2. MONOMER AND LINKAGE TYPE OF GALACTO- OLIGOSACCHARIDES AFFECT THEIR RESISTANCE TO ILEAL DIGESTION AND PREBIOTIC PROPERTIES IN RATS Oswaldo Hernández-Hernández 1, M. Carmen Marín-Manzano 2, Luis A. Rubio 2, F. Javier Moreno 3, M. Luz Sanz 1 and Alfonso Clemente 2* 1 Instituto de Química Orgánica General (CSIC), Madrid, Spain 2 Estación Experimental del Zaidín (CSIC), Granada, Spain 3 Instituto de Investigación en Ciencias de la Alimentación (CSIC-UAM), Madrid, Spain J Nutr 2012, 142 (7) Funded by the Spanish Research Council (PIF-SIALOBIOTIC 200870F010-1, -2 and 3), and ERDF-cofinanced grant from Junta de Comunidades de Castilla-La Mancha (POII10-0178-4685) and MICINN (AGL2011-27884) *To whom correspondence should be addressed. E-mail: alfonso.clemente@eez.csic.es

3. The endogenous microbiota establishes a symbiotic mutualistic relationship and has a major impact upon the nutrition and health of the host supply of nutrients, conversion of metabolites, control of epithelial cell proliferation/differentiation, pathogen exclusion stimulation of the immune system and likely many others…...

4. MODULATION OF INTESTINAL MICROBIOTA: PROBIOTICS AND PREBIOTICS Given the emergent evidence of the roles played by the human microbiota in health and disease, there is a growing interest in identifying live microorganisms (PROBIOTICS) and dietary compounds (PREBIOTICS) capable of modulating the composition and metabolic activities of the intestinal microbiota in order to confer beneficial effects on the host. PROBIOTICS PREBIOTICS Some examples of Inulin Galacto-oligosaccharides (GOS) Fruto-oligosaccharides (FOS) Lactulose Breast-milk oligosaccharides Bifidobacterium animalis Bifidobacterium breve Bifidobacterium lactis Lactobacillus casei Saccharomyces cerevisiae Escherichia coli Nissle 1917

5. Galacto-oligosaccharides (GOS) Usually derived from transgalactosylacion reactions of lactose (GOS-La) catalysed by β-galactosidases of fungal, bacterial or yeast origin 2-10 molecules of galactose (Gal) and a terminal glucose (Glu) A complex mixture of non-digestible carbohydrates with different glycosidic linkages and degrees of polymerization (DP) Lactose: Gal-β-(1→4)-Glc Lactulose: Gal-β-(1→4)-Fru Lactose-derived galactooligosaccharides GOS-La Lactulose-derived galactooligosaccharides GOS-Lu Commercially available Novel

6. Aims To carry out a comparative study in vivo regarding the ileal digestibility of GOS-La and GOS-Lu and to evaluate if their major components are fermented by the intestinal microbiota to promote the selective growth of beneficial bacteria in the large intestine of rats

7. GOS-Lu Lactulose and β-galactosidase from Aspergillus oryzae Size Exclusion Chromatography Removal of mono- and disaccharides, including lactose DP≥3; 35 % trisaccharides Activated charcoal treatment SYNTHESIS FRACTIONATION PRODUCTION OF GOS GOS-La Industrially available Removal of monosaccharides DP≥ 2; 14 % trisaccharides

8. IN VIVO EXPERIMENTAL DESIGN Animal model: Wistar rats, n=12 per treatment group Control diet: AIN-93G (Testdiet) Experimental diet: AIN-93G + 1 % (wt:wt) prebiotic Control (-): no prebiotics GOS-Lu GOS-La Treatments Control diet Days of treatment 0 6 20 Experimental diet Body weight (g) 40 ± 570 ± 5 130 ± 5 SACRIFICE Adaptation period Cr 2 O 3 (2g.kg -1 ) was included as an indigestible marker in all diets Samples Fecal samples: d0, 7 and 14 of experimental treatment Ileal samples: the ileal (last 20 cm of small intestine) content was collected

9. Gos-Lu disaccharides Gos-Lu trisaccharides Representative GC-MS profiles of TMS oxime of di- and trisaccharides present in dietary GOS-Lu, as well as in ileal and fecal samples of rats fed GOS-Lu for 14d. Ileal control is defined as ileal sample from rats fed control diet. Peak number correspond to the carbohydrates indicated in next slide.

10. Peak number 1 DisaccharidesITIT Ileal digestibility 2 (%) Total 0.0 1+2Gal-(1→4)-Fru (lactulose)2878-2887 3Gal-(1→1)-Gal + Gal-(1→4)-Gal E2903 4Gal-(1→5)-Fru 12915 5aGal-(1→3)-Gal E + Gal-(1→2)-Gal E2932 5bGal-(1→5)-Fru 22937 6Gal-(1→4)-Gal Z + unk2959 7Gal-(1→3)-Gal Z – Gal-(1→2)-Gal Z2979 8Gal-(1→6)-Fru 13003 9Gal-(1→6)-Fru 23012 10Gal-(1→1)-Fru 13029 11aGal-(1→6)-Gal E3046 11bGal-(1→1)-Fru 23049 12Gal-(1→6)-Gal Z3094 Trisaccharides Total 12.5 ± 2.6 13unknown3785 14unknown3794 15Gal-(1→6)-Gal-(1→4)-Fru 13809 16Gal-(1→6)-Gal-(1→4)-Fru 23826 17unknown3835 18Gal-(1→4)-Gal-(1→1)-Fru3841 Gal: galactose; Fru: fructose. GOS-Lu: lactulose-derived galacto-oligosaccharides. 1 Labelled peaks are described in previous slide. 2 Data are mean ± SD, n=3 (pools of samples from 4 rats); calculated as ileal digestibility of the whole fraction of di- or trisaccharides. Ileal digestibility, retention indices (I T ) and tentative structural identification of galacto- oligosaccharides (di- and trisaccharides) of GOS-Lu. FULLY RESISTANT HIGHLY RESISTANT

11. GOS-La disaccharides GOS-La trisaccharides Representative GC-MS profiles of TMS oxime of di- and trisaccharides present in dietary GOS-La, as well as in ileal and fecal samples of rats fed GOS-La for 14d. Ileal control is defined as ileal sample from rats fed control diet. Peak number correspond to the carbohydrates indicated in next slide

12. Peak number 1 Disaccharides 2 IT 1Gal-(1→1)-Gal or Gal-(1→1)-Glc2685 2Gal-(1→4)-Glc E + Gal-(1→3)-Glc E2699 3unknown2701 4Gal-(1→4)-Glc Z+ Gal-(1→4)-Gal E2709 5Gal-(1→3)-Glc Z + unk2727 6Gal-(1→2)-Glc E2736 7Gal-(1→2)-Glc Z2765 8Gal-(1→6)-Glc E2824 9Gal-(1→6)-Glc Z2868 Trisaccharides Individual ileal Ileal digestibility 3 (%) digestibility 3,4 (%) Total 52.9 ± 2.7 10(1→1) 5 36616.0 ± 1.2 11unknown367547.7 ± 2.6 12unknown371122.6 ± 1.8 13unknown372399.5 ± 2.9 14unknown3755100.0 ± 0.0 15unknown376630.2 ± 3.3 16Gal-(1→4)-Gal-(1→4)-Glc E377529.5 ± 4.8 17Gal-(1→6)-Gal-(1→4)-Glc E + Gal-(1→4)-Gal-(1→2)-Glc379477.0 ± 6.6 18Gal-(1→4)-Gal-(1→4)-Glc Z + unk380177.9 ± 4.6 19unknown3811100.0 ± 0.0 20Gal-(1→6)-Gal-(1→2)-Glc 1 + Gal-(1→4)-Gal-(1→2)-Glc 238260.0 ± 0.0 21unknown386254.1 ± 1.5 22unknown3880100.0 ± 0.0 23Gal-(1→6)-Gal-(1→6)-Glc 1388981.3 ± 3.6 24unknown396325.3 ± 8.6 25unknown399730.4 ± 3.5 26unknown401229.7 ± 1.9 Gal: galactose; Fru: fructose. GOS-Lu: lactulose-derived galacto-oligosaccharides. 1 Labelled peaks are described in previous slide. 2 Disaccharides were not present in GOS-La diet. 3 Data are mean ± SD, n=3 (pools of samples from 4 rats); 4 calculated as ileal digestibility of the whole fraction of trisaccharides. 5 Nonidentified monomers. Ileal digestibility, retention indices (I T ) and tentative structural identification of galacto- oligosaccharides (di- and trisaccharides) of GOS-La. PARTIALLY RESISTANT

13. Diets Time Pooled SEM P value 2 Control GOS- Lu GOS- La 0 7 14 Diets Time Interaction Bacterial Groups (log 10 copy number/g of freeze-dried fecal sample) All bacteria 10.5 a 10.7 b 10.8 b 10.6 a 10.9 b 0.007<0.001 NS 3 Bacteroides 10.6 a 10.7 b 10.9 c 10.7 b 10.6 a 10.8 c 0.007<0.001 NS Bifidobacteria 9.82 a 10.5 c 10.1 b 9.68 a 10.1 b 10.7 c 0.024<0.001 NS Lactobacilli 9.64 a 9.78 b 10.1 c 9.52 a 9.77 b 10.2 c 0.006<0.001 NS Eubacterium rectale / Clostridium coccoides group 9.47 a 10.1 b 10.2 b 9.59 a 9.83 b 10.2 c 0.019<0.001 NS Clostrodium leptum subgroup 9.33 a 9.48 b 9.62 c 9.57 c 9.38 a 9.47 b 0.007<0.001 NS Microbiota composition in fecal samples obtained from growing rats fed control, GOS- Lu or GOS-La diet on d 0, 7 and 14 1. 1 Data are mean and pooled SEM, n=3 (pools of samples from 4 rats), expressed as log 10 copy number/g of freeze-dried fecal sample. Within diet or time, means without a common letter differ, P≤0.05. GOS-La: lactose-derived galacto-oligosaccharides; GOS-Lu: lactulose-derived galacto-oligosaccharides. 2 Significance main effects (diet and treatment time) were determined by GLM REP (General Linear Model by Two-way Repeated Measures ANOVA). 3 NS: non-significant, P > 0.05.

14. Data are mean ± SD, n=3 (pools of samples from 4 rats). Effects of diet (P=0.004), time (P=0.001) and their interaction (P=0.008) were significant. For each time point, means without common letter differ, P<0.05. Asterisk indicates significant differences (P<0.05) in that dietary group from d 7. GOS-La: lactose-derived galacto-oligosaccharides; GOS-Lu: lactulose-derived galacto-oligosaccharides. Selectivity index (SI) scores of fecal samples obtained from growing rats fed control, GOS-Lu, or GOS-La diet on d 7 and 14.

15. Conclusions The disacharide fraction of GOS-Lu is fully resistant to digestion The trisacharide fraction of GOS-Lu is significantly more resistant than that from GOS-La (ileal digestibility 12.5 vs 52.9 %, respectively) Digestibility Great resistance of galactosyl-fructoses to gut digestion Differential digestibility of GOS-La trisaccharides was associated to difference on glycosidic linkages among galactose and glucose THESE DATA REVEALS THE KEY ROLE PLAYED BY THE MONOMER AND LINKAGE TYPE IN RESISTANCE TO GUT DIGESTION, a major criteria of prebiotic compounds

16. Conclusions The absence of GOS-Lu and GOS-La digestion-resistant oligosaccharides in fecal samples indicated that they were readily fermented within the large intestine. Fermentability Compared with controls, the GOS-Lu group has more bifidobacteria in fecal samples after 14 d of treatment The number of Eubacterium rectale was greater in the GOS-Lu and GOS-La groups than in controls THESE DATA SUPPORT A DIRECT RELATIONSHIP BETWEEN PATTERNS OF RESISTANCE TO DIGESTION AND PREBIOTIC PROPERTIES OF GALACTO- OLIGOSACCHARIDES In vivo prebiotic properties alfonso.clemente@eez.csic.es