Accepted Article
Received Date : 15-May-2013 Revised Date : 23-Jul-2013 Accepted Date : 29-Jul-2013 Article type : Research Paper Editor : Julian Marchesi
Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro
Karen P. Scott*, Jennifer C. Martin, Sylvia H. Duncan and Harry J. Flint
Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK.
Keywords: Faecalibacterium prausnitzii, Roseburia spp., Firmicutes, colonic anaerobes, inulin, fructans, bifidobacteria
Running title – Prebiotic substrates for dominant gut bacteria
*Corresponding author: Karen P. Scott, Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK. Tel: 01224 438730 Fax: 01224 438699 e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6941.12186 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Dietary macronutrients affect the composition of the gut microbiota and prebiotics are used to improve and maintain a healthy gut. The impact of prebiotics on dominant gut bacteria other than bifidobacteria however is under-researched. Here we report carbohydrate utilisation patterns for representative butyrate-producing anaerobes, belonging to the Gram-positive Firmicutes families Lachnospiraceae and Ruminococcaceae, by comparison with selected Bacteroides and Bifidobacterium species. Growth assessments using anaerobic Hungate tubes and a new rapid microtitre plate assay were generally in good agreement. The Bacteroides strains tested showed some growth on basal medium with no added carbohydrates, utilising peptides in the growth medium. The butyrate-producing strains exhibited different growth profiles on the substrates, which included starch, inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and xylo-oligosaccharides (XOS). Eleven were able to grow on short-chain FOS but this number decreased as the chain length of the fructan substrates increased. Long chain inulin was utilised by Roseburia inulinivorans, but by none of the Bifidobacterium species examined here. XOS was a more selective growth substrate than FOS, with only six out of the 11 Firmicutes strains able to use XOS for growth. These results illustrate the selectivity of different prebiotics and help to explain why some are butyrogenic.
Introduction The human large intestine is inhabited by a highly complex bacterial community dominated by hundreds of different species of obligate anaerobes (Flint et al., 2007). Cultureindependent 16S rRNA analysis has indicated that the two most abundant bacterial phyla in adults are the Bacteroidetes (normally between 10 – 50%) (Duncan et al 2008) and the Firmicutes (up to around 75%) (Eckburg et al., 2005; Tap et al., 2009; Qin et al., 2010; Walker et al., 2011). The dominant species of Firmicutes mainly belong to the families Lachnospiraceae and Ruminococcaceae (Duncan et al., 2007a). Members of the phylum Actinobacteria, especially Bifidobacterium spp. can also be abundant in the adult human colon (normally up to 10%) (Duncan et al., 2007a; Walker et al., 2011), but have often been underestimated by 16S rRNA sequence analysis if the correct primers are not used (Sim et al., 2012). Although at least two-thirds of bacterial phylotypes detected in the human intestinal microbiota by molecular approaches are not represented in culture collections, a recent study estimated that among the most abundant phylotypes, each representing more
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than 0.5 % of the total microbiota, more than 60 % were represented by cultured species (Walker et al., 2011; Flint et al., 2012a,b). These cultured bacterial representatives are an extremely valuable resource and can be used to test, for example, substrate utilisation and cross feeding interactions. Moreover, complete genome sequences of cultured bacteria provide a framework for metagenomic studies (Qin et al., 2010; Lepage et al., 2013).
There is considerable interest in using dietary approaches, including prebiotics, to modulate the composition of the gut microbiota. The most widely used prebiotics are fructans, both short-chain fructo-oligosaccharides (scFOS, degrees of polymerisation (DP) 2 – 9; β,1-2 linked fructose residues) and long-chain (lc) inulin (DP between 10 - 60); galactooligosaccharides (GOS) and xylo-oligosaccharides (XOS). Most of these prebiotics have been reported to increase numbers of bifidobacteria detected in faeces (Roberfroid et al., 1998; Macfarlane et al., 2008; Ramirez-Farias et al., 2009; Lecerf et al., 2012) and many previous studies have focused on changes in bifidobacteria abundance as a measure of prebiotic effects. Interestingly, lc-inulin has also been shown to enhance the production of butyrate by the faecal microbiota (Kleessen et al., 2001) and butyrate has the potential to benefit colonic health (Pryde et al., 2002; Hamer et al., 2008). Bifidobacteria, however, do not produce butyrate, leaving the mechanism of enhanced butyrate production unclear, although bacterial cross feeding is likely to play a role (Belenguer et al 2006). However, mixed lc-inulin/scFOS, and GOS supplementation have been shown to increase the abundance of Faecalibacterium prausnitzii in human volunteers (Ramirez-Farias et al., 2009; Davis et al., 2010; Dewulf et al., 2013) suggesting that certain butyrate-producing Firmicutes species might be directly stimulated by these prebiotics.
In the current study our aim was to investigate the ability of a range of dominant human colonic butyrate-producers (Flint et al., 2012b) and Bifidobacterium species, to utilise selected non-digestible dietary carbohydrates, including prebiotics, for growth. The most abundant butyrate-producing species detected in recent 16S rRNA studies of adults are Faecalibacterium prausnitzii, Eubacterium rectale, Eubacterium hallii and Anaerostipes hadrus (Tap et al., 2009; Walker et al., 2011). These species were included, together with additional species of human colonic butyrate-producers (Duncan et al., 2004a; Duncan et al., 2006; Louis & Flint, 2009).
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Materials and Methods Substrates The substrates studied included fructans of increasing chain length ranging from P95 short chain (sc)FOS (DP 2-8), Synergy1 (50:50 mixture of P95 scFOS and HP) and HP inulin (degree of polymerisation, (DP) >5; all gifted by BENEO-Orafti, Belgium); dahlia long chain (lc) inulin (DP ~25, purified from Dahlia tubers at RINH, A. Gordon); xylo-oligosaccharides (XOS; Suntory Ltd, Tokyo, Japan); and Vivinal GOS (FrieslandCampina Domo, The Netherlands) which has a dry matter content of 75% consisting of 59% GOS, 21% lactose, 19% glucose and 1% galactose. Amylopectin potato starch was purchased from SigmaAldrich, catalogue number A8515).
Bacterial strains and growth conditions Bacterial strains used are listed in Table 1. Routine culturing of bacterial strains was in M2GSC medium (Miyazaki et al., 1997), using the Hungate tube method and maintaining the media under CO2. Growth analyses on single carbon sources were performed in 9.5 ml basal YCFA medium (Lopez-Siles et al., 2012) supplemented with 0.5 % w/v of the specific substrate indicated. These tubes were inoculated in triplicate with 0.2 ml of a 16-18 h M2GSC culture and incubated anaerobically at 37 ºC. Growth was determined by following changes in optical density at 650 nm (OD650), taking hourly measurements. Maximum specific growth rates (μmax) were determined during exponential growth (Pirt, 1975). Growth was also monitored in microtitre plates, setting the plates up within an anaerobic cabinet and maintaining anaerobic conditions throughout. In this case, readings were obtained from six replicate samples. Overnight cultures (10 or 20 µl) were added to pre-reduced YCFA supplemented with the appropriate substrate (final volume 200 µl) in flat-bottom 96 well microtitre plates (Corning, Sigma Aldrich). Sample blanks containing uninoculated medium were used as controls. After inoculation, microtitre plates were sealed with PCR film to prevent evaporation and to maintain the anaerobic atmosphere (Cernat & Scott, 2012). Cells were incubated for 23 h at 37 °C in a Tecan Safire2 microplate reader (Tecan Group Ltd), with optical density readings at 650 nm taken automatically every hour using the Magellan software (Tecan Group Ltd), with low speed shaking for 5 seconds prior to each reading. Maintenance of anaerobic conditions was verified by performing control growth experiments using either glucose or maltose as an energy source, and including the anaerobic indicator dye resazurin in the growth media.
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Short chain fatty acid production (SCFA) SCFA production was assessed in culture supernatants (1 ml) after 24 hr growth at 37 ºC using gas chromatography as described previously (Richardson et al., 1989). After derivatisation of duplicate samples, 1 μl was analysed using a Hewlitt Packard gas chromatograph fitted with a fused silica capillary column with helium as a carrier gas. The SCFA concentrations were calculated from the relative response factor with respect to the internal standard (2-ethylbutyrate).
Results Comparison of growth of selected faecal anaerobes in tubes and microtitre plates Eleven of the predominant Firmicute bacteria isolated from human faeces (Barcenilla et al., 2000) were compared with some of the most prevalent Bifidobacterium and Bacteroides species from the human colon for their ability to utilise a range of carbohydrate substrates for growth under anaerobic conditions. Butyrate producing members of the families Ruminococcaceae (clostridial cluster IV) and Lachnospiraceae (cluster XIVa) were included in the study (Table 1). YCFA medium (Lopez-Siles et al., 2012) contains a mixture of short chain fatty acids (SCFA) and supported good growth of all of the strains tested, in the presence of their preferred carbohydrate energy sources (glucose or maltose). Only B. vulgatus 1447 and B. thetaiotaomicron B5482 gave significant growth in the absence of any added carbohydrates due to their ability to use peptides present in the casitone and yeast extract components of the basal YCFA medium (Fig. 1). In the remaining data presented, all such basal growth has been subtracted from the OD650 values for the carbohydrate substrates. The maximum OD650 achieved by the bacterial isolates during growth on selected substrates is shown in Fig. 1 (growth in tubes, detailed data in Table S1) and Table 2 and Table 2S (growth in high-throughput microtitre plates). Generally the results obtained using the two methods are comparable, although the maximum OD values attained in the microtitre plates tended to be lower, rarely exceeding an OD of 1.0 (Fig. 2). B. infantis 20088 for example grew well on scFOS in tubes, but less well in the microtitre plate system. This may be due to either substrate depletion in the micro-volumes involved or a build-up of bacterial fermentation products inhibiting further growth. Virtually all of the bacterial strains tested were able to grow well on scFOS (P95), indicating that it is not a particularly selective growth substrate (Fig. 1, Table 1S). However, as the
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complexity of the fructan substrate increased from scFOS to long chain inulin, fewer bacterial strains were able to grow to high ODs. Some of the bacteria were possibly able to utilise any short chain FOS present for initial growth and were unable to grow further once these were depleted, resulting in lower maximum ODs on Synergy1 (a 1:1 mixture of P95 scFOS and HP inulin) compared to scFOS (Table 1S, Table 2). Several Bifidobacterium isolates grew well on both scFOS and HP inulin, but generally higher ODs were achieved on scFOS (Fig. 1, Table 2). Only four of the Firmicutes, and none of the bifidobacteria, were able to grow utilising dahlia inulin (DP~25; Fig. 1), with Roseburia inulinivorans A2-194 achieving the highest OD and the highest specific growth rate (Tables 2S, 3). Growth in microtitre plates of strain SS2/1 belonging to the newly proposed species Anaerostipes hadrus (Allen-Vercoe et al., 2012) on long chain inulin substrates occurred only after a prolonged lag period of 10 h on HP and 15 h on Dahlia inulin (Table 2S). This suggests that for this bacterium, a switch in gene expression is a pre-requisite for growth, and also illustrates that the shorter chain fructans are more readily fermentable. The genes required for inulin utilization by R. inulinivorans have been shown to be inducible (Scott et al., 2011).
Only six bacterial species grew well in tubes on amylopectin starch (achieving an OD of >0.4, Fig. 1, Table 1S). Four of these strains were Roseburia species, which corresponds to published data indicating that the proportion of Roseburia sp. in the gut microbiota can increase as a consequence of carbohydrate, particularly starch, supplementation (Duncan et al., 2007b; Walker et al., 2011). B. breve 20213 was the only Bifidobacterium isolate tested able to utilise amylopectin starch for growth (Table 2). However, different Bifidobacterium species and strains, including B. adolescentis L2-32, have been shown to grow on starch (Duncan et al., 2004a; Belenguer et al., 2006).
Bacterial growth rates Similar maximal specific growth rates (μmax h-1) were achieved on the scFOS (P95) and the other substrates (Table 3). R. inulinivorans was the only Roseburia species tested to utilise all the substrates, and the growth rate on the HP fructan was similar to that on the shorter chain substrates, and was actually higher on dahlia inulin, although the final OD was lower. Interestingly when the growth rates of cultures in tubes and microtitre plates were compared, higher specific growth rates were attained in the microtitre plates, even though the maximal
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OD values were lower (Fig. 2). This may be due to the higher initial inoculation density in microtitre plates compared to Hungate tubes. The growth of the isolates was assessed on additional prebiotic sources, including XOS and GOS (microtitre plate method only; Tables 2, 2S). Most of the isolates tested grew efficiently on GOS in microtitre plates, including the bifidobacteria species (Fig. 3 a,b). In fact GOS was the only substrate to support good growth of B. pseudocatenulatum. The strains that grew more poorly (final OD650 0.3) of six isolates tested (Fig 3 c,d).
Bacterial metabolism The metabolic activities of the bacterial strains were compared by assessing the main SCFA fermentation products after 24 h. The fermentation products for a given bacterium were generally similar for amylopectin starch and fructan substrates (Fig. 4). The main fermentation product for most of the Firmicutes selected in this study was butyrate (1020mM), followed by formate (6-10mM) with many of these bacteria consuming acetate during growth, as previously reported (Barcenilla et al., 2000). Both E. rectale and R. inulinivorans also produced detectable amounts of lactate (6 - 2
HP DP>5
Dahlia DP~25
GOS
XOS
0.28 ±0.03 0.08 ±0.07 0.19 ±0.04 0.14 ±0.14 0.1 ±0.05 0.01 ±0.01 0.06 ±0.05 0.04 ±0.01
0.17 ±0.04 ng
0.51 ±0.13 0.16 ±0.05 0.37 ±0.08 0.64 ±0.02 0.73 ±0.09 0.24 ±0.03 1.16 ±0.08 ng
0.53 ±0.10 0.14 ±0.09 0.27 ± 0.2
0.5 ±0.11 0.15 ±0.05 0.3 ±0.11 0.29 ±0.01 0.42 ±0.06 0.44 ±0.03 0.12 ±0.03 ng
0.15 ±0.08 0.1 ±0.06 0.14 ±0.05 0.06 ±0.06 0.11 ±0.04 ng
0.83 ±0.02 0.80 ±0.06 ng
0.40 ±0.01 0.17 ±0.04 ng
0.77 ±0.02 0.78 ±0.01 0.98 ± 0.05 0.92 ±0.01 0.57 ±0.06
ng
ng ng 0.91 ±0.08 ng 0.12 ±0.06 ng
0.41 ±0.06 0.71 ±0.05 0.35 ±0.08 0.69 ±0.05 0.05 ±0.02
ng ng
0.13 ±0.04 0.15 ±0.04 ng 0.05 ±0.02
a
Data are average maximal OD650 readings from two independent experiments, with 4-6 replicates in each. ng – no growth (final OD after 24 hr 0.5 are shown in bold.
Table 3 - Maximum specific growth rates (h-1) of selected bacteria in Hungate tubes on starch and increasing chain lengths of fructan substrates (0.5%) Substrate
starch
P 95 scFOS
Synergy1
HP
Inulin
E. rectale A1-86
0.40 ±0.05
0.34 ± 0.01
0.38 ± 0.02
0.36 ± 0.07
0.41 ± 0.07
R. inulinivorans A2-194
0.40 ± 0.03
0.38 ± 0.06
0.40 ± 0.03
0.35 ± 0.06
0.54 ± 0.04
F. prausnitzii A2165
no growth
0.28 ± 0.02
0.25 ± 0.03
0.21 ± 0.16
no growth
E. hallii L2-7
no growth
0.54 ± 0.09
0.58 ± 0.04
0.46 ± 0.07
no growth
B. longum 20219
no growth
0.34 ± 0.06
0.38 ± 0.08
0.11 ± 0.08
no growth
Data are the average of triplicate results
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P95 scFOS
1.6
1.6
1.2
1.2
OD650
OD650
Basal YCFA
0.8
0.8
0.4
0.4
0
0
XOS
1.6
1.6
HP
1.2
OD650
OD650
1.2 0.8
0.8 0.4
0.4
0
0
Starch
1.6
1.6
Dahlia Inulin
1.2
0.8 0.4 0
OD650
1.2
OD650
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Fig. 1
0.8 0.4 0
Fig. 2 Bacterial growth on two fructan substrates (0.5%) in tubes compared to microtitre plates (A) 1.40 1.20 1.00 0.80
OD650 0.60
P95 scFOS - tubes P95 scFOS - plates
0.40 0.20 0.00
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(B) 1.40 1.20 1.00 0.80
OD650
Dahlia - tubes
0.60
dahlia - plates 0.40 0.20 0.00
Fig. 3 Growth of selected strains in microtitre plates on 0.5% GOS or 0.5% XOS a) Firmicutes on 0.5% GOS 1.0
0.8 R.faec R.inte
0.6
A.cacc
OD65
R.inul
0.4
E.rect E.hall
0.2
C.euta F.prau
0.0 0 -0.2
1
2
3
4
5
6
7
8
9
Time (hr)
10
11
12
13
14
15
16
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b) Other bacteria on 0.5% GOS 1.0
0.8 B.infa B.long
0.6
B.thet
OD65 0.4
B.adol B.angu
0.2
B.brev B.bifi
0.0 0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Time (hr)
-0.2
c) Firmicutes on 0.5% XOS 1 0.8 R.inte 0.6
R.faec
OD65
A.cacc
0.4
E.rect R.inul
0.2
R.homi 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -0.2
d) Other bacteria on 0.5% XOS
Time (hr)
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1
0.8
OD65 0.6 B.adol B.thet
0.4
B.angu B.brev
Time (hr)
0.2
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
-0.2
Firmicute bacteria: R.faec – Roseburia faecis; R.inte – Roseburia intestinalis; A.cacc – Anaerostipes caccae; R.inul – Roseburia inulinivorans; E.rect – Eubacterium rectale; E.hall – Eubacterium hallii; C.euta – Coprococcus eutactus; F.prau – Faecalibacterium prausnitzii; R.homi – Roseburia hominis. Others: B.infa - Bifidobacterium infantis; B.long – Bifidobacterium longum 20219; B.thet – Bacteroides thetaiotaomicron; B.adol – Bifidobacterium adolescentis ; B.brev – Bifidobacterium breve; B.bifi – Bifidobacterium bifidum; B.angu – Bifidobacterium angulatum
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Received Date : 15-May-2013 Revised Date : 23-Jul-2013 Accepted Date : 29-Jul-2013 Article type : Research Paper Editor : Julian Marchesi
Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro
Karen P. Scott*, Jennifer C. Martin, Sylvia H. Duncan and Harry J. Flint
Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK.
Keywords: Faecalibacterium prausnitzii, Roseburia spp., Firmicutes, colonic anaerobes, inulin, fructans, bifidobacteria
Running title – Prebiotic substrates for dominant gut bacteria
*Corresponding author: Karen P. Scott, Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK. Tel: 01224 438730 Fax: 01224 438699 e-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6941.12186 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Dietary macronutrients affect the composition of the gut microbiota and prebiotics are used to improve and maintain a healthy gut. The impact of prebiotics on dominant gut bacteria other than bifidobacteria however is under-researched. Here we report carbohydrate utilisation patterns for representative butyrate-producing anaerobes, belonging to the Gram-positive Firmicutes families Lachnospiraceae and Ruminococcaceae, by comparison with selected Bacteroides and Bifidobacterium species. Growth assessments using anaerobic Hungate tubes and a new rapid microtitre plate assay were generally in good agreement. The Bacteroides strains tested showed some growth on basal medium with no added carbohydrates, utilising peptides in the growth medium. The butyrate-producing strains exhibited different growth profiles on the substrates, which included starch, inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and xylo-oligosaccharides (XOS). Eleven were able to grow on short-chain FOS but this number decreased as the chain length of the fructan substrates increased. Long chain inulin was utilised by Roseburia inulinivorans, but by none of the Bifidobacterium species examined here. XOS was a more selective growth substrate than FOS, with only six out of the 11 Firmicutes strains able to use XOS for growth. These results illustrate the selectivity of different prebiotics and help to explain why some are butyrogenic.
Introduction The human large intestine is inhabited by a highly complex bacterial community dominated by hundreds of different species of obligate anaerobes (Flint et al., 2007). Cultureindependent 16S rRNA analysis has indicated that the two most abundant bacterial phyla in adults are the Bacteroidetes (normally between 10 – 50%) (Duncan et al 2008) and the Firmicutes (up to around 75%) (Eckburg et al., 2005; Tap et al., 2009; Qin et al., 2010; Walker et al., 2011). The dominant species of Firmicutes mainly belong to the families Lachnospiraceae and Ruminococcaceae (Duncan et al., 2007a). Members of the phylum Actinobacteria, especially Bifidobacterium spp. can also be abundant in the adult human colon (normally up to 10%) (Duncan et al., 2007a; Walker et al., 2011), but have often been underestimated by 16S rRNA sequence analysis if the correct primers are not used (Sim et al., 2012). Although at least two-thirds of bacterial phylotypes detected in the human intestinal microbiota by molecular approaches are not represented in culture collections, a recent study estimated that among the most abundant phylotypes, each representing more
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than 0.5 % of the total microbiota, more than 60 % were represented by cultured species (Walker et al., 2011; Flint et al., 2012a,b). These cultured bacterial representatives are an extremely valuable resource and can be used to test, for example, substrate utilisation and cross feeding interactions. Moreover, complete genome sequences of cultured bacteria provide a framework for metagenomic studies (Qin et al., 2010; Lepage et al., 2013).
There is considerable interest in using dietary approaches, including prebiotics, to modulate the composition of the gut microbiota. The most widely used prebiotics are fructans, both short-chain fructo-oligosaccharides (scFOS, degrees of polymerisation (DP) 2 – 9; β,1-2 linked fructose residues) and long-chain (lc) inulin (DP between 10 - 60); galactooligosaccharides (GOS) and xylo-oligosaccharides (XOS). Most of these prebiotics have been reported to increase numbers of bifidobacteria detected in faeces (Roberfroid et al., 1998; Macfarlane et al., 2008; Ramirez-Farias et al., 2009; Lecerf et al., 2012) and many previous studies have focused on changes in bifidobacteria abundance as a measure of prebiotic effects. Interestingly, lc-inulin has also been shown to enhance the production of butyrate by the faecal microbiota (Kleessen et al., 2001) and butyrate has the potential to benefit colonic health (Pryde et al., 2002; Hamer et al., 2008). Bifidobacteria, however, do not produce butyrate, leaving the mechanism of enhanced butyrate production unclear, although bacterial cross feeding is likely to play a role (Belenguer et al 2006). However, mixed lc-inulin/scFOS, and GOS supplementation have been shown to increase the abundance of Faecalibacterium prausnitzii in human volunteers (Ramirez-Farias et al., 2009; Davis et al., 2010; Dewulf et al., 2013) suggesting that certain butyrate-producing Firmicutes species might be directly stimulated by these prebiotics.
In the current study our aim was to investigate the ability of a range of dominant human colonic butyrate-producers (Flint et al., 2012b) and Bifidobacterium species, to utilise selected non-digestible dietary carbohydrates, including prebiotics, for growth. The most abundant butyrate-producing species detected in recent 16S rRNA studies of adults are Faecalibacterium prausnitzii, Eubacterium rectale, Eubacterium hallii and Anaerostipes hadrus (Tap et al., 2009; Walker et al., 2011). These species were included, together with additional species of human colonic butyrate-producers (Duncan et al., 2004a; Duncan et al., 2006; Louis & Flint, 2009).
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Materials and Methods Substrates The substrates studied included fructans of increasing chain length ranging from P95 short chain (sc)FOS (DP 2-8), Synergy1 (50:50 mixture of P95 scFOS and HP) and HP inulin (degree of polymerisation, (DP) >5; all gifted by BENEO-Orafti, Belgium); dahlia long chain (lc) inulin (DP ~25, purified from Dahlia tubers at RINH, A. Gordon); xylo-oligosaccharides (XOS; Suntory Ltd, Tokyo, Japan); and Vivinal GOS (FrieslandCampina Domo, The Netherlands) which has a dry matter content of 75% consisting of 59% GOS, 21% lactose, 19% glucose and 1% galactose. Amylopectin potato starch was purchased from SigmaAldrich, catalogue number A8515).
Bacterial strains and growth conditions Bacterial strains used are listed in Table 1. Routine culturing of bacterial strains was in M2GSC medium (Miyazaki et al., 1997), using the Hungate tube method and maintaining the media under CO2. Growth analyses on single carbon sources were performed in 9.5 ml basal YCFA medium (Lopez-Siles et al., 2012) supplemented with 0.5 % w/v of the specific substrate indicated. These tubes were inoculated in triplicate with 0.2 ml of a 16-18 h M2GSC culture and incubated anaerobically at 37 ºC. Growth was determined by following changes in optical density at 650 nm (OD650), taking hourly measurements. Maximum specific growth rates (μmax) were determined during exponential growth (Pirt, 1975). Growth was also monitored in microtitre plates, setting the plates up within an anaerobic cabinet and maintaining anaerobic conditions throughout. In this case, readings were obtained from six replicate samples. Overnight cultures (10 or 20 µl) were added to pre-reduced YCFA supplemented with the appropriate substrate (final volume 200 µl) in flat-bottom 96 well microtitre plates (Corning, Sigma Aldrich). Sample blanks containing uninoculated medium were used as controls. After inoculation, microtitre plates were sealed with PCR film to prevent evaporation and to maintain the anaerobic atmosphere (Cernat & Scott, 2012). Cells were incubated for 23 h at 37 °C in a Tecan Safire2 microplate reader (Tecan Group Ltd), with optical density readings at 650 nm taken automatically every hour using the Magellan software (Tecan Group Ltd), with low speed shaking for 5 seconds prior to each reading. Maintenance of anaerobic conditions was verified by performing control growth experiments using either glucose or maltose as an energy source, and including the anaerobic indicator dye resazurin in the growth media.
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Short chain fatty acid production (SCFA) SCFA production was assessed in culture supernatants (1 ml) after 24 hr growth at 37 ºC using gas chromatography as described previously (Richardson et al., 1989). After derivatisation of duplicate samples, 1 μl was analysed using a Hewlitt Packard gas chromatograph fitted with a fused silica capillary column with helium as a carrier gas. The SCFA concentrations were calculated from the relative response factor with respect to the internal standard (2-ethylbutyrate).
Results Comparison of growth of selected faecal anaerobes in tubes and microtitre plates Eleven of the predominant Firmicute bacteria isolated from human faeces (Barcenilla et al., 2000) were compared with some of the most prevalent Bifidobacterium and Bacteroides species from the human colon for their ability to utilise a range of carbohydrate substrates for growth under anaerobic conditions. Butyrate producing members of the families Ruminococcaceae (clostridial cluster IV) and Lachnospiraceae (cluster XIVa) were included in the study (Table 1). YCFA medium (Lopez-Siles et al., 2012) contains a mixture of short chain fatty acids (SCFA) and supported good growth of all of the strains tested, in the presence of their preferred carbohydrate energy sources (glucose or maltose). Only B. vulgatus 1447 and B. thetaiotaomicron B5482 gave significant growth in the absence of any added carbohydrates due to their ability to use peptides present in the casitone and yeast extract components of the basal YCFA medium (Fig. 1). In the remaining data presented, all such basal growth has been subtracted from the OD650 values for the carbohydrate substrates. The maximum OD650 achieved by the bacterial isolates during growth on selected substrates is shown in Fig. 1 (growth in tubes, detailed data in Table S1) and Table 2 and Table 2S (growth in high-throughput microtitre plates). Generally the results obtained using the two methods are comparable, although the maximum OD values attained in the microtitre plates tended to be lower, rarely exceeding an OD of 1.0 (Fig. 2). B. infantis 20088 for example grew well on scFOS in tubes, but less well in the microtitre plate system. This may be due to either substrate depletion in the micro-volumes involved or a build-up of bacterial fermentation products inhibiting further growth. Virtually all of the bacterial strains tested were able to grow well on scFOS (P95), indicating that it is not a particularly selective growth substrate (Fig. 1, Table 1S). However, as the
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complexity of the fructan substrate increased from scFOS to long chain inulin, fewer bacterial strains were able to grow to high ODs. Some of the bacteria were possibly able to utilise any short chain FOS present for initial growth and were unable to grow further once these were depleted, resulting in lower maximum ODs on Synergy1 (a 1:1 mixture of P95 scFOS and HP inulin) compared to scFOS (Table 1S, Table 2). Several Bifidobacterium isolates grew well on both scFOS and HP inulin, but generally higher ODs were achieved on scFOS (Fig. 1, Table 2). Only four of the Firmicutes, and none of the bifidobacteria, were able to grow utilising dahlia inulin (DP~25; Fig. 1), with Roseburia inulinivorans A2-194 achieving the highest OD and the highest specific growth rate (Tables 2S, 3). Growth in microtitre plates of strain SS2/1 belonging to the newly proposed species Anaerostipes hadrus (Allen-Vercoe et al., 2012) on long chain inulin substrates occurred only after a prolonged lag period of 10 h on HP and 15 h on Dahlia inulin (Table 2S). This suggests that for this bacterium, a switch in gene expression is a pre-requisite for growth, and also illustrates that the shorter chain fructans are more readily fermentable. The genes required for inulin utilization by R. inulinivorans have been shown to be inducible (Scott et al., 2011).
Only six bacterial species grew well in tubes on amylopectin starch (achieving an OD of >0.4, Fig. 1, Table 1S). Four of these strains were Roseburia species, which corresponds to published data indicating that the proportion of Roseburia sp. in the gut microbiota can increase as a consequence of carbohydrate, particularly starch, supplementation (Duncan et al., 2007b; Walker et al., 2011). B. breve 20213 was the only Bifidobacterium isolate tested able to utilise amylopectin starch for growth (Table 2). However, different Bifidobacterium species and strains, including B. adolescentis L2-32, have been shown to grow on starch (Duncan et al., 2004a; Belenguer et al., 2006).
Bacterial growth rates Similar maximal specific growth rates (μmax h-1) were achieved on the scFOS (P95) and the other substrates (Table 3). R. inulinivorans was the only Roseburia species tested to utilise all the substrates, and the growth rate on the HP fructan was similar to that on the shorter chain substrates, and was actually higher on dahlia inulin, although the final OD was lower. Interestingly when the growth rates of cultures in tubes and microtitre plates were compared, higher specific growth rates were attained in the microtitre plates, even though the maximal
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OD values were lower (Fig. 2). This may be due to the higher initial inoculation density in microtitre plates compared to Hungate tubes. The growth of the isolates was assessed on additional prebiotic sources, including XOS and GOS (microtitre plate method only; Tables 2, 2S). Most of the isolates tested grew efficiently on GOS in microtitre plates, including the bifidobacteria species (Fig. 3 a,b). In fact GOS was the only substrate to support good growth of B. pseudocatenulatum. The strains that grew more poorly (final OD650 0.3) of six isolates tested (Fig 3 c,d).
Bacterial metabolism The metabolic activities of the bacterial strains were compared by assessing the main SCFA fermentation products after 24 h. The fermentation products for a given bacterium were generally similar for amylopectin starch and fructan substrates (Fig. 4). The main fermentation product for most of the Firmicutes selected in this study was butyrate (1020mM), followed by formate (6-10mM) with many of these bacteria consuming acetate during growth, as previously reported (Barcenilla et al., 2000). Both E. rectale and R. inulinivorans also produced detectable amounts of lactate (6 - 2
HP DP>5
Dahlia DP~25
GOS
XOS
0.28 ±0.03 0.08 ±0.07 0.19 ±0.04 0.14 ±0.14 0.1 ±0.05 0.01 ±0.01 0.06 ±0.05 0.04 ±0.01
0.17 ±0.04 ng
0.51 ±0.13 0.16 ±0.05 0.37 ±0.08 0.64 ±0.02 0.73 ±0.09 0.24 ±0.03 1.16 ±0.08 ng
0.53 ±0.10 0.14 ±0.09 0.27 ± 0.2
0.5 ±0.11 0.15 ±0.05 0.3 ±0.11 0.29 ±0.01 0.42 ±0.06 0.44 ±0.03 0.12 ±0.03 ng
0.15 ±0.08 0.1 ±0.06 0.14 ±0.05 0.06 ±0.06 0.11 ±0.04 ng
0.83 ±0.02 0.80 ±0.06 ng
0.40 ±0.01 0.17 ±0.04 ng
0.77 ±0.02 0.78 ±0.01 0.98 ± 0.05 0.92 ±0.01 0.57 ±0.06
ng
ng ng 0.91 ±0.08 ng 0.12 ±0.06 ng
0.41 ±0.06 0.71 ±0.05 0.35 ±0.08 0.69 ±0.05 0.05 ±0.02
ng ng
0.13 ±0.04 0.15 ±0.04 ng 0.05 ±0.02
a
Data are average maximal OD650 readings from two independent experiments, with 4-6 replicates in each. ng – no growth (final OD after 24 hr 0.5 are shown in bold.
Table 3 - Maximum specific growth rates (h-1) of selected bacteria in Hungate tubes on starch and increasing chain lengths of fructan substrates (0.5%) Substrate
starch
P 95 scFOS
Synergy1
HP
Inulin
E. rectale A1-86
0.40 ±0.05
0.34 ± 0.01
0.38 ± 0.02
0.36 ± 0.07
0.41 ± 0.07
R. inulinivorans A2-194
0.40 ± 0.03
0.38 ± 0.06
0.40 ± 0.03
0.35 ± 0.06
0.54 ± 0.04
F. prausnitzii A2165
no growth
0.28 ± 0.02
0.25 ± 0.03
0.21 ± 0.16
no growth
E. hallii L2-7
no growth
0.54 ± 0.09
0.58 ± 0.04
0.46 ± 0.07
no growth
B. longum 20219
no growth
0.34 ± 0.06
0.38 ± 0.08
0.11 ± 0.08
no growth
Data are the average of triplicate results
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P95 scFOS
1.6
1.6
1.2
1.2
OD650
OD650
Basal YCFA
0.8
0.8
0.4
0.4
0
0
XOS
1.6
1.6
HP
1.2
OD650
OD650
1.2 0.8
0.8 0.4
0.4
0
0
Starch
1.6
1.6
Dahlia Inulin
1.2
0.8 0.4 0
OD650
1.2
OD650
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Fig. 1
0.8 0.4 0
Fig. 2 Bacterial growth on two fructan substrates (0.5%) in tubes compared to microtitre plates (A) 1.40 1.20 1.00 0.80
OD650 0.60
P95 scFOS - tubes P95 scFOS - plates
0.40 0.20 0.00
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(B) 1.40 1.20 1.00 0.80
OD650
Dahlia - tubes
0.60
dahlia - plates 0.40 0.20 0.00
Fig. 3 Growth of selected strains in microtitre plates on 0.5% GOS or 0.5% XOS a) Firmicutes on 0.5% GOS 1.0
0.8 R.faec R.inte
0.6
A.cacc
OD65
R.inul
0.4
E.rect E.hall
0.2
C.euta F.prau
0.0 0 -0.2
1
2
3
4
5
6
7
8
9
Time (hr)
10
11
12
13
14
15
16
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b) Other bacteria on 0.5% GOS 1.0
0.8 B.infa B.long
0.6
B.thet
OD65 0.4
B.adol B.angu
0.2
B.brev B.bifi
0.0 0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Time (hr)
-0.2
c) Firmicutes on 0.5% XOS 1 0.8 R.inte 0.6
R.faec
OD65
A.cacc
0.4
E.rect R.inul
0.2
R.homi 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -0.2
d) Other bacteria on 0.5% XOS
Time (hr)
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1
0.8
OD65 0.6 B.adol B.thet
0.4
B.angu B.brev
Time (hr)
0.2
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
-0.2
Firmicute bacteria: R.faec – Roseburia faecis; R.inte – Roseburia intestinalis; A.cacc – Anaerostipes caccae; R.inul – Roseburia inulinivorans; E.rect – Eubacterium rectale; E.hall – Eubacterium hallii; C.euta – Coprococcus eutactus; F.prau – Faecalibacterium prausnitzii; R.homi – Roseburia hominis. Others: B.infa - Bifidobacterium infantis; B.long – Bifidobacterium longum 20219; B.thet – Bacteroides thetaiotaomicron; B.adol – Bifidobacterium adolescentis ; B.brev – Bifidobacterium breve; B.bifi – Bifidobacterium bifidum; B.angu – Bifidobacterium angulatum
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