Influence of isomaltooligosaccharides on intestinal microbiota in rats

Journal of Applied Microbiology ISSN 1364-5072 Influence of isomalto-oligosaccharides on intestinalmicrobiota in ratsA. Ketabi1, L.A. Dieleman2 and M.G. Ga¨nzle1 1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada2 Department of Medicine, Center of Excellence for Gastrointestinal Inflammation and Immunity Research, University of Alberta, AB, Canada inulin, isomalto-oligosaccharides (IMO),lactobacilli, rat Aims: Isomalto-oligosaccharides (IMO) with a(1 fi 6) and a(1 fi 4) gluco-sidic linkages are produced by enzymatic conversion of starch. IMO are only partially digestible but data on their influence on intestinal microbiota are Michael Ga¨nzle, Department of Agricultural, limited. It was the aim of this study to investigate the effect of IMO diet on Food and Nutritional Science, University of intestinal microbiota and short-chain fatty acids production (SCFA) in rats.
Alberta, 4-10 Ag ⁄ For Centre, Edmonton, AB, Methods and results: Three groups of F344 rats, each consisting of six animals, Canada T6G 2P5.
E-mail: mgaenzle@ualberta.ca were fed IMO, inulin or a control diets for six weeks. A qualitative assessmentof the intestinal microbiota was achieved by PCR-denaturing gradient gel 2010 ⁄ 2317: received 20 December 2010, electrophoresis (DGGE). Major bacterial taxa were quantified by quantitative PCR (qPCR), and SCFA were measured using gas chromatography. Quantitative PCR demonstrated that lactobacilli were one of the dominant bacterial taxa infaecal samples from rats. IMO increased the number of lactobacilli and the total number of intestinal bacteria in rats fed IMO compared with animalsreceiving control and inulin diets. Furthermore, PCR-DGGE with lactobacilli-specific primers showed an altered biodiversity of lactobacilli in rats fed IMOcompared with control diet.
Conclusions: IMO selectively stimulates lactobacilli and increases their diversityin rats.
Significance and impact of study: Isomalto-oligosaccharides specifically stimu-late growth of intestinal lactobacilli in a rat model system.
of other oligosaccharides that are present in food or are used as food additives such as isomalto-oligosaccharides Dietary nondigestible oligosaccharides (NDO) modulate (IMO), soybean oligosaccharides and lactulose, is limited the composition and activity of intestinal microbiota and (Kolida and Gibson 2008). The diverse structure, mono- they may also exert health benefits in the host. They mer composition and degree of polymerization of dietary improve bowel function, may prevent overgrowth of oligosaccharides influence not only intestinal fermentation pathogenic bacteria through selective stimulation of non- and SCFA production, (Kleessen et al. 2001; Nilsson and pathogenic members of intestinal microbiota and increase Nyman 2005) but also affect their technological properties production of short-chain fatty acids (SCFA). SCFA reduce the luminal pH and provide energy for colonocytes IMO with a(1 fi 6) and a(1 fi 4) glucosidic linkages (Topping and Clifton 2001; Meyer and Stasse-Wolthuis are used as alternative low-calorie sweeteners in food 2009). Intestinal fermentation and health benefits of products (Kohmoto et al. 1992). IMO are only partially fructo-oligosaccharides and galacto-oligosaccharides have digested and the undigested portion is fermented in the been well documented in animal and human studies colon. The caloric content of a commercial IMO prepara- (Meyer and Stasse-Wolthuis 2009; Gibson et al. 2010).
tion was about 75% when compared with maltose However, information regarding intestinal fermentation (Kohmoto et al. 1992). IMO improve constipation similar ª 2011 The AuthorsJournal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology to other fibres (Wang et al. 2001). NDO and polysaccha- 55 mmol l)1 maltose as glucosyl-acceptor for synthesis of rides usually have a laxative effect when taken in high oligosaccharides. Bacterial cells were removed by centrifu- dosage. However, IMO are tolerated at higher dosages gation, and oligosaccharides in the supernatant were anal- compared with other NDO (Kaneko et al. 1994).
ysed by HPAEC-PAD as described earlier.
Commercial IMO preparations consist of isomaltose,isomalto-triose, panose and isomalto-tetraose as major compounds. Different products differ substantially intheir composition, particularly the proportion of digest- Five-week-old F344 rats were housed in specific patho- ible carbohydrates (maltose and glucose), the proportion gen-free conditions (SPF). Rats in three treatment groups of a-(1 fi 4) linkages and the degree of polymerization were fed either IMO (BioNeutra), commercial inulin (Kohmoto et al. 1991, 1992; Yen et al. 2010), and these (Raftiline HP, Raffinerie Tirlemontoise, Tienen, Belgium) differences in composition likely influence digestibility, or a control diet. Each treatment group consisted of six caloric content and their effect on intestinal microbiota.
rats and one or two animals were housed per cage. The Several human studies indicate bifidogenic properties base diet was a commercial laboratory rodent diet (5001; of IMO (Kohomto et al. 1988; Kohmoto et al. 1991, Lab Diet Inc., Leduc, Canada) with the following compo- 1992; Kaneko et al. 1994). However, the majority of these sition: 23% crude protein; 4Æ5% crude fat; 6% fibre; 8% studies relied on culture-dependent methods that targeted ash. Inulin or IMO were added to this base diet at 8 g only a few bacterial groups in the colon. Information (kg body weight))1 for 6 weeks. Rats were given free regarding the in vivo effect of IMO on intestinal microbi- access to water. Sampling of stool from individual ani- ota using culture-independent methods is limited (Yen mals was performed at 5, 8 and 11 weeks of age. Faecal et al. 2010). The aim of this study was to investigate the samples were immediately snap-frozen at )80°C for anal- effect of IMO on microbiota composition and SCFA pro- ysis of intestinal microbiota and SCFA. Experiments were duction in the intestine of rats using culture-independent approved by the University of Alberta Animal Policy and Welfare Committee (UAPWC) in accordance with theCanadian Council on Animal Care (CCAC) guidelines.
PCR-denaturing gradient gel electrophoresis (DGGE) Determination of IMO components with high- PCR-DGGE analysis with universal primers was per- performance anion exchange chromatography – pulsed formed as previously described (Tannock et al. 2000). In brief, DNA from faecal samples was extracted using the IMO were obtained from BioNeutra Inc. (Edmonton, Qiagen DNA extraction kit and the DNA concentration Canada). The composition of the IMO preparation was was adjusted to 50–70 mg l)1. Universal primers HAD1- specified by the supplier as IMO with predominantly a-(1 fi 6) linkages and a degree of polymerization (DP) of 2 (18–25%), DP 3 (15–23%), DP 4 (14–22%), DP 5 CAG CAG T-3¢) and HAD2 (5¢-GTA TTA CCTG CGG (8–10%), DP 6 (6–8%), DP 7 (2–4%) and DP 8 (2–3%).
CTG CTG GCA C-3¢) were used to amplify bacterial Isomalto-oligosaccharides were analysed by HPAEC-PAD rDNA. DGGE was performed by using a DCodeÔ Uni- with a Carbopac PA20 column coupled to an ED40 versal Mutation Detection System (Bio-Rad, Hercules, chemical detector (Dionex, Oakville, Canada) using water CA, USA) in 6% acrylamide gels with a denaturing gradi- (A), 200 mmol l)1 NaOH (B) and 1 mol l)1 Na-acetate ent of 30–55%. Electrophoresis was performed at 150 V (C) as solvents at a flow rate of 0Æ25 ml min)1 and a and 60°C for about 3 hours. Gels were stained with ethi- temperature of 25°C. The gradient was as follows: 0 min dium bromide and viewed by UV transillumination.
30Æ4% B, 1Æ3% C, 22 min 30Æ4% B and 11Æ34% C fol- Patterns were normalized by including PCR products lowed by washing and regeneration. Isomaltose, isomalto- from one sample on all gels. Cluster analysis was per- triose and panose were identified and quantified by use of formed by unweighted pair group method with arithmetic external standards (all obtained from Sigma, ON, mean (UPGMA) algorithm based on the Dice correla- Canada). Other peaks were tentatively identified by tion coefficient using an optimization coefficient of synthesizing oligosaccharides of the panose series with 1% (Bionumerics software, version 3; Applied Maths, dextransucrase of Weissella minor ATCC35912 (Galle et al. 2010). In brief, W. minor was grown for 24 hour in DGGE analysis of the diversity in Lactobacillus– modified de Man, Rogosa and Sharpe medium (MRS) Pediococcus–Leuconostoc–Weissella species was performed containing 230 mmol l)1 sucrose as glucosyl-donor and using the primers LAC1 (5¢- AGC AGT AGG GAA TCT Journal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology TCC A-3¢) and LAC2- GC (5¢- CGC CCG GGG CGC kinase were quantified using degenerate primers (Table 1).
PCR and calibration of qPCR were carried out on a Fast TYC ACC GCT ACA CAT G-3¢) with subsequent separa- Real-Time PCR unit (Applied Biosystems, Streetsville, tion of amplicons by DGGE (Walter et al. 2001). Selected Canada) as described previously (Metzler-Zebeli et al.
bands from DGGE gels were excised from the gel, used as 2010). Samples from individual animals were analysed in template for PCR amplification with primers Lac1 and at least duplicate. Results from samples obtained from the Lac2, and sequenced in the Molecular Biology Facility of same treatment group and time point were averaged and Department of Biological Sciences at the University of results are reported as log(gene copy number per g).
Alberta. Sequences were deposited with accession num-bers: HM765476 (Lactobacillus animalis), HM765477, Analysis of short-chain fatty acids in stool samples with HM765478 and HQ658983 (all Lactobacillus reuteri).
Sequences were matched to type strain sequences availableon RNA database project (http://rdp.cme.msu.edu/seq SCFA were extracted from 100 mg stool samples by add- ing 200 ll of 5% phosphoric acid. Solids were removedby centrifugation at 17 000 g and supernatants wereinjected on a Stabilwax-DA column (30 m, 0Æ53 mm ID, Quantification of microflora by quantitative PCR (qPCR) 0Æ5 lm df). The head pressure was 7Æ5 psi, and split vent Group-specific primers were used to quantify 16S rRNA flow was set to 20 ml min)1 or adjusted as required.
gene copy numbers (rDNA) of Lactobacillus–Pediococcus– Injector temperature was 170°C. The column temperature Leuconostoc–Weissella species (Lactobacillus group), Bifido- was held at 90°C for 0Æ1 min, increased to 170°C at bacterium spp., Bacteroides–Prevotella–Porphyromonas spp.
10°C min)1 and held for 2 min. The detector temperature (Bacteroides group), clostridial clusters I (Clostridium was 190°C. Concentration of acetate, propionate, buty- perfringens group), IV (Clostridium leptum group), XI rate, isobutyrate, isovalerate, valerate and caproate was (Clostridium difficile group) and XIV (Clostridium cocco- determined used external standards (obtained from ides group), Enterobacteriaceae and total bacteria. Genes Sigma, ON, Canada). Isocaproate was used as internal coding for butyrate CoA-CoA transferase and butyrate Table 1 Oligonucleotide primers used in qPCR of faecal samples *AT, annealing temperature in °C, F, forward; R, reverse.
ª 2011 The AuthorsJournal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology additional components of the IMO preparation, but these could not be identified with external or enzymatically A mixed procedure with repeated measures was used to analyse the effect of diet, time and the interaction of timeand diet using SAS software. Data from five-week-old rats Qualitative analysis of faecal microbiota with DGGE were used as covariates. Results were reported as meanvalues and their standard errors. The least significant dif- PCR-DGGE was employed to initially assess qualitative ference test was used to identify differences between treat- effects of IMO or inulin on the faecal microflora. Cluster ments. Differences were considered to be significant if analysis of PCR-DGGE at 8 weeks of age showed that almost all faecal samples from inulin-fed rats were sepa-rated from those fed control or IMO diets. There was noclear separation between treatment groups at 11 weeks of Quantitative analysis of faecal microbiota with qPCR Carbohydrates in the IMO preparation were separatedand quantified by HPAEC-PAD (Fig. 1). Isomaltose, Quantitative differences between bacterial taxa in faecal isomaltotriose and panose accounted for 11Æ3 ± 2Æ9, samples from IMO-fed and control animals were assessed 5Æ8 ± 1Æ2 and 5Æ6 ± 2Æ2% (w ⁄ w) of the IMO preparation, using qPCR and group-specific primers (Table 2). The respectively; glucose and maltose were essentially absent.
Lactobacillus group was one of the dominant bacterial 6¢Glucosylpanose and 6¢6¢diglucosylpanose were also taxa in the samples; feeding IMO significantly increased rDNA copy numbers of faecal organisms in the Lactoba- (Fig. 1). Dextransucrase from W. minor produces oligo- cillus group compared with rats on control diet (Table 2).
dextran from sucrose and maltose, a homologous series In contrast, the number of bifidobacteria in faecal sam- of linear oligosaccharides composed of a-(1 fi 6) linked ples from rats fed IMO was low and significantly different glucose moieties and a maltose residue at the reducing from animals fed the control diet at eleven weeks of age.
end. These oligosaccharides elute with increasing degrees of polymerization (Galle et al. 2010, Dols et al., 1997).
decreased over time in both treatment groups. The Other disaccharides and higher oligosaccharides were Bacteroides group as well as clostridial clusters I, IV andXIV was not affected by diet or time. Total number offaecal bacteria was increased in rats fed IMO compared Quantitative analysis of genes encoding key enzymes of bacterial butyrate metabolism demonstrated that genes encoding butyrate kinase were below the detection limit of 104 gene copies per g in all samples (data not shown).
Copy numbers of genes encoding butyrate CoA-CoAtransferase were unaffected by diet or time.
Qualitative assessment of organisms in the Lactobacillus group by PCR-DGGE with group-specific primers To determine whether the increased abundance of organ- isms in the Lactobacillus group in rats fed IMO was asso- ciated with an increased biodiversity, PCR-DGGE analysis with primers specific for the Lactobacillus group was pulsed amperometric detection separation of Isomalto-oligosaccha- performed (Fig. 3). The number of bands in faecal sam- rides (lower trace) and of oligosaccharides of the panose series (POS) ples of rats fed IMO diet after 8 or 11 weeks of age was synthesized with dextransucrase of Weissella minor ATCC 35912 with not significantly increased compared with those from the maltose as acceptor carbohydrate. Glucose, sucrose, isomaltose, isom- same time points in rats fed control diets, and cluster altotriose, maltose and panose were identified and quantified by use analysis did not clearly separate the banding patterns of external standards; 6¢glucosylpanose and 6¢6¢diglucosylpanose weretentatively identified by enzymatic synthesis of oligosaccharides of the according to the diet. However, one specific band was observed almost exclusively in rats fed IMO. Four bands Journal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology electrophoresis of faecal samples of rats at 8 weeks of age (Panel a) and at 11 weeks of age (Panel b) fed IMO, inulin or control diet correlation coefficient, Tol, position tolerance 1%; Opt, optimization 0Æ5%). IMO, isomalto- oligosaccharides; INU, inulin; Cont, control; #, differing in their abundance in IMO-fed and control polymerization as well as the ratio of a-(1 fi 4) to animals were subjected to sequence analysis. Band a, a-(1 fi 6) linkages of IMO. The composition of commer- identified as L. reuteri, was present in most animals. Band cial IMO preparations differs substantially; products b, also identified as L. reuteri, was present in most sam- contain up to 40% disaccharides and a substantial ples from rats fed IMO but was essentially absent in rats proportion of maltose and glucose (Kohmoto et al. 1992; fed the control diet. Band c, attributed to L. animalis, was Kaneko et al. 1994; Yen et al. 2010). The IMO prepara- present in few animals fed either IMO or control diets.
tion employed in this study did not contain maltose and Band d, also identified as L. reuteri, was present in most glucose, whereas 6¢glucosylpanose and 6¢6¢diglucosylpa- of the control rats but was absent in rats fed IMO diet nose were identified by enzymatic synthesis of oligosac- charides of the panose series (Dols et al. 1997; Galle et al.
2010). Isomaltose is hydrolysed by brush border enzymesin the intestinal epithelium, the digestibility of isomalto- triose and panose is unclear and longer-chain oligosac- SCFA were analysed in the faecal samples of rats fed IMO charides are considered nondigestible (Kohmoto et al.
or inulin diet to determine the effect of nondigestible car- 1992; Kaneko et al. 1995). The composition of commer- bohydrates in colonic carbohydrate fermentation. Acetate, cial IMO preparations thus affects digestibility and their butyrate and propionate were the dominant end products effect on the composition of intestinal microbiota.
of bacterial fermentation in faecal samples, whereas This study assessed the influence of an IMO prepara- isobutyrate, isovalerate, valerate and caproate were minor tion on intestinal microbiota of rats by PCR-DGGE and components of SCFA. Inulin did not change faecal SCFA qPCR targeting dominant bacterial groups of the rodent concentrations compared with rats on a control diet.
intestine (Benson et al. 2010). The effect of inulin on However, IMO significantly decreased faecal acetate com- intestinal microbiota of rodents is well established pared with rats fed control diet at 11 weeks of age. Total (Kleessen et al. 2001; Meyer and Stasse-Wolthuis 2009; SCFA was also decreased in rats fed IMO diet, compared Gibson et al. 2010), and samples from rats fed inulin with control and inulin treatments. Propionate, butyrate, were therefore analysed using DGGE, and qPCR quantifi- isobutyrate, isovalerate, valerate and caproate were not cation of bifidobacteria and lactobacilli only. In keeping affected by dietary intervention treatments (Fig. 4a,b).
with previous studies, inulin significantly increased num-bers of bifidobacteria from 5Æ8 to 6Æ5 log copy numbers,whereas the abundance of the Lactobacillus group remained unchanged (data not shown, Kleessen et al. 2001). Dietary Isomalto-oligosaccharides are produced commercially by IMO exhibited a remarkable specificity towards the stim- transglycosylation of maltodextrins obtained by starch ulation of the Lactobacillus group. Lactobacillus species hydrolysis (Pan and Lee 2005). Starch hydrolysis and colonize the rodent forestomach (Walter 2008) and are a dominant bacterial groups in faecal microbiota of rodents ª 2011 The AuthorsJournal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology Table 2 Effect of diet, time and interaction IMO, isomalto-oligosaccharides; ns, not significant; SEM, standard error of mean.
*Significant (P < 0Æ05).
(Benson et al. 2010). Previous studies in rodent models um spp. have extracellular enzymes hydrolysing polymeric also reported increased numbers of lactobacilli as a result a-(1 fi 4) and a-(1 fi 6)-linked glucans (Ryan et al.
of dietary intervention with IMO (Kaneko et al. 1990).
2006). In contrast, enzymes for IMO metabolism in lacto- Lactobacillus animalis, Lactobacillus johnsonii and L. reuteri bacilli are unknown; however, lactobacilli have only few, are dominant Lactobacillus species in the rodent intestine.
if any, extracellular glycosyl hydrolases and preferentially Other Lactobacillus spp., pediococci, Leuconostoc spp. and metabolize disaccharides using intracellular hydrolases or Weissella spp., which are also detected by the Lactobacillus phosphorylases (Ga¨nzle et al. 2007). Lactobacilli are thus group primers, are substantially less abundant (Walter expected to preferentially metabolize low molecular 2008; Benson et al. 2010). Analysis of PCR-DGGE pat- weight IMO, whereas bifidobacteria are capable of hydro- terns generated with primers specific for the Lactobacillus lysis of larger polymeric glucans. Similarly, lactobacilli group indicates that a strain of L. reuteri was specifically and bifidobacteria exhibited preference towards metabo- lism of low and high molecular weight galacto-oligosac- The number of bifidobacteria decreased in rats fed an charides, respectively (Gopal et al. 2001).
IMO diet. However, previous reports indicated that IMO The total number of faecal bacteria increased in rats increased faecal bifidobacteria in BALB ⁄ c mice (Kaneko fed IMO diet compared with control diet. Other groups et al. 1990). This discrepancy can be attributed to the low of bacteria were not affected by the IMO diet. Faecal numbers of bifidobacteria in rodent intestines, in contrast Enterobacteriaceae decreased at 11 weeks of age in all to lactobacilli, a stable and more abundant genus in animals irrespective of the diet. Inulin or fructo- rodent intestinal microbiota (Walter 2008). Bifidobacteri- oligosaccharides altered the numbers of organisms in the Journal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology Figure 3 Denaturing gradient gel electrophoresis of faecal samples of rat at 8 and 11 weeks of age fed IMO or control diets run with lactobacil-li-specific primers. Band assignment was carried out with BIONUMERICS with a 1% tolerance for the position of the band in the gels; presence (+) orabsence ()) of bands that were identified by sequencing is shown to the right of the gel. IMO, isomalto-oligosaccharides; Cont, control; #, ratnumber, (age of rats in weeks). Selected bands were excised from the gel and identified by sequencing, a, Lactobacillus reuteri; b, Lactobacillusreuteri; c, Lactobacillus animalis; d, Lactobacillus reuteri.
C. coccoides cluster as well as enterococci in the rat lactobacilli have a questionable selectivity (Mikkelsen et al. 2003; Simpson et al. 2004) and do not allow the Differences between the microbiota of rats and humans quantification of other major bacterial groups in the relate to the physiology of the digestive tract. Rats have a intestine. The use of fluorescent-in-situ-hybridization forestomach with nonsecretory epithelium which is absent with four group-specific probes indicated that dietary in humans; moreover, fibre fermentation occurs in the IMO stimulated bifidobacteria and particularly lactobacilli caecum in rats and in the colon in humans (Tiihonen in elderly, constipated patients, whereas the abundance of et al. 2008). Bifidobacteria occupy narrow environmental Bacteroides spp. and Clostridium spp. decreased (Yen et al.
niches compared with lactobacilli, belong to the dominant 2010). In summary, an increased abundance of lactobacilli bacteria in humans, and colonize the intestine of infants as observed in this study corresponds to human studies, shortly after birth (Biavati et al. 2000; Lamendella et al.
whereas the effect of IMO on the abundance of bifidobac- 2008). In contrast, lactobacilli are abundant throughout teria appears to differ between rodent models and human the rodent digestive tract, but are much less abundant in human intestines (Walter 2008; Walter et al. 2008). Initial Although studies in humans demonstrated increased numbers of lactobacilli and bifidobacteria seem to be an SCFA concentrations after consumption of up to 10 g per important factor in stimulation of bacteria by NDO day of IMO (Chen et al. 2001; Yen et al. 2010), this study regardless of the host (Tiihonen et al. 2008). Despite these found decreased acetate and total SCFA in rats fed IMO.
differences between rodent models and humans, the bifid- Ninety-five per cent of SCFA produced by intestinal bac- ogenic effect of fructans in rats (Kleessen et al. 2001; teria are rapidly absorbed by the colon, only unabsorbed Rodriguez-Cabezas et al. 2010) matches results in human SCFA are detected in the faeces (Topping and Clifton studies (Tuohy et al. 2001; Bouhnik et al. 2004; Whelan 2001; Wong et al. 2006). IMO likely stimulates lactate et al. 2005). Studies in humans to determine the effect of and SCFA production in the upper intestine of rats, dietary IMO on intestinal microbiota relied predomi- resulting in SCFA absorption in the intestine and nantly on culture-dependent methods. Isomalto-oligosac- charides were bifidogenic at a dose of 10 g per day and In conclusion, IMO exhibited a remarkable selectivity stimulated lactobacilli in a dose-dependent fashion and particularly increased the abundance and biodiversity (Kohmoto et al. 1991; Kaneko et al. 1994). However, of lactobacilli. Structural differences in nondigestible car- cultivation media for enumeration of bifidobacteria and bohydrates substantially influence their effect on the ª 2011 The AuthorsJournal of Applied Microbiology 110, 1297–1306 ª 2011 The Society for Applied Microbiology acknowledges funding from the Research Chairs of Canada; L.A. Dieleman is supported by Canadian Insti- tutes of Health and Research and by the Alberta IBD Consortium, a team grant of the Alberta Heritage Foun- dation for Medical Research. Ghader Manafi-Azar isacknowledged for assistance in statistical analysis.
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