• Development of microbiological techniques to identify and quantify intestinal bacteria
• The fermentation of GOS/FOS in vitro
• The influence of short chain fatty acids (SCFA) on gut barrier and pathogens.
• The prebiotic effect in vitro and in vivo
• Possible direct effects on pathogens
• Effects of GOS/FOS on vaccination (i.e. non-virulent infection) in mouse models
• Effect of GOS/FOS on allergy in mouse models

Characterisation of the microbiota and its fermentation products

Firstly, methods for the reliable identification and detection of intestinal microorganisms as an alternative to the plating technique were developed using the molecular detection of 16S rRNA or its encoding gene. Specific 16S rRNA-based oligonucleotide probes were developed and used to detect groups of bacteria directly in faecal samples by means of fluorescent in situ hybridisation (FISH). Duplex 5’nuclease assays (QRT-PCR) were developed for the accurate quantification of Bifidobacterium species; namely B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. dentum, B. infantis, and B. longum. Also, based on the same methodology, assays were developed to identify and quantify Lactobacillus species. Thus, the tools to investigate the quantitative composition of the microbiota in mammalian faecal samples were made available for further research¹.

The fermentation properties of the new GOS/FOS mixture were investigated in an in-vitro semi-dynamic fermentation system using an inoculum of fresh faeces from breast-fed (BF) infants aged less than one year. HMOS or a mixture of GOS/FOS was added and glucose fermentation served as reference. The samples were taken at different time points and analysed for their short chain fatty acid (SCFA) content by gas chromatography. Gas production was also measured. At the time of complete fermentation and highest SCFA production at 48h, the fermentation products derived from GOS/FOS were not different from those produced by the fermentation of purified HMOS. The predominant SCFA with both substrates

Effect of GOS/FOS on the gut barrier

In order to investigate the effects of GOS/FOS on the gut barrier, a model was developed in which epithelial cells (T 84 cell line) were combined with myofibroblast cells (CDD-18 Co cell line) in a co-culture. After 24 hours incubation with a SCFA mixture identical to that found in the faeces of breast-fed infants, PGE1 and PGE2 were measured in the supernatant (ELISA method) and the mucin expression was investigated (Dotblot Muc-2). The results showed that the SCFA mixture, as found in the faeces of breast-fed infants and in the faeces of infants fed a formula supplemented with GOS/FOS, stimulates mucin 2 production and improves gut barrier integrity.

Suppression of pathogens by GOS/FOS

In another series of experiments the effects of SCFA and pH on the microbiota (growth of pathogens and commensals) were investigated in culture media. The test pathogenic microorganisms were Escherichia coli (ETEC), Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and the commensal bacteria used were Lactobacillus rhamnosus, Lactobacillus plantarum and Bifidobacterium breve³. The results showed that SCFA at concentrations found in the faeces of breast-fed infants, especially acetate, decrease the growth of pathogens in a dose dependent manner but do not affect the growth of commensals. This effect is only seen at pH 5.5 (the pH found in the faeces of breast-fed infants and infants fed a formula supplemented with GOS/FOS), but not at pH 7.5 (the pH usually found in formula-fed infants)⁴.

The possible direct effects of oligosaccharides on infection were investigated by observing the influence of different oligosaccharides isolated from human milk and of GOS/FOS on enteroinvasive Escherichia coli (EIEC) 4608-58 in in vitro infection assays. This particular strain of Escherichia coli is a clinical isolate that resembles Shigella flexneri according to its infection cycle. The bacterial cell counts of EIEC 4608-58 after infection of Caco-2 cells were analysed as well as changes in their proteome, as revealed by MALDI-MS peptide mass fingerprinting. HMOS (especially a neutral fraction) as well as GOS/FOS exert an influence on bacterial protein expression and also on the infection process of enterocytes. The differentially expressed proteins from EIEC 4608-58 identified so far comprise enzymes of energy metabolism⁵.

Studies using animal models

Finally the immunomodulatory effect of GOS/FOS (at different concentrations in the diet) was analysed in in vivo animal models for infection and allergies. In the vaccination model, C57Bl/6 mice were vaccinated with a commercially available influenza vaccine. The primary parameter was the T-helper 1 dependent delayed-type hypersensitivity response (DTH). In addition, cytokine levels and antibody titres were analysed. In GOS/FOS fed mice the vaccine-specific immune response was significantly enhanced, as reflected by increased DTH responses to the vaccine (Figure 1). Also the Th1/Th2 ratio based upon cytokine profiles in supernatants from spleen cell cultures was enhanced. The effects were specific for GOS/FOS because other types of fibres/oligo saccharides failed to modulate vaccination responsiveness. GOS/FOS exposure during distinct phases of the study indicated that supplementation during the priming phase of the immune response in particular was crucial. Recently preliminary experiments with C57Bl/6 pups indicated that GOS/FOS can induce similar effects in mice having an immature immune system. The conclusion from these
data is that GOS/FOS can modulate the immune system systemically in infant as well as in adult mice.⁶ In GOS/FOS fed mice, cytomegaly virus DNA copy numbers were significantly reduced in multiple organs one day after infection, indicating an improved resistance to viral infections⁷.

Figure 1. Improvement of the vaccination responsiveness (non-virulent infection) in the Vaccination Mouse Model (Vos et al. Int Immunopharmacol 2006)


The effects of GOS/FOS on allergies were investigated by sensitising animals with two consecutive ovalbumin injections followed by three ovalbumin aerosol exposures. GOS/FOS feeding significantly inhibited several markers for respiratory allergy in this model, such as serum IgE levels, airway hyperresponsiveness and pulmonary inflammation⁸ (Figure 2).


Figure 2. Decrease of the allergic response (expressed as IgE levels) in an allergy mouse model after feeding GOS/FOS (Garssen et al. 29th World Allergy Organisation Congress, Munich 2005)


Conclusion

Pre-clinical studies have clearly demonstrated that a specific mixture of oligosaccharides comprising GOS/FOS in a ratio of 9:1 can affect the immune system beneficially leading to less severe allergy and improved resistance to systemic infection.

References

1. Haarman M, Knol J. Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula. Appl Environmental Microbiol 2005; 71:2318-24.
2. Govers M, Buco A, Hendrich T, et al. Comparison of human milk oligosaccharides with oligosaccharides for use in infant nutrition on in vitro fermentation using faeces from breast-fed and formula-fed infants. J Pediatr Gastroenterol Nutr 2005; 40:694.
3. Willemsen LE, Koetsier MA, van Deventer SJ et al. Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts. Gut 2003; 52:1442-47.
4. Van Limpt C, Crienen A, Vriesema A, et al. Effect of colonic short chain fatty acids, lactate and pH on the growth of common gut pathogens. Pediatr Res 2004;56:487.
5. Spory A, Mank M, Schmitt JJ, et al. Effects of Neutral and Acidic Oligosaccharides on Invasion, Viability, and Protein Expression of Enteroinvasive Escherichia coli, 2005 abstract WP159, 4th HUPO annual world congress in Munich, Germany.
6. Vos AP, Haarman M, Buco A, et al. A specific prebiotic oligosaccharide mixture stimulates delayed type hypersensitivity in a murine influenza vaccination model. International Immunopharmacology 2006; 6, 1277-86.
7. Stassen F, Vos AP, Grauls G, et al. Cytomegalovirus infection in mice is affected by dietary oligosaccharides. Abstract NVVI. 8-9 December 2005.
8. Garssen J, Vos P, M`Rabet L, et al. Oral exposure to a mixture of galacto-oligosaccharides and long chain fructo-oligosaccharides as a new concept for allergy prevention. 29th World Allergy Organisation Congress, Munich 2005, Abstract 1591.