1. VII WORKSHOP PROBIÓTICOS, PREBIÓTICOS Y SALUD
Sevilla, 28 y 29 Enero 2016
University of Reading
ASSESSMENT OF THE IN VITRO PREBIOTIC POTENTIAL OF NOVEL OLIGOSACCHARIDES ENZYMATICALLY SYNTHESIZED USING MICROBIAL TRANSGLYCOSIDASES ACTING ON SUCROSE
M. Díez-Municioa, S. Kolidab, M. Herreroa, A. Olanoa, R. A. Rastallb, F. J. Morenoa
(a) Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Madrid, Spain.
(b) Department of Food and Nutritional Sciences, The University of Reading, Reading, United Kingdom.
INTRODUCTION
A wide range of non-digestible oligosaccharides with potential of being selectively fermented in the human gut is emerging, which can be considered as candidate functional food ingredients. Among the strategies used to obtain new prebiotic candidates, enzymatic synthesis has attracted special attention as this process commonly exhibits high yields, substrate specificity and regio- and stereo-specificity. Once the challenges of production, structural characterization and purification have been met, evaluation of the prebiotic potential of dietary carbohydrates is an essential research target. In this work, fermentation profiles of five enzymatically synthesized purified oligosaccharides (4-galactosyl-kojibiose, kojibiose, lactulosucrose, and sets of lactosyl- (DP 4-6) and raffinosyl-oligofructosides (DP 4-7)) were studied using an in vitro batch-culture fermentation system inoculated with human faecal slurries, simulating the events in the distal colon.
MATERIALS & METHODS
In vitro batch-culture fermentation, enumeration of bacterial populations by fluorescence in situ (FISH) hybridization,
and short chain fatty acid (SCFA) analysis
Synthesized oligosaccharides employed in this study to investigate the fermentation selectivity
Oligosaccharide
Chemical structure DP
Synthesis (reaction type, substrate, enzyme involved)
Purity
Other components
4-galactosyl-kojibiose (Gal-koj)
β-Gal-(1→4)-α-Glc-(2→1)-α-Glc 3
Transglucosylation of lactose by
dextransucrase from L. mesenteroides B-512F
96%
Lactose (1.5%), unknown trisaccharide (2.5%)
Kojibiose
(Koj)
α-Glc-(1→2)-α-Glc 2
Transglucosylation of lactose by dextransucrase from L. mesenteroides B-512F followed by S. cerevisiae yeast treatment and hydrolysis with β-galactosidase from K. lactis
84%
Yeast metabolites (16%)
Lactulosucrose
(Lusc)
β-Gal-(1→4)-β-Fru-(2→1)-α-Glc
Transglucosylation of lactulose by
92%
Unknown trisaccharide (8%)
Lactosyl-oligofructosides(LFOS)
β-Gal-(1→4)-α-Glc-(1→2)-
[β-Fru-(1→2)]n; n = 2-4
4-6
Transfructosylation of lactosucrose by
inulosucrase from L. gasseri DSM 20604
90%
Lactosucrose (0.5%)
FOS (DP4-6) (9.5%)
Raffinosyl-oligofructosides (RFOS)
β-Gal-(1→6)-α-Glc-(1→2)-
[β-Fru-(1→2)]n; n = 2-5
4-7
Transfructosylation of raffinose by
87%
Melibiose (11.5%)
Raffinose (1.5%)
Gal-Koj
Kojibiose
Lusc
LFOS
RFOS
3 healthy donor (24-37 years old)
Controls:
Anaerobic
pH-controlled
batch cultures
Lactulose
FOS
Without CH
37ºC, pH , time points: 0,5,10,24 h
Bif164
Lab158
Bac303
Erec482
Chis150
Ato291
Prop853
Fpra655
DAPI (total)
Acetate
Propionate
Butyrate
2-5
2-4
Bacterial counts – FISH
Selectivity index
SCFA analysis - HPLC
RESULTS & DISCUSSION
Effect of synthesized oligosaccharides on bacterial populations
Selectivity Index (SI) scores for Bifidobacteria and Lactobacilli
SCFA production during fermentation
Bifidobacterium
𝐒𝐈= 𝐁𝐢𝐟 𝐭 𝐁𝐢𝐟 𝟎 𝐋𝐚𝐛 𝐭 𝐋𝐚𝐛 𝟎 − 𝐀𝐭𝐨 𝐭 𝐀𝐭𝐨 𝟎 − 𝐁𝐚𝐜 𝐭 𝐁𝐚𝐜 𝟎 − 𝐄𝐫𝐞𝐜 𝐭 𝐄𝐫𝐞𝐜 𝟎 − 𝐏𝐫𝐨𝐩 𝐭 𝐏𝐫𝐨𝐩 𝟎 − 𝐅𝐩𝐫𝐚 𝐭 𝐅𝐩𝐫𝐚 𝟎 − 𝐂𝐡𝐢𝐬 𝐭 𝐂𝐡𝐢𝐬 𝟎 𝐓𝐨𝐭𝐚𝐥 𝐜𝐨𝐮𝐧𝐭 𝐭 𝐓𝐨𝐭𝐚𝐥 𝐜𝐨𝐮𝐧𝐭 𝟎
Acetate
Most prevalent
Statiscally significant differences from the 0h value, for all the oligosaccharides and fermentation times
Concentration (mM)
Lactobacillus *
Slow rate of fermentation by Bif & Lab
Dendogram analysis:
Insights into the structure-microbial growth relationship
Results showed a strong bifidogenic effect for all oligosaccharides tested, with similar Bifidobacterium levels compared to those produced by well-established prebiotics (lactulose and FOS). Accordingly, acetic acid was the most prevalent SCFA produced.
Selectivity index (SI) for bifidobacteria and lactobacilli was calculated for each oligosaccharide to obtain a comparative relationship between the growth of these beneficial bacteria with respect to the other bacterial groups studied.
Oligosaccharides sharing structural features such as monomer composition and type of glycosidic linkage were grouped by dendrogram analysis, thus, underlining the potential correlation between the structural features of prebiotic candidates and their impact on the composition and metabolism of the human faecal microbiota.
Kojibiose / galactosyl-kojibiose
FOS / RFOS
Lactulose / Lactulosucrose / LFOS AA
Koj
Gal-Koj
FOS
RFOS
Lusc
 LFOS
Koj
Gal-Koj
FOS
RFOS
Lusc
LFOS Lu
Control
Koj
Gal-Koj
LFOS
FOS
RFOS Lu
Lusc
Control
SI scores for Bif and Lab at 24 h
Mean acetate levels (5, 10, 24 h)
CONCLUSIONS
With the exception of kojibiose, this work provides the first evidence on the in vitro prebiotic potential of the enzymatically synthesized oligosaccharides, i.e., 4-galactosyl-kojibiose,lactulosucrose, and sets of lactosyl- (DP 4-6) and raffinosyl-oligofructosides (DP 4-7). All these carbohydrates exerted a strong bifidogenic activity and were high producers of acetate following their fermentation on human faecal samples.
Lactulosucrose/lactulose/LFOS, kojibiose/4-galactosyl-kojibiose and FOS/RFOS were respectively clustered together according to their SI scores and levels of acetate production. This finding might point out towards a potential relationship between the molecular structure of prebiotic compounds and their impact on the composition and metabolism of the human gut microbiota and their subsequent physiological effects on the host.
Acknowledgements: This work has been financed by project AGL from the Spanish Ministry of Science and Innovation (MICINN). M. Díez-Municio is supported by CSIC through JAE-Pre Programme co-funded by European Social Fund (ESF) and she conducted a predoctoral short-stay at the University of Reading through a CSIC International Programme (project i-LINK ).