MONOMER AND LINKAGE TYPE OF GALACTO-OLIGOSACCHARIDES AFFECT THEIR RESISTANCE TO ILEAL DIGESTION AND PREBIOTIC PROPERTIES IN RATS

Oswaldo Hernández-Hernández1, M. Carmen Marín-Manzano2, Luis A. Rubio2, F. Javier Moreno3, M. Luz Sanz1 and Alfonso Clemente2*

1Instituto de Química Orgánica General (CSIC), Madrid, Spain2Estación Experimental del Zaidín (CSIC), Granada, Spain

3Instituto 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

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…...

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

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-LaLactulose-derived galactooligosaccharides

GOS-LuCommercially available Novel

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

GOS-LuLactulose and β-galactosidase from Aspergillus oryzae

Size Exclusion ChromatographyRemoval of mono- and

disaccharides, including lactoseDP≥3; 35 % trisaccharides

Activated charcoal treatment

SYNTHESIS FRACTIONATION

PRODUCTION OF GOS

GOS-LaIndustrially available

Removal of monosaccharidesDP≥ 2; 14 % trisaccharides

IN VIVO EXPERIMENTAL DESIGNAnimal 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 ± 5 70 ± 5 130 ± 5

SACRIFICE

Adaptation period

Cr2O3 (2g.kg-1) was included as an indigestible marker in all diets

SamplesFecal samples: d0, 7 and 14 of experimental treatment

Ileal samples: the ileal (last 20 cm of small intestine) content was collected

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.

Peak number1 Disaccharides IT Ileal digestibility2 (%)

Total 0.01+2 Gal-(1→4)-Fru (lactulose) 2878-2887

3 Gal-(1→1)-Gal + Gal-(1→4)-Gal E 2903

4 Gal-(1→5)-Fru 1 2915

5a Gal-(1→3)-Gal E + Gal-(1→2)-Gal E 2932

5b Gal-(1→5)-Fru 2 2937

6 Gal-(1→4)-Gal Z + unk 2959

7 Gal-(1→3)-Gal Z – Gal-(1→2)-Gal Z 2979

8 Gal-(1→6)-Fru 1 3003

9 Gal-(1→6)-Fru 2 3012

10 Gal-(1→1)-Fru 1 3029

11a Gal-(1→6)-Gal E 3046

11b Gal-(1→1)-Fru 2 3049

12 Gal-(1→6)-Gal Z 3094

Trisaccharides

Total 12.5 ± 2.613 unknown 3785

14 unknown 3794

15 Gal-(1→6)-Gal-(1→4)-Fru 1 3809

16 Gal-(1→6)-Gal-(1→4)-Fru 2 3826

17 unknown 3835

18 Gal-(1→4)-Gal-(1→1)-Fru 3841

Gal: galactose; Fru: fructose. GOS-Lu: lactulose-derived galacto-oligosaccharides.1Labelled peaks are described in previous slide.2Data 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 (IT) and tentative structural identification of galacto-oligosaccharides (di- and trisaccharides) of GOS-Lu.

FULLY RESISTANT

HIGHLY RESISTANT

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

Peak number1 Disaccharides2 IT

1 Gal-(1→1)-Gal or Gal-(1→1)-Glc 2685

2 Gal-(1→4)-Glc E + Gal-(1→3)-Glc E 2699

3 unknown 2701

4 Gal-(1→4)-Glc Z+ Gal-(1→4)-Gal E 2709

5 Gal-(1→3)-Glc Z + unk 2727

6 Gal-(1→2)-Glc E 2736

7 Gal-(1→2)-Glc Z 2765

8 Gal-(1→6)-Glc E 2824

9 Gal-(1→6)-Glc Z 2868

Trisaccharides

Individual ileal Ileal digestibility3 (%) digestibility3,4 (%)

Total 52.9 ± 2.7

10 (1→1)5 3661 6.0 ± 1.2

11 unknown 3675 47.7 ± 2.6

12 unknown 3711 22.6 ± 1.8

13 unknown 3723 99.5 ± 2.9

14 unknown 3755 100.0 ± 0.0

15 unknown 3766 30.2 ± 3.3

16 Gal-(1→4)-Gal-(1→4)-Glc E 3775 29.5 ± 4.8

17 Gal-(1→6)-Gal-(1→4)-Glc E + Gal-(1→4)-Gal-(1→2)-Glc 3794 77.0 ± 6.6

18 Gal-(1→4)-Gal-(1→4)-Glc Z + unk 3801 77.9 ± 4.6

19 unknown 3811 100.0 ± 0.0

20 Gal-(1→6)-Gal-(1→2)-Glc 1 + Gal-(1→4)-Gal-(1→2)-Glc 2 3826 0.0 ± 0.0

21 unknown 3862 54.1 ± 1.5 22 unknown 3880 100.0 ± 0.0

23 Gal-(1→6)-Gal-(1→6)-Glc 1 3889 81.3 ± 3.6

24 unknown 3963 25.3 ± 8.6

25 unknown 3997 30.4 ± 3.5

26 unknown 4012 29.7 ± 1.9

Gal: galactose; Fru: fructose. GOS-Lu: lactulose-derived galacto-oligosaccharides.1Labelled peaks are described in previous slide. 2Disaccharides were not present in GOS-La diet.3Data are mean ± SD, n=3 (pools of samples from 4 rats); 4calculated as ileal digestibility of the whole fraction of trisaccharides. 5Nonidentified monomers.

Ileal digestibility, retention indices (IT) and tentative structural identification of galacto-oligosaccharides (di- and trisaccharides) of GOS-La.

PARTIALLY RESISTANT

Diets Time

Pooled SEM

P value2

Control

GOS- Lu

GOS- La 0

7

14

Diets

Time

Interaction

Bacterial Groups

(log10 copy number/g of freeze-dried fecal sample)

All bacteria 10.5a 10.7b 10.8b 10.6a 10.6a 10.9b 0.007 <0.001 <0.001 NS3

Bacteroides 10.6a 10.7b 10.9c 10.7b 10.6a 10.8c 0.007 <0.001 <0.001 NS

Bifidobacteria 9.82a 10.5c 10.1b 9.68a 10.1b 10.7c 0.024 <0.001 <0.001 NS

Lactobacilli 9.64a 9.78b 10.1c 9.52a 9.77b 10.2c 0.006 <0.001 <0.001 NS

Eubacterium rectale / Clostridium coccoides group

9.47a 10.1b 10.2b 9.59a 9.83b 10.2c 0.019 <0.001 <0.001 NS

Clostrodium leptum subgroup 9.33a 9.48b 9.62c 9.57c 9.38a 9.47b 0.007 <0.001 <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 141.

1Data are mean and pooled SEM, n=3 (pools of samples from 4 rats), expressed as log10 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.2Significance main effects (diet and treatment time) were determined by GLM REP (General Linear Model by Two-way Repeated Measures ANOVA).3NS: non-significant, P > 0.05.

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.

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

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