© Horizon Scientific Press. Offprints from www.ciim.net

*For correspondence: r.a.rastall@reading.ac.uk

Curr. Issues Intestinal Microbiol. 7: 13–18. Online journal at www.ciim.net

In vitro Three-stage Continuous Fermentation of

Gluco-oligosaccharides Produced by Gluconobacter oxydans NCIMB 4943 by the Human Colonic

Microflora

S. Wichienchot1, P. Prasertsan1,

T. Hongpattarakere1, G.R. Gibson2, and

R.A. Rastall2*

1Department of Industrial Biotechnology, Prince of Songkla University, Hatyai, Thailand2School of Food Biosciences, The University of Reading, Whiteknights, Reading, UK

Abstract

Gluco-oligosaccharides produced by Gluconobacter

oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in

situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the gluco-oligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

Introduction

There has been sustained interest in recent times in the concept of functional foods for gut health (Rastall et al., 2005). This has encompassed probiotic bacteria, prebiotic carbohydrates and symbiotic combinations of the two. There are several prebiotics on the world market with varying properties (Rycroft et al., 2001; Palframan et al., 2002) when evaluated using in vitro fermentation assays. Recent developments in glycotechnology have led to the view that it may be possible to design enhanced forms of prebiotic carbohydrate with specific functional enhancements (Rastall and Maitin, 2002). One highly desirable attribute would persistence of a selective, prebiotic, type of bacterial fermentation towards the distal regions of the colon. The distal colon tends to have higher concentrations of toxic compounds, often resulting from proteolysis (Macfarlane et al., 1992). This region is also the site of onset of colon cancer and ulcerative colitis (Ghosh et al., 2000) and the targeting of functional food ingredients towards this region might be expected to have health benefits.

Oligodextrans of varying molecular weight have been previously investigated as prebiotics in vitro (Olano-Martin et al., 2000). They were found to be selective for bifidobacteria and lactobacilli and also to result in the production of high levels of butyrate. This latter property

seemed to be related to an undesirable increase in clostridial populations.

In this study, novel gluco-oligosaccharides have been manufactured from maltodextrins by the action of dextran dextrinase (Mountzouris et al., 1999). Such materials consist of glucan chains with 1 6 and 1 4 linkages and are classed as dextrans. They have been previously shown to be selective towards bifidobacteria and lactobacilli in faecal batch culture (Wichienchot et al., unpublished). These novel gluco-oligosaccharides are of higher molecular weight than those tested by Olano-Martin et al. (2000). The molecular weight range of the materials tested in this study was 7.8–65.6 kDa and hence these materials may be more slowly fermented and thus persist for longer in the colon. However, it is unclear whether the prebiotic effect would be maintained in concurrence with persistence and therefore these traits were evaluated in an in vitro three-stage continuous culture system which simulated different colonic regions.

Materials and methods

Carbohydrate substrates

G19 Gluco-oligosaccharides were prepared by the action of Gluconobacter oxydans NCIMB 4943 using Glucidex 19 commercial maltodextrin as substrate (Mountzouris

et al., 1999). Maltodextrin Glucidex 19 was supplied by Roquette Ltd, France.

Basal medium for three-stage continuous system

(gut model)

The basal medium for the three-stage continuous system contained (g/l): soluble potato starch, 5; peptone water, 5; tryptone, 5; yeast extract, 4.5; NaCl, 4.5; KCl, 4.5; pig porcine mucin, 4; milk casein, 3; pectin, 2; larch wood xylan, 2; arabinogalactan, 2; NaHCO

3, 1.5; MgSO

4, 1.25;

guar gum, 1; cysteine-HCl, 0.8; KH2PO

4, 0.5; K

2HPO

4,

0.5; bile salts, 0.4; CaCl2.6H

2O, 0.15; FeSO

4.7H

2O,

0.005; haemin, 0.05; Tween 80, 1 ml; vitamin K1, 10 l;

resazurin, 4 ml. 135 ml of basal media was aliquoted into 3 Duran bottles and sterilized. 5 litres of basal medium was sterilized in a 10 l glass bottle and used as a medium reservoir (Sghir et al., 1998).

Oligodextran (10 g/l) produced from maltodextrin Glucidex 19 was added into basal medium aseptically before feeding started from day 10 to day 20.

Three-stage continuous system (gut model) fermentation

The system comprised of a cascade of three fermenters connected in series. The system was initially operated in batch culture mode in basal medium for 24 h. Basal medium for the gut model was pumped into vessel (V1), which sequentially fed V2, then V3 and ultimately

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14 Wichienchot et al.

a waste unit. V1 and V2 had operating volumes of 280 ml, whist the volume in V3 was 300 ml. V1, V2 and V3 simulated proximal, transverse and distal colon conditions respectively. The pH set points ( 0.1) of V1, V2 and V3 were 5.5, 6.2 and 6.8, respectively. Each vessel was magnetically stirred and continually sparged with oxygen-free nitrogen gas. Temperature (37 C) was maintained by a water-cooling system and culture pH was controlled automatically through the addition of 1 N NaOH or HCl. Flow rate of basal medium was controlled by a pump set to 28 2 ml/h to give an overall system dilution rate of 0.10 h-1. Dilution rates of V1, V2 and V3 were 0.10, 0.10 and 0.093 h-1, respectively. After 10 days of fermentation, the basal medium was supplemented with G19 oligodextran (10 g/l final concentration) and fed from day 10 to day 20 of fermentation. Samples (6 ml) were taken in triplicate at days 0, 9, 11 and 20 of the fermentations, then samples were analysed for bacterial enumeration by FISH and for SCFA by HPLC. Prebiotic index (PI) of oligodextran produced from maltodextrin (Glucidex 19) at each time point in vessels 1, 2 and 3 was calculated using the equation given in Section 2.3.

Prebiotic index (PI) equation

To quantify the prebiotic effect (selective fermentation), a prebiotic index was calculated as follows:

Prebiotic index (PI) = (Bif/Total)–(Bac/Total)+(Lac/Total)–(Clos/Total)

Where Bif is bifidobacterial numbers at sample time/number at inoculation; Bac is bacteroides numbers at sample time/numbers at inoculation; Lac is lactobacilli numbers at sample time/number at inoculation; Clos is clostridia numbers at sample time/number at inoculation; Total is total bacteria numbers at sample time/numbers at inoculation.

The equation assumes that an increase in the populations of bifidobacteria and/or lactobacilli has a positive effect. In contrast, an increase in bacteroides and clostridia was deemed negative (Palframan et al., 2003).

Short-chain fatty acid (SCFA) analysis

Samples (1.5 ml) was centrifuged (17,000xg) for 15 min to remove particulate materials and cells, then the supernatant (20 l) was injected onto an HPLC system attached to a UV detector set at 210 and 214 nm. The column was an ion-exclusion Aminex HPX-87H 150 7.8 mm ID (BioRad, Watford, Herts) maintained at 50 C with a column heater. The eluent, 0.005 M sulphuric acid in HPLC-grade water, was pumped through the column at a flow rate of 0.6 ml/min. Data from the UV detector were integrated using the ValueChromTM software package (Bio-Rad). Using external calibration curves, lactate, formate, acetate, propionate and butyrate were quantified in the sample (Rycroft et al., 2001).

Fluorescent in situ hybridization (FISH)The changes in the populations of selected bacterial groups were monitored by fluorescent in situ hybridization (FISH). Bacterial groups were selected for their ability to act as biomarkers of a healthy colon. Genus-specific

16S rRNA-targeted oligonucleotide probes labelled with fluorescent dye Cy 3 (supplied by Eurogentec Ltd., UK) were used for enumeration of Bifidobacterium (Bif 164), Bacteroides (Bac 303), Lactobacillus/Enterococcus spp. (Lab 158), Clostridium perfringens/histolyticum subgroup (His 150) and Clostridium coccoides/Eubaterium rectale

group (Erec 482). The DNA sequences of Bif 164, Bac 303, Lab 158, His 150 and Erec 482 probes were 5’-CAT CCG GCA TTA CCA CCC-3’, 5’-CCA ATG TGG GGG ACC TT-3’, 5’-GGT ATT AGC A(T/C)G TGT TTC CA-3’, 5’-TTA TGC GGT ATT AAT CT(C/T) CCTTT-3’ and 5’-GCT TCT TAG TCA GGT ACC G-3’, respectively (Rinne

et al., 2005). To obtain total bacterial counts, the nucleic acid stain 4’, 6-diamidino-2-phenylindole (DAPI) was added to each sample. Cells were fixed on a slide and those stained with DAPI or hybridized with probe were enumerated under a microscope as described below.

Samples (375 l) were removed and added to 1.125 ml filtered 4% (w/v) paraformaldehyde solution (pH 7.2), mixed and stored at 4 C overnight to fix the cells. The fixed cells were washed twice in filtered PBS (pH 7) and resuspended in 150 l filtered PBS. Ethanol (150 l) was added and the sample mixed and stored at –20 C for at least 1 h or until needed, but no longer than for 3 months. The fixed cells (16 l) were added to 264 l prewarmed hybridization buffer then filtered (0.2 m membrane). An appropriate volume of this mixture and probe (50 ng/ l) were mixed and placed into a hybridization oven at appropriate temperatures overnight (Bif 164, Erec 482 and His 150 at 50 C; Bac 303 and Lab 158 at 45 C; DAPI at any temperature.

The hybridization samples (5–120 l) were washed in 7 ml prewarmed, filtered, wash buffer and filtered through 0.2 m filter) containing 20 l DAPI solution (500 ng/ l) for 30 min at appropriate hybridization temperatures. Samples were vacuum filtered onto a 0.2 m isopore membrane filter (Millipore Corporation, UK) and the filters mounted in ‘SlowFade’ (Molecular Probes, The Netherlands) on clean slides. Microscope (Nikon Eclipse, E400, Japan) fitted with appropriate filters for the DAPI stain (excited at 550 nm and emits at 461 nm) and the Cy3 dye (excited at 550 nm and emits at 564 nm). A minimum of 15 fields, each containing 10–100 cells, was counted for each slide.

Results

Microbial changes

Changes in microbial populations in vessel 1 (V1) of the three-stage continuous system are presented in Fig. 1, V2 in Fig. 2 and V3 in Fig. 3. Generally, there was an increased total bacterial count after the additional carbohydrate substrate was fed. Increases in bifidobacteria, the C. coccoides/E. rectale group and the Lactobacillus/Enterococcus group were seen together with a decrease in C. perfringens/C. histolyticum subgroup.

Short-chain fatty acid analysis

Short-chain fatty acid (SCFA) concentrations generated in the three vessels are shown in Table 1. Feeding with the gluco-oligosaccharides resulted in elevated levels of acetate, propionate and butyrate. It must be remembered,

Gluco-oligosaccharide Fermentation in Gut Models 15

Fig. 1 Changes in bacterial populations enumerated using FISH in V1 of the three-stage continuous system. Error bars represent SD of triplicate assay samples. 1% (w/v) gluco-oligosaccharides was supplemented from day 10 to 20.

Fig. 2 Changes in bacterial populations enumerated using FISH in V2 of the three-stage continuous system. Error bars represent SD of triplicate assay samples. 1% (w/v) gluco-oligosaccharides was supplemented from day 10 to 20.

16 Wichienchot et al.

Fig. 3 Changes in bacterial populations enumerated using FISH in V3 of the three-stage continuous system. Error bars represent SD of triplicate assay samples. 1% (w/v) gluco-oligosaccharides was supplemented from day 10 to 20.

Table 1. SCFA* production on fermentation of gluco-oligosaccharides† in V1, V2 and V3 of the three-stage continuous system.

V1 V2 V3

Day Day Day

0 9 11 20 0 9 11 20 0 9 11 20

Lactate 9.181.25

11.254.50

17.044.40

5.041.10

0.000.00

8.201.20

5.411.10

4.391.12

0.000.00

4.221.10

4.971.08

4.420.88

Formate 7.512.20

13.655.56

21.073.45

7.672.00

9.531.56

7.192.12

2.200.86

1.220.29

8.222.00

1.270.20

1.410.89

1.140.10

Acetate 48.109.00

49.629.09

81.7314.40

73.9211.18

57.438.80

65.5615.03

71.909.86

96.0918.89

60.609.90

66.618.80

69.489.00

101.0216.69

Propionate 23.017.69

29.918.05

31.998.98

33.458.90

35.405.60

50.9211.05

56.3712.20

55.5413.80

35.784.59

52.016.60

57.628.90

53.829.78

Butyrate 0.000.00

0.000.00

23.423.88

19.474.50

0.000.00

2.720.99

24.985.50

24.288.50

0.000.00

0.000.00

19.655.20

27.534.50

Total SCFA 87.808.24

104.437.66

175.2512.20

139.5512.89

102.369.24

134.5914.56

160.8613.33

181.5218.24

104.607.45

124.1112.67

153.1318.69

187.9316.89

* Values were means SD from triplicate samples in mM concentrations at day 0, 9, 11 and 20 of fermentation.† 1% (w/v) gluco-oligosaccharides were supplemented in basal medium during day 10 to 20.

however, that cumulative levels of SCFA present in V2 and V3 are not representative of those that would be present in vivo as the gut model system does not model absorption of SCFA by the colonic epithelium.

Calculation of prebiotic index

The persistence of the prebiotic effect was determined by calculation of the prebiotic index in the three vessels as a function of time (Fig. 4). The data show a clear persistence of the prebiotic effect throughout the three vessels of the system with maximum PI in the second vessel.

Discussion

Typically, prebiotic substances are low molecular weight oligosaccharides carbohydrates (Gibson et al., 2004) which are relatively rapidly fermented by members of the colonic microflora, particularly bifidobacteria and lactobacilli. There is, however, potential for increasing colonic persistence of the prebiotic effect by manipulation of the molecular weight distribution of the carbohydrates. However, if the molecular weight is too high then this may compromise the prebiotic effect, in terms of microbial selectivity.

Gluco-oligosaccharide Fermentation in Gut Models 17

Fig. 4 Prebiotic index (PI) scores in V1, V2 and V3 of the three-stage continuous system before (day 9) and after supplemented with 1% (w/v) gluco-oligosaccharides at day 11 and 20 of fermentation.

Colonic persistence was investigated using an in vitro model of the human colon. This has been validated with reference to the colonic contents of sudden death victims and is a good model of the luminal microbiology of the human colon (Macfarlane et al., 1998). Generally, the majority of carbohydrate is fermented in vessel 1 leading to the highest levels of saccharolytic bacteria (Macfarlane

et al., 1998) and generating high concentration of short-chain fatty acids.

Previous studies have shown that gluco-oligosaccharides with a molecular weight range of 7.8–65.6 kDa, are candidate prebiotics (Wichienchot et al., unpublished). Moreover, the gluco-oligosaccharides are potentially prebiotic as determined using in vitro faecal bacterial batch cultures.

Fermentation, in the three-stage colonic model, of a low molecular weight (64.2% DP1–10, 35.8% DP 11) oligodextran stimulated the growth of bifidobacteria and lactobacilli in all vessels (Olano-Martin et al., 2000). However, the highest numbers of both bifidobacteria (9.31 log) and lactobacilli (8.85 log) were found in V1 whereas fermentation of gluco-oligosaccharides in this study resulted in the highest numbers of bifidobacteria (8.92 log) and the Lactobacillus/Enterococcus group (8.41 log) in V3. The highest numbers (log) of bifidobacteria in V1 and V2 were 8.83 and 8.84, respectively. The highest numbers (log) of lactobacilli in V1 and V2 were 8.27 and 8.38, respectively. An important difference between this study and that of Olano-Martin et al. (2000) is that the published study utilized selective media to enumerate bacterial populations. The current study has used FISH which will

enumerate non-culturable bacteria, but the probes used for FISH do not match exactly with the selectivities of the media used by Olano-Martin et al. (2000).

The PI values obtained in the three vessels (Fig. 4) clearly show an increased persistence into vessel two of the gut model. It is not clear at the current time why the PI should peak in this vessel and then decrease in vessel three. The vessels are, however, maintained at different pH values and have different dilution rates. Conceivably the conditions of pH and substrate availability in the third vessel are not favourable to maintain a high PI.

This study has shown that the gluco-oligosaccharides tested have potential application as persistent prebiotics. This must, however, be evaluated using in vivo human volunteer trials.

Acknowledgements

We would like to thanks to National Centre for Genetic Engineering and Biotechnology (BIOTEC), Thailand.

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