family environment. Maternal stress, anxiety, and depression are important risk factors. Moreover, violence episodes are more recurrent in families with colicky infants.8


The difference between breastfeeding and bottle-feeding for colicky infants is controversial. Many studies have shown contrasting results,17 but the majority of the authors agree to attribute an important role to bottle-feeding.9

A melatonin role was assumed too. This hormone is not secreted in infants, but only in adults, and has a hypnotic and relaxing role on the gastrointestinal smooth muscle. Its concentration shows a clear circadian rhythm, with a pick during night hours. Its presence in breast milk may be related to the lower occurrence of colics in breastfed infants compared with the bottle-fed infants.9

Thus far, no treatment is really effective for this problem, but recent literature shows an increasing attention toward probiotics,10 for the intestinal microbiota modulation.

Some Lactobacillus reuteri strains were studied, with contrasting results in different studies; other probiotics as bifidobacteria showed in vitro anti-inflammatory properties and the ability to inhibit coliforms growth, whose presence is significant in colicky infants.

Some probiotics exert a direct action on the bacterial growth, through bacteriocins production and final fermentation products, inhibiting pathogens or feeding commensals.11

Bifidobacterium breve is a species isolated by Reuter in 1963.

Strain B632 was isolated from healthy infants’ feces.12 Aloisio et al13 tested in vitro ability of this strain and of other 45 bifidobacteria strains to oppose the growth of several microorganisms such as Escherichia coli, Salmonella enteriditis, Clostridium difficile, Campylobacter jejuni, Klebsiella pneumoniae, and Enterobacter cloacae.

B. breve BR03, in a randomized clinical study, proved to have a beneficial effect on constipation in adults, it also seemed effective for the reduction of gas formation and for abdominal distension, and no side effects were shown during the treatment, while the beneficial effects lasted for up to 15 days after the end of the treatment.14

This strain also proved effective, combined with L. salivarius LS01, in the treatment of patients with atopic dermatitis, displaying an immunomodulant effect and reducing the microbial translocation and the permeability of intestinal walls, factors that seem to play a role in the genesis of this pathology.15

Both bifidobacteria strains showed, during an in vitro study, the ability to oppose 4 strains of E. coli (ATCC 8739, ATCC 10536, ATCC 35218, and ATCC 25922); in particular, BR03 displayed an activity against E. coli O157:H7, an enterohemorrhagic strain that through Shiga toxin causes a potentially lethal infection.16

The primary endpoint of the study was the assessment of the effectiveness of B. breve B632 and BR03 association in the reduction of infants crying over time. Both breastfed and bottle-fed babies were studied.

The second endpoint was to observe the effect of these probiotics on daily evacuations and on the number of regurgitations and vomits.


Type of Study

Interventional single-center, double-blind, randomized study, with treatment and placebo controlled.

Only healthy babies were accepted. They were not treated with antibiotics, enrolled within 15 days from birth, and with an informed consent signed by the parents.

A total of 83 subjects were enrolled, 60 breastfed infants and 23 bottle-fed infants, at the nursery department, during the postdischarge visit.

Twenty-three subjects dropped out, whereas 60 of them carried out the study: 29 infants were given probiotics, whereas 31 placebo. In particular, 43 babies were breastfed and 17 were bottle-fed.

Parents were asked to make an initial recruitment examination within 15 days from birth and a second one at the 91st day of age, at the clinic of Pediatric Gastroenterology.

During the 90 days of the study, parents were asked to give 5 drops of active product (108 viable cells/strain) or placebo and to daily take note of minutes of crying, number, color, and consistency of evacuations, and number of regurgitations or vomits.

Probiotical S.p.A. (Novara, Italy) supplied the active product and the placebo.

All data were collected in a database. Randomization was performed by an external operator of the study.

Categorical variables were analyzed with the Fischer exact test.

Comparisons between numeric variables between various groups were performed with the Kruskal-Wallis test (in the case of >2 groups) and with the Wilcoxon test for independent samples (in the case of only 2 groups being compared).

A probability value of P<0.05 was considered as the limit of statistical significance.


From a statistic point of view, no significant differences were detected in the infants treated with probiotics, compared with placebo group (P=0.75). Among breastfed subjects, the difference between the 2 groups (probiotic or placebo) is not significant (P=0.64). Among bottle-fed infants, the group administered with probiotics cried for an average of 18.89 minutes (SD=19.22; median=12.46); the placebo group cried for an average of 47.08 minutes (SD=58.8; median=31.44); a difference of 28.19 minutes is not statically significant (P=0.14).

The analysis of the 3 months of treatment demonstrated that during the first month, bottle-fed babies treated with probiotics cried 24.23 minutes on average (SD=17.76; median=21.75), whereas placebo group cried 48.88 minutes (SD=70.54; median=29.33) (P=0.50). During the second month, the probiotic group cried 20.30 minutes on average (SD=24.59; median=9.2), whereas the placebo cried 45.71 minutes on average (SD=58.12; median=31.33) (P=0.14). During the third month, the probiotic group cried 12.14 minutes on average (SD=19.82; median=4.88), and the placebo group cried 46.65 minutes (SD=52.83; median=29.66). This difference is statistically significant (P=0.016) (Fig. 1).

Minutes of daily crying in the probiotic group compared with placebo during the first, second, and third month of treatment, respectively.

No significant difference was observed in the number of evacuations, regurgitations, or vomits.


The evidence of the usefulness of some probiotic strains in the treatment and prevention of infant colics is growing, and therefore their use in clinical practice is spreading. In addition, the most recent findings on the pathophysiology of the infant intestinal microbiota, particularly on the maturation of the “mucosa” and of the intestinal immune “system barrier,” opens up to new and exciting prospects and opportunities.17,18

The use of 2 strains of B. breve B632 and BR03 to rebalance intestinal microbiota in bottle-fed infants is based also on the demonstration that these children lack B. breve.19

These 2 strains were also selected for their important in vitro activity against coliforms and Klebsiella13,16 which, according to Savino et al20 and Rhoads et al,21 are involved in the etiology of infant colics.

The third reason to use these 2 strains is due to their absolute harmlessness, safety, and tolerability, also demonstrated by 3 previous clinical studies that reported no adverse events of any kind.13–15

Previous studies on colics treatment did not give univocal results and most times were carried out both on breastfed and bottle-fed infants. A study by Indrio et al,22 for example, shows a significant reduction in crying time, whereas Sung et al23 did not show any effectiveness. Both studies used L. reuteri DSM 17938.

Savino used L. reuteri DSM 17938 in 46 babies (double-blind, randomized, controlled study). All the infants were breastfed. The treated infants showed a statistically significant decreased crying time compared with placebo.

In our pilot study, bottle-fed infants showed a decreased crying time already during the first month. The decreased crying time becomes more and more evident over time, until becoming statistically significant during the third month (Fig. 1). These data could represent a valid reason to reflect about a possible reinforcement of the dose used.

Even though the reported data refer to a limited group of subjects, the use of B. breve B632 and BR03 showed that bottle-fed infants treated with probiotics cried about 57% less compared with the placebo. These data encourage to expand clinical records, to confirm the effectiveness of this product, with particular reference to bottle-fed infants.


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bifidobacteria; colic; infants; crying time

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