Prebiotics in Infant Nutrition

Introduction
The concept of prebiotics was introduced less than ten years ago and was first used to define as a "non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health" (1). Accordingly, a prebiotic should resist host digestion and absorption in the stomach and the small intestine and reach the large bowel unmodified, where it is fermented by the microflora colonizing the gastrointestinal system. Once in the large bowel, it might selectively stimulate the growth or activity of one or a limited number of potentially beneficial bacteria, particularly bifidobacteria and lactobacilli, while decreasing the number of facultative anaerobic strains such as Escherichia coli and Clostridia (2,3).

Although undigested nitrogen- and lipid-containing compounds may also have prebiotic effects, the compounds most commonly studied for their proposed prebiotic nature are non-digestible carbohydrates. In particular, oligosaccharides, molecules containing a small number (three to about ten) of monosaccharide residues connected by glycosidic linkages, are considered the main units among prebiotics, which include fructooligosaccharides (FOS), inulin, lactulose and galactooligosaccharides (GOS). Other compounds with potential for development, but not as yet well known, are xylooligosaccharides, acidic oligosaccharides, soyoligosaccharides, pecticoligosaccharides, glucooligosaccharides, and isomaltooligosaccharides (4). All of these molecules seem to play a role in the accretion of intestinal flora, while improving health status and reducing risk for diseases (5). Recently Roberfroid has critically re-evaluated all mentioned oligosaccharides, coming to the conclusion that only two substances can be considered to be prebiotics: fructo-oligosaccharides from the inulin type and galacto-oligosaccharides. For all the other the data set available is insufficient (6).

In the large bowel, the breakdown of the prebiotic molecules by bacterial enzymes, such as ß-fructosidase and ß-galactosidase, determine the production of lactate and short-chain fatty acids (SCFA), acetate, propionate and butyrate, as end-products of fermentation. These molecules decrease the intraluminal pH, directly inhibiting the growth and activities of harmful microrganisms, and contributing to stimulation of the growth of Bifidobacteria, which compete with the enteropathogens for nutrients and epithelial adhesion sites. Several important factors influence the total oligosaccharide effects in the bowel, particularly the nature of the oligosaccharides, the dose, the duration of the treatment, the place where their fermentation mainly occurs (proximal or distal colon) and the initial composition of intestinal flora.

Although there is no recommended daily dose of prebiotics, doses of 4-20 g/day have shown efficacy in adults. Roberfroid et al. (7) suggested for adults that a minimum daily dose of 4 g/day of inulin or FOS would be needed to observe an increase in gut bifidobacteria, while doses higher than 20 g/day might induce some side effects, such as increased flatulence or abdominal bloating. These side effect are mainly attributed to the short chain fructo-oligosaccharides. Thus, the same quantity of inulin will have fewer side effects than a similar amount of short chain fructo-oligosaccharides, whose oral doses should be limited (8).

Human milk oligosaccharides
Oligosaccharides are one of the main components of human milk, which contains, on average, 10 g/L of neutral oligosaccharides and 1 g/L of acidic oligosaccharides (9). The concentration of these compounds in breast milk changes according to different lactation phases, being higher in colostrum than in transitional and mature milk. The composition of human milk oligosaccharides is very complex and more than 100 different oligosaccharide-like structures are known. They express an essentially bifidogenic effect and confer anti-infective properties to human milk.

Human milk or formula feeding in the neonatal period may have different effects on the colonization of the gastrointestinal tract, which is sterile at birth but becomes colonized during vaginal delivery with mainly the intestinal flora of the mother. Breast-fed infants show a predominance of bifidobacteria and/or lactobacilli in comparison to formula-fed infants, who develop an intestinal microflora richer in Enterobacteria and gram-negative organisms (10). The predominance of bifidobacteria in breast-fed infants is thought to contribute to a lower risk of enteric infections. Oligosaccharides might contribute to the natural defence against infections in two ways, that is, either directly, acting as receptor analogues to prevent attachment of enteropathogens on the epithelial surface and interacting with immune cells, or indirectly, altering the commensal gut microflora toward a more healthier composition (11-12).

Review of clinical trials in infants
Because of variety, variability, complexity and polymorphism of the oligosaccharide composition and structure, it is currently not feasible to reproduce the oligosaccharide components of human milk in a strictly structural fashion (13). Nevertheless, current research in infant nutrition is aimed at reproducing their biologic effect in infant and follow-on formulae (11). To date, only few published clinical trials have evaluated prebiotic substances in dietetic products for term and preterm infants, respectively.

Moro et al. (14) have tested a synergistic mixture of neutral galacto-oligosaccharides (GOS, derived from lactose) and long-chain fructo-oligosaccharides (FOS, derived from chicory). After 28 days of feeding, the term infants fed formula supplemented with the GOS/FOS mixture, at concentration of 0.4 g/100 mL or 0.8 g/100 mL, respectively, exhibited a dose-dependent stimulating effect on the growth of bifidobacteria and lactobacilli in the intestine. This combination resulted also in an increase of stool frequency and a reduction of stool consistency, closer to reference breast-fed infants. Boehm G et al. (15) tested in preterm infants a mixture of 90 percent GOS and 10 percent FOS, with a distribution of molecules and a concentration of total oligosaccharides close to human milk, added to a standard preterm formula. The supplementation with the mixture of oligosaccharides resulted in a clearly bifidogenic effect, accompanied by more frequent softer stools. Furthermore, the Ca/P ratio in the urine was similar to that observed in breast-fed infants, suggesting also an influence of prebiotics on calcium absorption.

A study with term infants has evaluated the nutritional efficacy and bifidogenic characteristics of a new infant formula containing partially hydrolysed whey proteins, modified fats with high beta-palmitate contents and prebiotics with starch (16). According to the results, the new formula supported satisfactory growth, led to higher counts of bifidobacteria in the feces, and was well tolerated. A recent prospective study, suggesting that formula-fed infants with "minor" gastrointestinal symptoms (such as colic, regurgitation and constipation) improve within two weeks of feeding the same type of formula, still requires confirmation from randomized controlled trials (RCTs) (17).

Other data from clinical trials, which have been communicated but are still not yet published, consider the bifidogenic effects of either prebiotic-enriched formulas or solid weaning foods with added prebiotic oligalacto-oligosaccharidesaccharides (18-21). Accordingly, a reduction of pathogens has been associated with the consumption of prebiotics. Other communications suggest that the GOS/FOS are detectable in stools in amounts similar to those displayed in infants given human milk oligosaccharides (22). Furthermore, the pattern of fecal short-chain fatty acids in infants fed the oligosaccharide mixture is similar to that of breast-fed infants but was significantly different from that of a group of infants fed with an unsupplemented formula (23).

Safety
Although short-term safety and effects on the total number of bifidobacteria in stools have been repeatedly demonstrated, there are no data on long-term benefits and safety. Although the induction of softer stools may be beneficial in infants with constipation, nobody knows whether the risk of dehydration in some infants is increased. The risk may exist for infants with renal immaturity and a poor ability to concentrate urine, particularly if an additional stress on water balance is induced during the first months of life by fever, respiratory distress, infectious diarrhoea, high dietary renal solute loads or refusal of the infant to accept appropriate quantities of fluids. These concerns are probably overestimating animal data obtained under non physiological conditions. In the meantime, many babies across Europe have been fed with GOS/FOS without complaints of any side effect.

Prebiotics in adults
Knowledge of the clinical effects of prebiotic oligosaccharides in adults is very limited. The consumption of fructo-oligosaccharides in these subjects, as in infants, is associated with an increase of bifidobacteria in faeces and laxative effects, which are useful in treatment of constipation. Nevertheless, studies in adults indicate that dietary fructo-oligosaccharides, because of the osmotic effects of fermentation in the intestinal lumen, may lead to side effects such as flatulence, bloating, abdominal pain, diarrhoea (especially after assumption of large daily doses), and gastroesophageal reflux. (24).

Conclusion
To improve the composition of infant and follow-up formulas, prebiotic oligosaccharides may represent nutrients of great interest for the investigators. A mixture closely mimicking the effects of breastfeeding on intestinal microbiology has been recently proposed and tested in clinical trials. The medium- and long-term consequences of these effects should be followed up and described in future studies. However, in spite of recent important advances, data are still not conclusive as far as the global health effects of prebiotics. As a general, final conclusion, we suggest that a compound be called a prebiotic only after its prebiotic effects have consistently been shown.