Oats have been cultivated since prehistoric times, and oatmeal porridge is a traditional dish in several North European countries( Reference Moore-Coyler 1 ). Anecdotically, Samuel Johnson (1709–1784) defined oat as ‘a grain, which in England is generally given to horses, but in Scotland supports the people’ – whereupon Patrick Murray, 5th Lord Elibank (1703–1778), replied: ‘Yes, and where else will you see such horses and such men?’ (cited by Fitzsimmons( Reference Fitzsimmons 2 )). Indeed, consumption of oats has several health benefits( Reference Rasane, Jha and Sabikhi 3 ). A role of oat β-glucans in prevention of the metabolic syndrome has been established( Reference Cloetens, Ulmius and Johansson-Persson 4 ), and oats may protect against both organic diseases and functional disorders affecting the gastrointestinal tract( Reference Thies, Masson and Boffetta 5 ). In vitro fermentation studies( Reference Hughes, Shewry and Gibson 6 ), and in vivo animal studies( Reference Drzikova, Dongowski and Gebhardt 7 , Reference Snart, Bibiloni and Grayson 8 ), suggest that certain constituents of oats may influence the gut microbiota. However, as recently emphasised by Rose( Reference Rose 9 ), the impact of eating wholegrain oats, containing dietary fibres, lipids and phenolics in a unique combination, has been sparsely studied on man.
The gut microbial community can be evaluated either by assessing its composition or by measuring its functions. The term ‘microflora-associated characteristic’ (MAC) has been introduced to designate the latter approach, being defined as ‘the recording of any anatomical structure or physiological, biochemical, or immunological function in a macroorganism, which has been influenced by the microflora in either an anabolic or catabolic way’( Reference Midtvedt, Björneklett and Carlsten-Duke 10 ). In the present pilot study, we aimed to explore the effect of eating oatmeal porridge every day for 1 week on healthy subjects by assessing the following MAC: lactulose-induced intestinal gas production, faecal excretion of SCFA and faecal levels of β-galactosidase and urease. In addition, rectal levels of PGE2 were analysed, as a measure of host inflammatory state.
Methods
Subjects
Healthy subjects were recruited from the hospital staff at Haukeland University Hospital (Bergen, Norway) and from students at the University of Bergen (Bergen, Norway). They were included if they considered themselves to be healthy, did not use any medications and were not pregnant. The participants were not further medically examined, but they were excluded if they fulfilled the Rome III criteria for irritable bowel syndrome( Reference Longstreth, Thompson and Chey 11 ) or had used antibiotics during the past month. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Regional Committee for Medical Research Ethics (REK Vest no. 030.08) and the Norwegian Social Science Data Service (no. 18685). Written informed consent was obtained from all subjects.
Dietary intervention
The subjects were instructed to avoid eating products containing oats for 2 weeks before the study, but otherwise maintained their habitual diet throughout the study period. For 7 d they ingested a daily portion of oatmeal porridge, made from 60 g oatmeal (1573 kJ/100 g (376 kcal/100 g); ‘AXA Bjørn Store Havregryn’; Lantmännen Cerealia), boiled with 150 ml tap water. The oatmeal contained (per 100 g) 13 g protein, 7 g fat, 61 g carbohydrate and 8·5 g dietary fibre, including 4·7 g β-glucans. Adherence to the dietary intervention was documented on a Norwegian diary that the participants were required to fill in.
Assessment of lactulose-induced intestinal gas production
The subjects underwent a lactulose breath test at the day before and the day after the dietary intervention. They were asked not to take any food or drink for 10 h before the challenge and not to use tobacco, eat, drink, sleep or walk around during the test. At 08.30 hours, baseline breath samples were collected, and the subjects ingested a solution of 10 g lactulose dissolved in 120 ml tap water, immediately followed by 60 ml of pure tap water for mouth cleaning. Breath sampling was then performed using collection bags from QuinTron Instrument Company at every 15 min for 3 h following lactulose ingestion. Pulmonary excretion of H2 and methane was quantified after correction for alveolar quality, based on CO2 concentration, using a QuinTron Model SC Gas Chromatograph (QuinTron Instrument Company). Individuals with methane excretion >1 parts per million (ppm) above the atmospheric methane concentration (i.e. approximately 1·8 ppm( Reference Levitt, Furne and Kuskowski 12 )) were classified as methane producers.
Assessment of faecal levels of SCFA, β-galactosidase and urease
Faecal samples were collected before and after the dietary intervention. The subjects collected all faeces that passed during a 72-h period, using one plastic box per d (volume 1000 ml; diameter 133 mm; reg. codes 257077 and 257078; Corporate Express). The upper edge of these boxes was equipped with a rim, making it easy to hold the box with both hands while defaecating directly into it. The subjects were carefully instructed on how this procedure to be performed and were told to store the boxes at −20°C immediately after voiding faeces and bring the frozen boxes to the hospital. Total (72 h) faecal wet weight was noted, and the collected faeces was homogenised with distilled water at a volume corresponding to two times the faecal wet weight and stored at −20°C until analysis of SCFA, β-galactosidase and urease.
SCFA analyses were performed at Karolinska Institutet (Stockholm, Sweden). The faecal material was homogenised after addition of distilled water containing 3 mmol/l of 2-ethylbutyric acid (as internal standard) and 0·5 mmol/l of H2SO4; 2 ml of the homogenate was vacuum distilled, according to the method of Zijlstra et al.( Reference Zijlstra, Beukema and Wolthers 13 ), as modified by Høverstad et al.( Reference Høverstad, Fausa and Bjørneklett 14 ). The distillate was analysed with GLC and quantified using internal standardisation. Flame ionisation detection was employed. The results were expressed in mmol/kg wet weight. The following SCFA were analysed: acetic, propionic, iso-butyric, n-butyric, iso-valeric, n-valeric, iso-caproic and n-caproic acids. Excretions of individual SCFA were calculated by multiplying the concentrations with the diluted 72 h faecal wet weight. Proportions of individual SCFA were calculated as percentages of total SCFA. On the basis of the work of Tjellström et al.( Reference Tjellström, Högberg and Stenhammar 15 ), we calculated two fermentation indices as follows: fermentation index A, reflecting the fermentation of carbohydrates and conceivably the pro-inflammatory properties of SCFA, was calculated as acetic acid concentrations minus propionic and butyric acid concentrations divided by the total concentration of SCFA. Fermentation index B, reflecting the fermentation of proteins and conceivably the anti-inflammatory properties of SCFA, was calculated as the sum of iso-butyric and iso-valeric acids.
Analysis of β-galactosidase was performed in the pre-homogenised samples that were diluted and homogenised once more with distilled water at a volume corresponding to eight times the original faecal wet weight. After this second homogenisation, a detergent (Triton X-100; CAS reg. no. 9002-93-1; Sigma-Aldrich) was added to the solution at 1 % concentration, and the samples were homogenised once more. Enzyme activity was then assayed fluorimetrically from the supernatants as described by Peters et al.( Reference Peters, Heath and Wansbrough-Jones 16 ) and modified by Andersen et al.( Reference Andersen, Schjonsby and Skagen 17 ).
Analysis of urease was performed in the pre-homogenised samples by a modified ‘rapid urease test’( Reference Nysaeter, Berstad and Weberg 18 ), principally similar to the routine clinical test used to detect the presence of Helicobacter pylori in gastric mucosal biopsies. Briefly, a working substrate solution was made from 0·1 ml 42 mm-HCl and 20 ml of a solution containing 2 g urea, 20 ml distilled water and 10 drops of fenol red. Homogenised faeces of 10 µl (diluted 1:8 with distilled water) was added to 200 ml of this working solution, and the time to colour change into red was noted. A standard curve, developed at the University of Bergen and based on Jack bean urease measurements, was then used to convert the time to colour change into urease concentration in mg/ml.
Assessment of rectal levels of PGE2
Rectal dialysis was performed in continuation with the lactulose breath test before and after the dietary intervention. After finishing the 3-h breath sampling period, the subjects were offered a 30 min lunch break. Taking advantage that food intake triggers the gastrocolic reflex, the subjects were asked to evacuate their bowels completely during this lunch period. Rectal dialyses were then performed for 4 h. The procedure has been described in detail by Egan et al.( Reference Egan, Sandborn and Mays 19 ). Briefly, dialysis bags (12 cm, 4 ml) were made of cellulose membrane tubing (Visking code DVT12000.01.000; molecular weight cut-off 12–14 kDa; Medicell International Ltd), filled with rheomacrodex (10 % dextran (mean molecular weight 40 kDa) in saline; Meda A/S) and inserted into the empty rectum. Dialysis bags were stored overnight at 4°C and bathed in rheomacrodex solution; there was no need for additional lubrication upon insertion. The dialysis bags were left for 4 h to obtain equilibrium with rectal fluid( Reference Lauritsen, Laursen and Bukhave 20 ), and the dialysates were thereafter aspirated from the bags using a syringe and a needle, transferred into plastic vials and stored at −80°C until analysis. PGE2 levels were measured using an enzyme immunoassay kit (Cat. no. 514010; Cayman Chemical Company), following the manufacturer’s instructions. The intra-assay CV was 5·6 %.
Statistical analyses
Data were analysed using GraphPad Prism version 6.0 (GraphPad Software Inc.). Data are expressed as mean values and standard deviations, unless otherwise stated. We used paired t tests for comparisons of means before and after the dietary intervention. All tests were two-tailed, and P values <0·05 was considered statistically significant.
Results
Subjects
Ten healthy subjects – eight females and two males, aged 22–49 years with a mean BMI of 23·0 (sd 2·9) kg/m2 – agreed to participate. All subjects completed the dietary intervention and underwent the prescribed study procedures. However, because of logistical reasons, SCFA levels were not analysed in faecal samples from one participant, and one individual refused to undergo rectal dialysis.
Lactulose-induced intestinal gas production
Neither basal nor lactulose-induced excretion of H2 changed significantly following the dietary intervention (Fig. 1(a)). Three individuals were classified as methane producers, but neither basal nor lactulose-induced excretion of methane changed significantly following the dietary intervention (Fig. 1(b)). H2 levels were similar in methane producers and methane non-producers (data not shown).
Fig. 1 Intestinal gas excretion following ingestion of a 10 g lactulose solution in healthy subjects (n 10) before and after eating oatmeal porridge daily for 1 week. The results are shown as mean values with error bars representing the standard errors. (a) Excretion of H2 in all subjects (n 10). (b) Excretion of methane in methane-producing subjects (n 3). 
, After; 
, before. ppm, Parts per million.
Faecal levels of SCFA, β-galactosidase and urease
Faecal wet weight (72 h) did not change following the dietary intervention. It varied between 90 and 666 g before the intervention, with a mean value of 394·5 (sd 191·4) g, and between 100 and 693 g after the intervention, with a mean value of 365·1 (sd 173·0) g.
Excretions and proportions of SCFA did not change following the dietary intervention (Table 1), and fermentation indices A and B remained unchanged (P=0·6 and 0·6, respectively).
Table 1 SCFA excretion (mmol/72 h) and proportions (percentage of total SCFA) in healthy subjects (n 9) before and after eating oatmeal porridge daily for 1 week (Mean values and standard deviations)
Faecal β-galactosidase activity was reduced during the study period (Fig. 2), from 14·6 (sd 14·8) to 5·3 (sd 3·7) mU/ml (P=0·049).
Fig. 2 Levels of β-galactosidase in faecal samples from healthy subjects (n 10) before and after eating oatmeal porridge daily for 1 week.
Faecal urease levels decreased from 4·5 (sd 3·0) to 3·7 (sd 2·8) mg/ml (P=0·031) following the dietary intervention (Fig. 3).
Fig. 3 Levels of urease in faecal samples from healthy subjects (n 10) before and after eating oatmeal porridge daily for 1 week.
Rectal levels of PGE2
Concentrations of PGE2 in rectal dialysates decreased from 298·0 (sd 314·1) to 163·1 (sd 70·5) pg/ml following the dietary intervention (Fig. 4). However, this reduction was not statistically significant (P=0·168).
Fig. 4 Levels of PGE2 in rectal dialysates from healthy subjects (n 9) before and after eating oatmeal porridge daily for 1 week.
Discussion
The present study aimed to explore the effect of oatmeal porridge on gut MAC. Microbial adaptions to dietary changes are known to occur rapidly( Reference David, Maurice and Carmody 21 ). Consistently, the results of our short dietary intervention suggest that certain microbial functions are modified within 1 week by eating oatmeal porridge. In the following, we discuss possible implications of these findings.
Microbial fermentation was evaluated both by measuring production of intestinal gas following lactulose ingestion and by assessing faecal excretion of SCFA. Lactulose is a disaccharide resistant to hydrolysis by human digestive enzymes and therefore unabsorbable within the small intestine but readily fermentable by colonic microbiota. Hence, gas excretion following lactulose ingestion may be considered as a measure of colonic fermentation capacity. Similar gas excretion curves before and after the dietary intervention thus suggest that eating oatmeal porridge does not alter the colonic ability to ferment carbohydrates. Regarding the effect of oat consumption on SCFA excretion, our results are comparable to previous studies by others( Reference Kashtan, Stern and Jenkins 22 – Reference Noakes, Clifton and Nestel 24 ). However, it should be emphasised that SCFA produced within the colon are readily absorbed, and only small amounts are excreted in the faeces. Faecal excretion of SCFA is therefore an uncertain estimate of colonic SCFA production. The pattern of gut microflora fermentation, as assessed by considering the relative distributions of individual SCFA, as well as indices of saccharolytic v. proteolytic fermentation( Reference Tjellström, Högberg and Stenhammar 15 ), may be more relevant, but neither of these parameters was changed during the present study.
β-Galactosidase is a microbial enzyme with similar catalytic activity as human lactase and is therefore often denoted as ‘microbial lactase’. Measurement of this microbial function was encouraged by previous studies suggesting that induction of β-galactosidase may be associated with improvement of abdominal symptoms in individuals with lactose intolerance( Reference Hertzler and Savaiano 25 , Reference Szilagyi, Rivard and Fokeeff 26 ). However, increased β-galactosidase levels should probably not be considered as a general marker of a favourable gut microflora( Reference Brigidi, Vitali and Swennen 27 ), as demonstrated by our finding of decreased levels of β-galactosidase activity in faeces following oat consumption. A reasonable interpretation is that this enzyme is simply not needed by the gut microflora when eating oatmeal porridge, and hence the microbiota adapt accordingly. It should be emphasised, however, that faecal levels of β-galactosidase may not properly reflect levels of β-galactosidase in the proximal colon, where most of the saccharolytic activity takes place.
Urease is a microbial enzyme that catalyses the hydrolysis of urea into ammonia. Ammonia exerts several toxic effects upon the host( Reference Mobley and Hausinger 28 ), and urease has previously been advocated as a general marker of an unfavourable gut microflora( Reference Brigidi, Vitali and Swennen 27 ). Our results of decreased faecal levels of urease following the dietary intervention are therefore interesting and suggest that oatmeal porridge has prebiotic actions. Furthermore, the use of oatmeal porridge in the treatment of hyperammonaemia, to suppress intestinal urease levels and thereby blood ammonia levels( Reference Shen, Albenberg and Bittinger 29 ), may be considered. Decreased levels of urease following ingestion of oatmeal porridge is probably a consequence of microbial adaption( Reference Langran, Moran and Murphy 30 ). As for the other faecal parameters assessed in this study, the levels of urease in faeces may not properly reflect the levels throughout the gastrointestinal tract.
Oats seem to have antioxidant capacity and anti-inflammatory activity( Reference Chu, Wise and Gulvady 31 ). The β-glucan components may be particularly important( Reference Raa 32 ), as demonstrated in a recent animal study showing positive effects of β-glucans on colonic tissue of both healthy rats and rats with lipopolysaccharide-induced enteritis( Reference Wilczak, Blaszczyk and Kamola 33 ). In the present study, we analysed PGE2 levels of the rectum as a marker of inflammatory activity of the gastrointestinal tract. Although the levels were not significantly altered after eating oatmeal porridge, concentrations of PGE2 were markedly reduced in some of the subjects. Thus, further studies on the effect of oatmeal porridge on inflammatory diseases of the colon should be encouraged, both in conditions characterised by low-grade (e.g. irritable bowel syndrome) and overt (e.g. inflammatory bowel disease) intestinal inflammation.
Taken together, ingestion of oatmeal porridge daily for 1 week in healthy subjects reduced faecal levels of β-galactosidase and urease, whereas colonic fermentation capacity, excretion of SCFA and rectal inflammation, assessed by PGE2 levels, were unaltered. The results thus suggest that oatmeal porridge may modulate gut microbial functions. These findings should encourage further studies to investigate the potential prebiotic properties of oatmeal porridge.
Acknowledgements
The authors thank Behzad Gharehnia, Tove Berstad, Elisabeth Norin and Anna-Karin Persson for invaluable technical assistance.
The study was funded by the University of Bergen, Norway.
The authors’ contributions are as follows: J. V. designed the study, performed the data collection, carried out the data analyses and wrote the manuscript; N. G. P. designed the study, performed the data collection and carried out the data analyses; T. M. supervised the study and contributed to data interpretation; A. B. designed the study, supervised the study and contributed to data interpretation. All authors read and approved the final version of the manuscript.
The authors have no financial or personal conflicts of interest to declare.
