Development of a bread delivery vehicle for dietary prebiotics to enhance food functionality targeted at those with metabolic syndrome

Abstract

Prebiotics are dietary carbohydrates that favourably modulate the gut microbiota. The aims of the present study were to develop a functional prebiotic bread using Bimuno®, (galactooligosaccharide (B-GOS) mixture), for modulation of the gut microbiota in vitro in individuals at risk of metabolic syndrome. A control bread, (no added prebiotic) and positive control bread (containing equivalent carbohydrate to B-GOS bread) were also developed. A 3-stage continuous in vitro colonic model was used to assess prebiotic functionality of the breads. Bacteria were quantified by fluorescence in situ hybridization and short chain fatty acids by gas chromatography. Ion-exchange chromatography was used to determine GOS concentration after bread production. Following B-GOS bread fermentation numbers of bifidobacteria and lactobacilli were significantly higher compared to controls. There was no significant degradation of B-GOS during bread manufacture, indicating GOS withstood the manufacturing process. Furthermore, based on previous research, increased bifidobacteria and butyrate levels could be of benefit to those with obesity related conditions. Our findings support utilization of prebiotic enriched bread for improving gastrointestinal health.

Introduction

The colonic microbiota exists in symbiosis, with benign, potentially beneficial and pathogenic organisms existing together within the human host.¹ It has been postulated that modification of this complex microbial environment may alter risk factors associated with obesity; indeed current research has indicated that colonic microbiota dysbiosis may be positively associated with the current obesity epidemic.^2-6 Recent studies report an aberrant gut microbiota and alteration of gut microbial metabolic activities in subjects at risk of metabolic syndrome, with an important influence of a number of human physiological functions. Many studies have shown that diet impacts on the gut microbiota composition.^7-10 Moreover, the gut microbiota has been shown to be receptive to host weight loss.¹¹ As such, dietary modulation of the gut microbiota and its metabolic output could positively influence host metabolism and, therefore, constitute a potential coadjutant approach in the management of obesity and weight loss. The prebiotic concept has attracted interest as an approach for modulation of the colonic microbiota. It is known that prebiotics are proven functional food ingredients that have a positive selective effect on the gut microbiota of humans and animals.^12,13 Many food components, especially non-digestible oligosaccharides and polysaccharide dietary fibers, have been claimed to have prebiotic activity, but few can authentically demonstrate such activity in vivo. Fructo-oligosaccharides (FOS, inulin and oligofructose) and galactooligosaccharides (GOS) have been demonstrated to fulfil the criteria for prebiotic classification repeatedly in human intervention studies. FOS and GOS improve the gut microbiota at the genus level and numerous intervention studies have shown their bifidogenic properties in vivo.¹⁴ To date, different studies have shown the positive effects of a B-GOS, as being selective toward the beneficial genus Bifidobacterium.^12,15,16,17 Despite the prebiotic oligosaccharides on the market, incorporation of prebiotics into different foods and the development of novel forms of oligosaccharides as prebiotics could enable increased consumption and improved ease of use.¹⁸ The idea of using bread as a delivery vehicle means that a food consumed by 99% of UK households may be functionally adjusted to give rise to added health effects. The incorporation of prebiotics into existing foods would make prebiotics more readily available to consumers. When considering a prebiotic to use, GOS provides a good option as they are stable over time in acidic conditions (6 months, pH3), thus use within fruit beverages is an option. Furthermore, high temperature treatments do not denature the structure of GOS (160°C for 10 minutes; 100 for 10 minutes at pH2)¹⁹, rendering the possibility of baking GOS into a variety of food products. Its low calorific value and sweet taste means that GOS is potentially useful as a sweetener. The present study aimed to investigate the impact of bread containing prebiotic GOS on the human intestinal microbial ecosystem of metabolic syndrome volunteers, using an in vitro 3-stage continuous culture system simulating the human large intestine (colon model). These results were compared to that of 2 control breads. Changes in the faecal microbiota were evaluated using 16S rRNA-based fluorescence in situ hybridization (FISH), whereas the potential biological effects of bread intervention on metabolic end products were assessed by short chain fatty acid (SCFA) analysis. Ion-exchange chromatography was also used to analyze whether the mono and oligosaccharide concentrations were maintained after the production processes, thus to access stability following the production process.

Results

HPAEC-PAD Analysis of B-GOS

Ion-exchange chromatography was used in order to determine the concentration of mono, di and oligosaccharides in breads. On average, the quantity of B-GOS recovered in the B-GOS bread was ˜1.43 g per slice (expected value was 1.38 g per slice) indicating that 2 slices (80 g) provided very close to the target 2.75 g GOS. Results showed that B-GOS concentrations were maintained after the production processes. Therefore, no loss of B-GOS content was observed as a result of dough processing, baking and storage.

Monitoring prebiotic functionality within a supplemented bread product in gut models

Numbers of the main bacterial groups constituting the core of the human intestinal microbiota were assessed by FISH before and after supplementation with B-GOS enriched bread (B-GOS bread), a control bread, which had no added prebiotic and a positive control bread containing added mono and disaccharides and maltodextrin, equivalent to those in B-GOS bread, (Figs. 1, 2 and 3, respectively). Following B-GOS bread fermentation, a significant increase in numbers of Bifidobacterium spp. (Bif164) in all the stages of the colonic model system (from 7.814 to 8.878 log CFU ml/ml in V1 [p = 0.0005], from 7.912 to 8.805 log CFU ml/ml in V2 [P = 0.004]) and from 8.096 to 8.866 CFU/ml [p = 0.002] in V3). The Lactobacillus-Enterococcus group (Lab158) significantly increased in V1, simulating the proximal colon, following administration of this bread (from 8.353 to 8.696 log CFU ml/ml in V1 [p = 0.01]. An increase of Clostridium cluster IX (Prop853) was seen in V2 when B-GOS bread was added into the system (from 8.484 to 8.630 log CFU ml/ml in V2 [p = 0.04]).

Figure 1.

Bacterial groups detected by FISH in the culture broth recovered from each vessels (V1, V2 and V3) of the colonic model system before (SS1) and after (SS2) the daily administration of B-GOS bread. Results are reported as mean of the data of 3 colonic models (Log₁₀ CFU/ml) ± standard deviations (SD).

Figure 2.

Bacterial groups detected by FISH in the culture broth recovered from each vessels (V1, V2 and V3) of the colonic model system before (SS1) and after (SS2) the daily administration of control bread. Results are reported as mean of the data of 3 colonic models (Log₁₀ CFU/ml) ± standard deviations (SD).

Figure 3.

Bacterial groups detected by FISH in the culture broth recovered from each vessels (V1, V2 and V3) of the colonic model system before (SS1) and after S2) the daily administration of positive control bread. Results are reported as mean of the data of 3 colonic models (Log₁₀ CFU/ml) ± standard deviations (SD).

Similarly to B-GOS bread, the positive control bread induced a smaller, but significant increase in bifidobacteria in all the stages of the colonic model system (from 8.102 to 8.534 log CFU ml/ml in V1 [p = 0.002], from 8.047 to 8.494 log CFU ml/ml in V2 [p = 0.002]) and from 7.998 to 8.518 CFU ml/ml [p < 0.001] in V3). Furthermore, an overall decrease of E. rectale/Clostridium cluster XIVa group in V1 and V2 of the entire colonic model system was observed during supplementation of this bread (from 8.987 to 8.359 log CFU ml/ml in V1 [p = 0.04], from 8.892 to 8.691 log CFU/ml in V2 [p = 0.03]) and from 8.924 to 8.651 CFU/ml [p =0.008]) in V3, as well as a decrease of Clostridium cluster IX (from 8.714 to 8.506 log CFU/ml in V3 [p = 0.04]). The positive control bread led to a decrease in concentration of Roseburia/E. rectale groups in the first stage (from 8.684 to 8.284 log CFU/ml in V1 [p = 0.001], whereas Clostridium clusters I and II (Chis150) concomitantly decreased in V2 and V3, respectively (from 8.363 to 7.924 log CFU/ml in V2 [p = 0.03], from 8.447 to 8.447 to 7.764 log CFU/ml in V3 [p = 0.001]).

The administration of control bread did not mediate any significant modification in total bacterial counts (EUB338 I, EUB338 II, and EUB338 III) during the intervention. FISH analysis showed that the subdominant lactic acid bacteria (Lab158) concomitantly decreased in V1 and V2, respectively (from 8.607 to 8.181 log CFU/ml in V1 [p = 0.009], from 8.605 to 8.184 log CFU/ml in V2 [p = 0.02]). A similar trend was demonstrated for Clostridium clusters I and II (Chis150) in V3 (from 8.171 to 7.770 log CFU/ml [p = 0.02]).

Impact of breads on production of SCFAs

SCFAs in the 3 different stages of the colonic model systems, at SS1 and SS2, were detected and quantified by gas chromatography (Figs. 4, 5 and 6).

Figure 4.

Short-chain fatty acids concentrations in the culture broths recovered from the vessel 1 of the colonic model before (SS1) and after (SS2) daily administration of B-GOS bread, positive control bread and control bread. Results are reported as means (mmol/l) of the data of three colonic models ± standard error of mean. For each colonic model, measurements were performed in triplicate at SS1 (days 17, 18 and 19) and SS2 (days 27, 28 and 29).

Figure 5.

Short-chain fatty acids concentrations in the culture broths recovered from the vessel 2 of the colonic model before (SS1) and after (SS2) daily administration of B-GOS bread, positive control bread and control bread. Results are reported as means (mmol/l) of the data of three colonic models ± standard error of mean. For each colonic model, measurements were performed in triplicate at SS1 (days 17, 18 and 19) and SS2 (days 27, 28 and 29).

Figure 6.

Short-chain fatty acids concentrations in the culture broths recovered from the vessel 3 of the colonic model before (SS1) and after (SS2) daily administration of B-GOS bread, positive control bread and control bread. Results are reported as means (mmol/l) of the data of three colonic models ± standard error of mean. For each colonic model, measurements were performed in triplicate at SS1 (days 17, 18 and 19) and SS2 (days 27, 28 and 29).

The administration of B-GOS bread in V1 induced a significant increase in butyrate (17.12 to 35.11 mmol/l; p = 0.0093) and acetate (22.24 to 34.78 mmol/l; p = 0.0068) over the course of the experiment (Fig. 4) Conversely, a significant decrease in propionate occurred in V1 (22.95 to 8.816 mmol/l; p = 0.0181).

Butyrate decreased significantly from 22.21 to 8.322 mmol/l; p = 0.0032) in V1 with control bread and as well in V1 with the positive control bread (35.95 to 20.77 mmol/l; p=0.0284) compared to SS1 (Fig. 4) When positive control bread was added into the colonic system, propionate levels decreased significantly at SS2 in V2 and as well in V3 (Figs. 5 and 6) None of the changes in concentrations of SCFAs in all vessels were significant with control bread when compared to SS1 (Figs. 4, 5 and 6)

Discussion

The incidence of obesity has reached alarming levels worldwide, thus increasing the risk of development of metabolic disorders (e.g. type 2 diabetes, coronary heart disease (CHD) and cancer). In the search for new therapeutic targets for treatment of obesity and related disorders, the gut microbiota and its activities have been investigated.

The human gut microbiota has already been shown to influence total energy intake and lipid metabolism, particularly through colonic fermentation of un-digestible dietary constituents. A characteristic of potential relevance between the colonic microbiota and obesity-related metabolic diseases is the ability of some bacteria to produce the 4-carbon short-chain fatty acid (SCFA) butyrate as a product of fermentation. Butyrate has shown potential beyond being a colonic energy source, anti-inflammatory effects have been reported.³³ Furthermore, recently it has been observed a lower abundance of several butyrate-producing bacteria in faecal samples from patients with type 2 diabetes, compared with healthy controls, suggesting a potential protective role of butyrate in obesity-related metabolic diseases.³³ To date, it has been suggested that butyrate, at least partly, explains the association between the intestinal ecosystem and host metabolic health. Furthermore, also in mouse models of obesity, dietary supplementation of butyrate can prevent and treat diet-induced obesity and insulin resistance.³⁴ In this context, the overall aim of this study was to focus on the evaluation of the impact of a bread vehicling a B-GOS prebiotic as a potential application in the prevention and treatment of metabolic syndrome in humans.

Prebiotics and the maintenance of their functionality within different food matrices has not been well researched, thus this study provided an in vitro approach to assess this. The bread recipe development was based on incorporating 2.75g B-GOS into an acceptable serving, this was successfully achieved. Alongside the production of B-GOS enriched bread, a control bread, which had no added prebiotic and a positive control bread which contained a mixture of mono and di-saccharides to provide carbohydrates equivalent to B-GOS bread were developed. Our findings in the current study showed that the carbohydrate concentrations were maintained after the production processes. In order to confirm the effect of food processing on prebiotic functionality, an in vitro 3-stage continuous fermentative colonic model system was used.

B-GOS bread fermentation resulted in positive modulation of the microbiota composition and metabolic activity. B-GOS bread mediated significant increases in bifidobacteria numbers in all the stages of the colonic model system, furthermore lactic acid bacteria significantly increased in vessel 1, simulating the proximal colon. These data show that functionality of B-GOS was maintained within the bread product. The positive control bread also led to increases in bifidobacteria in the 3 vessels of the model system, however, these changes were of a lesser magnitude to those seen in the B-GOS bread (0.4 log, v 1.05 log, vessel 1; 0.45 log, v 0.9 log, vessel 2; 0.53 log, v 0.77 log vessel 3). The ability of the positive control bread to invoke a bifidogenic effect is likely to be due to carbohydrates that remained after the in vitro pre-digestion process, which perhaps would not persist in vivo, e.g., glucose, galactose, lactose and maltodextin added to this bread preparation. The matrix of bread is complex, with carbohydrates that can be frequently bound to other compounds. It is therefore likely that the initial pre-digestion procedure was less complete for the bread.²⁰ The magnitude of the bifidogenic effect with the B-GOS bread indicates the additional presence of a food source for bifidobacteria and also lactobacilli within the B-GOS products.²¹

To be a prebiotic the fermentation must be selective, the positive control bread led to a reduction of bacteria within the E. rectales group, a group associated with butyrate production, a SCFA that has been shown to exert beneficial effects to the colonocytes.^22,35,36 Overall a more significant beneficial modulation was observed after fermentation of B-GOS bread compared to the positive control bread, indicating that B-GOS was having a positive stimulatory effect. The mechanisms need to be explored further, but there is evidence to consider a butyrate-increasing diet could offer benefits against obesity related metabolic diseases.

A bifidogenic effect is potentially of benefit to those at risk of metabolic syndrome as such changes have been linked to reduced levels of circulating lipopolysaccharide, a component of the Gram-negative bacteria cell wall, seen to be raised in obese volunteers.²³ These bifidogenic changes have coincided with reduced weight-gain and dis-lipidemia in murine studies.^2,3 Furthermore, bifidobacteria numbers have been observed to be low in type II diabetic volunteers, when compared to healthy counterpants.²⁴ Thus modulation of the microbiota to yield higher numbers of bifidobacteria could be beneficial for those with metabolic syndrome.

Following B-GOS bread administration, a significant increase in faecal butyrate was observed (Fig. 4) This was not observed following the positive control or control bread administration. Butyrate is a major intestinal epithelial cell energy source, associated with potential anticancer activities, such as reducing cellular malignancy through stimulation of apoptosis in malignant cells.²⁵ Furthermore, butyrate has also been observed to beneficially effect oxidative stress in the human colonic mucosa and to induce immune-modulatory effects.^26,27 It is generally accepted that carbohydrate fermentation results in beneficial effects for the host because of the generation of short chain fatty acids, whereas protein fermentation is considered detrimental to host health.²⁸ For all types of breads, a trend for reduced iso-valerate and reduced combined branched fatty acids, valerate and caproate concentrations was observed (Figs. 4, 5 and 6) which can therefore be viewed as a potentially positive effect as these organic acids are associated with protein fermentation.²⁸

Bread naturally contains fructans, which can be appropriate for bifidobacterial growth. However, these are unlikely to have impacted on the microbiota as the control bread did not lead to microbial modulations including bifidogenic effects. Furthermore the control bread lead to lactobacilli decreases, these were not observed in the B-GOS or mixed sugar products.

The versatility of B-GOS has previously been noted through its stability at high temperatures and also at low pH. Indeed, in the current study positive changes observed in vitro with the bread preparation have also been observed with the use of B-GOS powder in vitro and in vivo.^29,17 Therefore, the in vitro data obtained here indicate that the food matrix did not have a detrimental effect on the availability of B-GOS for bacterial fermentation. This is an important finding, as it shows the potential of B-GOS to be used within baked goods.

Following B-GOS bread production, beneficial modulations were seen in terms of bifidogenic and butyrogenic effects, which were significant and additional to what is seen in control bread. Future work will therefore explore the role such changes play in human health with regards to metabolic syndrome through an in vivo trial.

Materials and Methods

Galacto-oligosaccharide mixture (B-GOS)

The prebiotic GOS mixture (B-GOS; Bimuno®) was provided in powder form consisting of (wt:wt) 48% galactooligosaccharides with a degree of polymerization between 2–5, 22% lactose 18% glucose and 12% galactose and supplied by Clasado Ltd, Milton Keynes, United Kingdom.

Bread preparation

Bread products were prepared according to the Chorleywood Bread Process (CBP). The products were B-GOS enriched bread, control bread, which had no added prebiotic, and a positive control bread containing a mixture of simple fermentable sugars to provide a carbohydrate content equivalent to that found in Bimuno®. All breads were made with flour standardized to 85% extraction rate. Ingredients used for the bread preparation are reported in Table 1 The required dose of B-GOS was 2.75 g, thus recipe development was based on incorporating this dosage into an acceptable serving of bread (2 slices, ˜80 g) through adding 5.5 g Bimuno® (Patent n°5,118,521). A mixture of glucose, galactose, lactose and maltodextrin to provide a carbohydrate content equivalent to 5.5 g of Bimuno® was added to the positive control bread. All ingredients were weighed and, with the exception of water, mixed for 30 seconds at low speed in an industrial kitchen mixer. Then, the water was added and all ingredients mixed for a further 100 seconds at low speed, followed by a high speed mix on a 180 Watt/h setting. Afterwards, the dough was removed and placed into a clean bowl. Aliquots of 460 g portions were prepared and placed into greased tins at room temperature for 10 min. The dough pieces were shaped and transferred into a proving set oven at 40°C, high humidity for 10 min. The dough pieces were re-shaped and placed into the proving oven for a further 45 min. Subsequently, loaves were baked for 30 min at 230°C and then cooled on wire racks. The weights and dimensions of the loaves were recorded prior to storage into plastic food bags at −18°C.

Table 1.

Ingredients for bread preparation

	Control bread	Positive control bread	B-GOS bread
Strong white bread flour (g)	1000	1000	1000
Salt (g)	18	18	18
Vegetable fat (g)	7	7	7
Dried yeast (g)	14	14	14
Water (ml)	630	630	630
Bimuno® (g)	0	0	110
Glucose (g)	0	7.26	0
Galactose (g)	0	4.84	0
Maltodextrin (g)	0	12.1	0
Lactose (g)	0	85.8	0

Analysis of B-GOS by high performance anion exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

To determine whether the mono, di and oligosaccharide concentrations were maintained after bread production, the AOAC 2001.02 (method 32–33).³⁰ Carbohydrate composition of the reaction mixture was determined by high performance anion exchange chromatography coupled with pulsed amperometric detector (HPAEC-PAD). A Dionex system (Dionex corporation, Surrey, UK) consisting of a GS50 gradient pump, an ED50 electrochemical detector with a gold working electrode, an LC25 chromatography oven, and an AS50 autosampler was used. Separation was performed using a pellicular anion-exchange resin based column, CarboPac PA-1 analytical column (4 mm × 250 mm), connected to a CarboPac PA1 Guard column (4 mm × 50 mm) (Dionex corporation, Surrey, UK). The column temperature was maintained at 25°C; elution was performed at a flow rate of 1ml/min using gradient concentrations of sodium hydroxide and sodium acetate solutions (0.2 mol/l). All chromatographic analyses were performed in triplicate using Bimuno, glucose, galactose and lactose at a range of concentrations 0.1–1mmol/l.

Simulated in vitro human digestion

Prior to being added into the colon model system, the breads were digested in vitro under conditions mimicking the upper gastrointestinal tract (stomach, small intestine).³¹ Dialysis was conducted using a membrane of 100–200 Daltons cut-off (Spectra por 100–200 Da MWCO dialysis membrane, Spectrum Laboratories Inc.., UK) to remove monosaccharides from the pre-digested breads. Ion-exchange chromatography was performed to monitor flour digestion, thus ensuring efficiency of the digestion procedure.

Three-stage continuous culture gut model system

Three glass fermenters were connected in series, with an increasing working volume, simulating the proximal (V1, 280 ml), transverse (V2, 300 ml) and distal colon (V3, 320 ml). The system was kept at 37°C, pH maintained at 5.5 (V1), 6.2 (V2) and 6.8 (V3) under anaerobic conditions by continuously sparging with O2-free N2 (15ml/min). V1 was fed by means of a peristaltic pump with an a culture medium, consisting of the following chemicals (g/l) in distilled water: starch, 5.0; pectin (citrus), 2.0; guar gum, 1.0; mucin (porcine gastric type III), 4.0; xylan (oatspelt), 2.0; arabinogalactan (larch wood), 2.0; inulin, 1.0; casein (BDH Ltd.), 3.0; peptone water, 5.0; tryptone, 5.0; bile salts No. Three, 0.4; yeast extract, 4.5; FeSO₄ × 7H₂O, 0.005; NaCl, 4.5; KCl, 4.5; KH₂PO4, 0.5; MgSO₄ × 7H2O, 1.25; CaCl₂ × 6H₂O, 0.15; NaHCO₃, 1.5; cysteine, 0.8; hemin, 0.05; Tween 80, 1.0.³² Human faecal samples were collected on site, kept in an anaerobic cabinet (10% H2, 10% CO2, 80% N2) and used within 15 min of collection. This experiment was carried out in triplicate using faecal samples from 3 different volunteers at risk of developing metabolic syndrome (2 males aged 31 and 1 female aged 42 y old). None of the volunteers had received antibiotics or probiotics for at least 3 months before sampling. A 1:5 (w/w) faecal dilution in anaerobic PBS [0.1 g/l (pH 7.4)] was prepared and the samples homogenized in a stomacher (Seward, Worthing, UK) for 2 min at 460 paddle beats per minute. Each stage of the colonic model was inoculated with 100 ml faecal slurry. The total system transit time was set at 48 h. Following inoculation, the colonic model was run as a batch culture for a 24 h period in order to stabilize the bacterial populations prior to the initiation of medium flow. The system was then ran for 8 full volume turnovers when steady state conditions were achieved (SS1). Taking into account the operating volume (900 ml) and the retention time (48 h) of the system, the post in vitro digestion breads were added daily into V1 at 1% (w/v), for a further 8 volume turnovers upon which steady-state 2 (SS2) was achieved. Each steady state was confirmed by stabilization of SCFAs profiles over 3 consecutive days.

In vitro enumeration of bacterial population by FISH

Samples for FISH were immediately fixed in 4% paraformaldehyde.²¹ Intestinal bacterial groups of interest and total bacterial populations were evaluated in samples from the colonic model system as previously described by Martin-Pelaez et al.³⁵ The probes used are reported in Table 2 and were commercially synthesized and 5′-labeled with the fluorescent Cy3 dye (Sigma).

Table 2.

Oligonucleotide probes used in this study for FISH analysis

Target genus or group	Probe	Sequence (5′ to 3′)	Pre-treatment/%Formamide	Hybridization-Washing Temperature (°C)
Most Bacteria	EUB338a	GCTGCCTCCCGTAGGAGT	None 30% formamide	46 – 48
Most Bacteria	EUB338IIa	GCAGCCACCCGTAGGTGT	None 30% formamide	46 – 48
Most Bacteria	EUB338IIIa	GCTGCCACCCGTAGGTGT	None 30% formamide	46 – 48
Most Bacteroides sensu stricto and Prevotella spp; all Parabacteroides; Barnesiella viscericola and Odoribacter splanchnicus	Bac303	CCAATGTGGGGGACCTT	None	46 – 48
Most Bifidobacterium spp	Bif164	CATCCGGCATTACCACCC	None	50 – 50
Most members of Clostridium cluster XIVa; Syntrophococcus sucromutans, [Bacteroides] galacturonicus and [Bacteroides] xylanolyticus, Lachnospira pectinschiza and Clostridium saccharolyticum	Erec482	GCTTCTTAGTCARGTACCG	None	50 – 50
Faecalibacterium prausnitzii and related sequences	Fprau655	CGCCTACCTCTGCACTAC	None	58 – 58
Most Lactobacillus, Leuconostoc and Weissella spp.; Lactococcus lactis; all Vagococcus, Enterococcus, Melisococcus, Tetragenococcus, Catellicoccus, Pediococcus and Paralactobacillus spp	Lab158	GTATTAGCAYCTGTTTCCA	Lysozymeb	50 – 50
Most members of Clostridium cluster I; all members of Clostridium cluster II; Clostridium tyrobutyricum; Adhaeribacter aquaticus and Flexibacter canadensis (family Flexibacteriaceae); [Eubacterium] combesii (family Propionibacteriaceae)	Chis150	TTATGCGGTATTAATCTYCCTTT	None	50 – 50
Clostridium cluster IX	Prop853	ATTGCGTTAACTCCGGCAC	None	50 – 50
Roseburia sub cluster	Rrec584	TCAGACTTGCCG(C/T) ACCGC	None	50 – 50

^a,bThese probes were used together in equimolar concentrations. * Lysozyme (100U; 20 μl of 1 mg/ml solution of 50,000 U/mg⁻ protein)

Short chain fatty acids (SCFAs) analysis by gas chromatography

Aliquots of 1 ml gut model supernatant were centrifuged at 13000 g for 5 min. The supernatants were transferred into fresh microcentrifuge tubes and stored at −20°C until use. Samples were derivatized as described by Richardson et al.³⁶ Briefly, the supernatants were thawed on ice and centrifuged at 13000 g for 10 min. 500 µl of supernatant was transferred into a fresh microcentrifuge tube with 25 µl of internal standard (2-ethyl butyric acid) followed by 250 µl of concentrated HCl and 1 ml of ether. Tubes were vortexed for 1 min and centrifuged at 3000 g for 10 min. Aliquots (400 µl) of the ether extract were pipetted into a Wheaton vial and 50 µl of N-tertButyldimethyl silyl N-methyltrifluoroacetamide (MTBSTFA) were added and the vials closed tightly. The vials were heated at 80°C for 20 min in a water bath. Samples were transferred to Agilent crimp cap vials for gas chromatography analysis. Vials were capped with Crimp top natural rubber/PTFE seal type 7 aluminum silver 11 mm Chromacol caps and sealed using a crimper. The capped vials were left at room temperature for 48 h for derivatization. Calibration was achieved using standard solutions of derivitized acetic, propionic, i-butyric, n-butyric, i-valeric, n-valeric, and n-caproic acids as described for test samples. The final concentration of each standard was 25, 10, 5, 1, and 0.5 mmol/l. The derivatized samples were run through a 5890 series II GC system (HP, Crawley, West Sussex, UK) fitted with SGE-HT5 column (0.32 mm × 25 m × 0.1 µm; J &W Scientific, Folsom, CA, USA) and flame ionization detector. Helium was used as a carrier gas and delivered at a flow rate of 14 ml/min. The head pressure was set at 10 psi with a split ratio 10:1. Injector, column and detector were set at 275, 250 and 275°C respectively. One micro liter of sample was injected with a run time of 10 min. Peaks were integrated using the Atlas Lab managing software (Thermo Lab Systems, Mainz, Germany). Organic acid concentrations were quantified by comparing their response factors to the internal standard within the standards and expressed in mmol/l.

Statistical analysis

Data were analyzed by one-way ANOVA, using Tukey's post-test analysis when the overall P value of the experiment was below the value of significance (p < 0.05). An additional paired t-test was applied in order to assess the significance of results of single pairs of data. Analyses were performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

The authors thank the visiting students Sandrine Lecomte and Mathilde Pauleau for their technical assistance.

Funding

BBSRC was supported this study through their Diet, Research and Industry Club (DRINC) scheme (BB/HOO4734/1).