Effect of arabinoxylo-oligosaccharides on proximal gastrointestinal motility and digestion in healthy volunteers

e-SPEN, the European e-Journal of Clinical Nutrition and Metabolism (2008) 3, e220ee225

e-SPEN, the European e-Journal of Clinical Nutrition and Metabolism http://intl.elsevierhealth.com/journals/espen

ORIGINAL ARTICLE

Effect of arabinoxylo-oligosaccharides on proximal gastrointestinal motility and digestion in healthy volunteers* Lieselotte Cloetens a, Katrien Swennen b, Vicky De Preter a, Willem F. Broekaert b, Christophe M. Courtin b, Jan A. Delcour b, Paul Rutgeerts a, Kristin Verbeke a,* a Department of Gastrointestinal Research and Leuven Food Science and Nutrition Research Centre (LFoRCe), University Hospital Gasthuisberg, K.U. Leuven, Herestraat 49, B-3000 Leuven, Belgium b Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), K.U. Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium

Received 21 December 2007; accepted 30 May 2008

KEYWORDS Arabinoxylooligosaccharides; Proximal motility; Lipid digestion; Prebiotic; Protein digestion

Summary Background & aims: Before evaluating the effect of arabinoxylo-oligosaccharides (AXOS), a newly proposed prebiotic, on colonic metabolism, we evaluated its influence on proximal gastrointestinal motility [gastric emptying (GE) and oro-caecal transit time (OCTT)] and digestion of proteins and lipids. Methods: Ten healthy volunteers each performed 2 13CO2-breath tests for assessing protein digestion and 2 for lipid digestion, each in the absence and presence of 2.2 g AXOS. To each test meal, 14C-labelled octanoate and inulin-14C-carboxylic acid were added for simultaneous measurement of GE and OCTT, respectively. Breath samples were collected and analysed with isotope ratio mass spectrometry (13C) or liquid scintillation (14C). Results: Addition of AXOS to the lipid test meal did not influence lipid digestion. However, GE but not OCTT of the lipid test meal was significantly accelerated by ingestion of AXOS (p Z 0.028). In the presence of AXOS, the rate of protein digestion proceeded slower but the overall extent of protein digestion after 5 h was not significantly different. GE and OCTT of the protein digestion test meal remained unchanged.

Abbreviations: AXOS, arabinoxylo-oligosaccharides; AXOS-15-0.26, AXOS with an average DP of 15 and an average DAS of 0.26; DP, degree of polymerisation; DAS, degree of arabinose substitution; GE, gastric emptying; GEt1=2 , gastric half emptying time; IQR, interquartile range; OCTT, oro-caecal transit time. * Conference presentation: UEGW 2006. Gut 2006; 55 (Suppl V) A178. * Corresponding author. Tel.: þ32 16 34 43 97; fax: þ32 16 34 43 99. E-mail address: [email protected] (K. Verbeke). 1751-4991/$ - see front matter ª 2008 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.eclnm.2008.05.008

Effect of AXOS on motility and digestion

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Conclusions: Addition of a low dose of AXOS to a meal does not influence the supply of lipids and proteins to the colon. Therefore, possible effects of AXOS on the colonic metabolism should not be due to differences in proximal motility and digestion. ª 2008 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

Introduction

AXOS preparation

Arabinoxylo-oligosaccharides (AXOS) are degradation products of arabinoxylan, the main hemicellulose polysaccharide in the cell wall of cereals. The structure of AXOS consists of a main chain of b-1,4-xylosepyranosyl units to which arabinosefuranosyl units are linked as side chains at the O-2 or O-3 position. AXOS with varying degree of polymerisation (DP) and degree of arabinose substitution (DAS; arabinose to xylose ratio) can be obtained by enzymatical treatment and/or chemical extraction of arabinoxylan.1 Isolated AXOS can be further fractionated using graded ethanol precipitation and ultrafiltration.2 In vitro studies as well as experimental animal studies have demonstrated the bifidogenic effect of AXOS, indicating the prebiotic potential of AXOS.3,4 A prebiotic has been defined as a selectively fermented ingredient that allows specific changes, both in the composition and/or activity of the microbiota, that confers benefits upon host well-being and health.5 However, the effects of AXOS preparations in humans are still largely unknown. Experiments are being conducted in healthy people to evaluate the prebiotic potential of AXOS by investigating their effect on colon fermentation.6 Importantly, colon fermentation depends on the availability of substrates to the colonic microbiota, besides the composition of the colonic microbiota and the transit time trough the colon. Substrate availability on its own is influenced by the dietary intake as well as by the digestive processes in the proximal gastrointestinal tract, including the oro-caecal transit time (OCTT). For instance, factors that reduce the gastric emptying (GE) rate may result in an overall slower rate of digestion and absorption. Alternatively, factors that enhance small bowel transit may induce maldigestion and malabsorption through a shorter contact time with digestive enzymes and absorbing surface, and result in an increased supply of nutrients to the colon. In the present study, breath tests have been performed to investigate the influence of AXOS with an average DP of 15 and an average DAS of 0.26 (AXOS-15-0.26) on the extent of protein digestion and lipid digestion in healthy volunteers. Simultaneously, GE and OCTT were measured to assess the effect of AXOS-15-0.26 on gastrointestinal motility.

AXOS-15-0.26 were prepared as previously described.1 Table 1 presents the properties of the AXOS preparation. In this study, 3.2 g of the crude product was administered which corresponds to 2.2 g pure AXOS after correction for the content in polymeric xylose and arabinose (72.3%) and moisture content (3.7%). The selected dose of AXOS-150.26 was based upon a previous doseeresponse study, in which was shown that a dose of 2.2 g AXOS-15-0.26 administered to healthy volunteers exerted beneficial effects on colonic bacterial metabolism upon a dose of 2.2 g.6 AXOS-15-0.26 was dissolved in water for administration.

Methods Subjects Ten healthy volunteers (median age 22 y; median BMI 22.4 kg/m2) participated in this study. None of the subjects were taking any medication or had a history of gastrointestinal disease or surgery. The study protocol was approved by the Ethics Committee of the Katholieke Universiteit Leuven and all subjects gave written informed consent.

Test meals The test meal for measurement of protein digestion consisted of a pancake prepared from 7.5 g of lyophilized 13Cleucine labelled egg white, 17.2 g of lyophilized 13C-leucine labelled egg yolk, 3.75 g of lyophilized unlabelled egg white, 3.0 g of milk powder, 7.0 g of sugar, 17.0 g of flour and 130 mL of water. 13C-leucine (Euroiso-Top, Saint Aubin, France) was incorporated in the egg white and yolk by feeding hens a 13C-leucine-supplemented diet as described previously.7,8 Briefly, laying hens were given free access to food which was supplemented with 3 g/kg 13C-leucine (99 mol%, Euriso-Top, Saint Aubin, France). By the hen’s metabolism, the dietary 13C-leucine was incorporated in

Table 1

Characterisation of the AXOS preparation AXOS-15-0.26

Moisture (%) Ash (%dm) Protein content (%dm) Monosaccharide composition (%dm) L-Arabinose D-Xylose D-Mannose D-Galactose D-Glucose Arabinoxylan content (%dm)a Arabinose to xylose ratiob Molecular weightpeakc DPpeakd

3.7 8.2 5.0 17.4 64.7 0.1 0.8 1.4 72.3 0.26 1600 15

dm Z dry matter. a Arabinoxylan Z 0.88  (% L-Arabinose þ % D-Xylose). b Arabinose to xylose ratio Z L-arabinose/D-xylose. c Molecular weightpeak Z the average molecular weight deduced from high performance size exclusion chromatography. d DPpeak Z molecular weightpeak/132.

e222 the egg protein. Eggs were collected daily and separated into white and yolk fractions. The fractions were freezedried and pooled. The isotopic enrichment of the egg white and yolk used in this study was 1.384 and 1.164 atom percentage, respectively. Using the formula described by Geboes et al.,8 the efficiency of 13C-leucine incorporation was calculated to be 28.04%. A total amount of 198.85 mg 13 C-leucine was administered in the protein test meal. The mole percentage of labelled leucine in the test meal was 19.46%, making the mole percentage excess of 13C-leucine in the test meal 18.08%. The caloric content of the meal amounted to 327 kcal (18.7 g of protein, 26.8 g of carbohydrates and 16.2 g of fat). The test meal to measure lipid digestion consisted of two slices of white bread (6.4 g of protein, 37.6 g of carbohydrate, and 1.0 g of fat; and 185 kcal) smeared with 30 g chocolate paste (1.3 g of protein, 12.7 g of carbohydrate, and 14.9 g of fat; 190 kcal), which contained 250 mg 13Cmixed triglyceride (1,3-distearyl-2-[carboxyl-13C]-octanoyl glycerol (Euriso-Top, Saint Aubin, France)). The caloric content of the complete test meal amounted to 375 kcal. To each test meal, 18.5kBq 14C-labelled sodium octanoate (ARC, Saint Louis, MO, USA) and 74kBq inulin-14C-carboxylic acid (Amersham Biosciences, Buckinghamshire, UK) were added to allow simultaneous measurement of GE and OCTT, respectively. Recently, inulin-14C-carboxylic acid has been validated as an alternative substrate to lactose-13C-ureide to measure OCTT.9

Study design Each of the subjects performed four tests with at least one week between each test. For all subjects, the order of the test meals was the same. In the two first tests, protein digestion was evaluated, in the absence and presence of AXOS-15-0.26, while in the second two tests lipid digestion was evaluated, again in the absence and the presence of AXOS-15-0.26. GE rate and OCTT were measured at each test occasion. All tests were performed after an overnight fast. Basal breath samples were obtained to determine the baseline values of 13CO2 and 14CO2. Consequently, the volunteers received a dedicated test meal (without or with AXOS-15-0.26), which they consumed in less than 15 min. From then on, breath samples were collected every 15 min up to 6 h for 13C-analysis and up to 10 h for 14C-analysis. After 6 h, the subjects received one sandwich with cheese or ham and were allowed to freely drink water until the end of the breath test. No physical exercise was allowed during the tests.

Analysis of breath samples Breath samples for 13C analysis were collected in Exetainers (PDZ, Cheshire, UK). The 13C-enrichment of CO2 in breath was analysed using isotope ratio mass spectrometry (ABCA, Europa Scientific, Crewe, UK). CO2-production was assumed to be 300 mmol/m2 of body surface per hour. The body surface area was calculated using the weighteheight formula of Haycock et al.10 Breath test results for protein digestion and lipid digestion were expressed as the percentage of the administered dose of 13C

L. Cloetens et al. excreted per hour and the cumulative percentage of the administered dose of 13C excreted over 6 h (%cum 6h). The maximum excretion rate of 13C (%max) and the time of maximum excretion rate of 13C (tmax) were also calculated. Breath samples for 14C analysis were collected by blowing through a pipette into a vial containing 2 mmol hyamine hydroxide until the thymolphtaleine indicator became discoloured, corresponding to the capture of 2 mmol CO2. 14CO2 was measured by b-scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer, model 3375, Packard Instruments Inc., Downers Grove, IL, USA) after addition of 10 mL Hionic flour (PerkinElmer, Boston, USA). A two-peaked breath 14C-excretion curve was obtained representing GE and OCTT. To interpret both peaks, it was assumed that the 14C-excretion in the first 4 h was solely derived from octanoic acid. The 14C-excretion data were mathematically analysed using the curve fitting techniques developed to calculate the gastric half emptying time (GEt1=2 ). After subtraction of the fitted GE curve, OCTT was calculated as the time to the appearance of 10% of the maximal 14C-excretion using the curve fitting described by Morrison et al.11

Statistics All results were expressed as median plus interquartile range (IQR). The statistical analysis was performed with SPSS Software (SPSS 14.0 for Windows, SPSS Inc., 1989e 2003, Chicago, USA). Because of the small number of subjects non-parametric Wilcoxon tests (paired) or Manne Witney tests (unpaired) were used to compare baseline values and values after the intake of AXOS-15-0.26. p-values less than 0.05 were considered significant.

Results Influence of AXOS-15-0.26 on lipid digestion Fig. 1A and B show the median 13CO2-pattern obtained after consumption of the lipid test meal with and without 2.2 g AXOS-15-0.26, whereas Table 2 summarizes the lipid digestion and motility characteristics. No significant differences in lipid digestion characteristics were observed if 2.2 g AXOS-15-0.26 was added to the lipid test meal. Fig. 2 shows a typical example of the 14CO2-excretion after consumption of the lipid test meal in the presence of AXOS-15-0.26. OCTT could be determined in all volunteers in both test conditions. GE was significantly accelerated by addition of 2.2 g AXOS-15-0.26 to the test meal (p Z 0.028); although tmax of lipid digestion was not different. No significant differences in OCTT were found between both test conditions.

Influence of AXOS-15-0.26 on protein digestion Fig. 3A and B show the median 13CO2-pattern obtained after consumption of the protein test meal with and without 2.2 g AXOS-15-0.26, whereas Table 2 summarizes the protein digestion and motility characteristics. In the presence of AXOS-15-0.26, protein digestion proceeded somewhat slower between 135 and 150 min than in the absence of AXOS-15-0.26 (Fig. 3A), which resulted in a significantly

Effect of AXOS on motility and digestion

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e223 allowing calculation of the OCTT value. No significant differences in motility characteristics, neither in GE nor in OCTT, were observed if 2.2 g AXOS-15-0.26 was added to the protein test meal.

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Figure 1 Median 13CO2-excretion curves, expressed as % dose/h (1A) and cumulative % dose (1B) after consumption of the lipid digestion test meal with (-) and without (,) 2.2 g AXOS-15-0.26. At no time point, a statistically significant difference was observed in the digestion of the test meal with and without AXOS-15-0.26.

lower cumulative excretion of 13CO2 between 135 and 285 min (Fig. 3B). However, the arrears were made up from 210 min on (faster protein digestion in the presence of AXOS-15-0.26) resetting in a similar cumulative 13CO2-excretion from 300 min on. Only in nine volunteers after consumption of the test meal without AXOS-15-0.26 and in eight volunteers after the consumption of the test meal with AXOS-15-0.26, the fitted OCTT curve (i.e. after subtraction of the fitted GE curve from the total 14CO2-excretion) showed a peak,

AXOS have recently been proposed as a new promising prebiotic substrate and have some particularly attractive characteristics. First, structurally different types of AXOS (varying DP and DAS) can be obtained by changing the conditions of hydrolysis of arabinoxylan.12 Since the structure of a carbohydrate influences its fermentation properties, this might allow to choose the structure with optimal prebiotic characteristics. Secondly, AXOS can be generated in situ in processed cereal-based food products such as bread, pasta, cookies and beer trough interaction with endoxylanases with the cereal-derived AX. Thirdly, stimulation of bifidobacteria in chickens has been observed at a lower dose as compared to inulin.13 These promising properties prompted us to thoroughly evaluate the characteristics of AXOS in humans. In our opinion, it is important not only to evaluate the effect of a prebiotic in the colon but to characterise as well its properties in the proximal gastrointestinal tract. Dietary interventions with prebiotics generally aim at increasing the saccharolytic fermentation in the colon and concomitantly decreasing the proteolytic fermentation, which can be achieved by stimulating the growth and/or activity of saccharolytic bacteria such as bifidobacteria and lactobacilli. Since the type and amounts of fermentation metabolites depends at least partly, on the substrate availability, it is important to investigate the possible influence of the dietary intervention on the supply of nutrients to the colonic microbiota. Mechanisms, by which indigestible carbohydrates alter the digestion and absorption of other nutrients, are often related to their physicochemical properties such as viscosity, water-holding capacity and osmolarity.14 For example, the disaccharide lactulose is known to accelerate OCTT which is attributed to its osmotic effect. Lactulose draws fluid into the intestinal lumen, in this way distending the small intestine and stimulating small intestinal motility and peristalsis. A shorter OCTT might lead to a shorter

Table 2 Results of protein digestion and lipid digestion test meal in the absence and presence of 2.2 g AXOS-15-0.26: digestion characteristics, GE and OCTT (median and IQR, Wilcoxon or ManneWitney test, a Z 0.05) Lipid digestion test meal

Digestion %max tmax(min) %cum 6h Motility GE (t1/2(min)) OCTT (min) a

Protein digestion test meal

Without AXOS-15-0.26

With AXOS-15-0.26

Without AXOS-15-0.26

With AXOS-15-0.26

8.4 (5.9e10.4) 165 (135e199) 35.2 (25.3e43.9)

8.4 (4.9e9.6) 173 (139e195) 33.5 (29.0e41.8)

6.1 (4.0e7.0) 143 (131e150) 14.7 (13.0e17.5)

5.2 (4.4e6.6) 128 (116e150) 13.9 (11.1e17.4)

67 (56e76) 397 (379e479) n Z 10

54 (36e62)a 405 (376e414) n Z 10

62 (58e72) 344 (336e411) nZ9

69 (55e87) 429 (326e496) nZ8

Significantly different from the lipid digestion test meal without AXOS-15-0.26 (p Z 0.028).

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14C-excretion

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400

300

200

OCTT

100

0

0

100

200

-100

300

400

500

600

time (min) measured dpm values

fitted GE curve

netto OCTT curve

fitted OCTT curve

Figure 2 14CO2-excretion pattern in one volunteer after the consumption of the lipid test meal with 2.2 g AXOS-15-0.26. The 14Cexcretion data were mathematically analysed using the curve fitting techniques developed to calculate the GEt1=2 . After subtraction of the fitted GE curve, OCTT was calculated as the time to the appearance of 10% of the maximal 14C-excretion. (dpm Z desintegrations per minute).

contact time of food with digestive enzymes and a lower degree of digestion. Indeed, Holgate and Read showed a reduced absorption of fat, carbohydrate, protein, water and electrolytes after the administration of lactulose to 14 patients with terminal ileostomies.15 They suggested that the

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16 14 12 10

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Figure 3 Median CO2-excretion curves, expressed as % dose/ h (3A) and cumulative % dose (3B) after consumption of the protein digestion test meal with (-) and without (,) 2.2 g AXOS-15-0.26. * Z Significant difference at this time point between the protein test meal with and without AXOS-15-0.26.

absorption of food constituents was impaired due to accelerated small intestinal transit but also due to osmotic inhibition of fluid absorption, resulting in dilution of food particles and reduced digestion. Another example is that of water-holding polysaccharides. They increase the viscosity of the intestinal content which might interfere with mixing of the intestinal contents and diffusion of nutrients towards the epithelial cells, resulting in a higher supply of nutrients to the colon. In addition, viscous polysaccharides such as gums and pectins have been shown to prolong GE resulting in an overall slower rate of digestion.16,17 To the contrary, carbohydrates such as oligofructose and inulin, that do not significantly influence viscosity, are not expected to induce pronounced effects on transit and small intestinal digestion. Indeed, Geboes et al.18 found no effect of Raftilin HP (long-chain inulin) on GE, OCTT, lipid digestion and protein digestion. Similarly, a non-viscous type-3 resistant starch (CoActistar, Cerestar, Belgium) did not influence protein digestion.19 The intrinsic viscosity of AXOS-15-0.26 amounts to 0.52 dL/g which is slightly higher than that of oligofructose (0.04 dL/g) but clearly lower than that of native arabinoxylans (2.81e4.23 dL/g) (unpublished results). The breath test results obtained in this study in healthy volunteers showed that protein digestion proceeded slightly slower in the presence of 2.2 g AXOS-15-0.26. Nevertheless, provided that the OCTT of a meal is at least 5 h, the cumulative amount of digested protein is similar in both occasions, suggesting that the addition of AXOS will not significantly alter the supply of dietary proteins to the colon. The results indicate that lipid digestion was not slowed down by addition of AXOS-15-0.26 to the test meal. However, it is possible that a slower digestion of lipids was compensated by the observed faster GE, resulting in a similar lipid digestion curve in the absence and presence of AXOS-15-0.26. It is not clear by what physiological mechanism AXOS-15-0.26 would accelerate GE of the lipid digestion test meal

Effect of AXOS on motility and digestion whereas the GE of the protein digestion test meal remained unchanged. In addition, in previous experiments performed at our laboratory, no influence on GE was observed upon the addition of AXOS in a dose varying between 0 g and 4.9 g to another test meal (8.4 g of proteins, 11.2 g of fat and 26.7 g of carbohydrates; 242 kcal).20 Factors that might affect GE of a meal include the volume of the meal, the caloric content and the viscosity. However, it is rather unlikely that the influence of AXOS on one of these parameters would be different in the protein test meal as compared to the lipid test meal. Furthermore, even in young healthy volunteers, GE is characterized by a large intra-individual variability. A mean coefficient of variation of 26.7% (range 8.7e41.2%) was found upon three repeated measurements of GEt1=2 in healthy volunteers.21 Similar results were obtained in healthy children22 or in studies using scintigraphic methods.23 This large day-to-day variability is an important problem in interpreting GE data. Therefore, it is more likely that the observed effect of AXOS-15-0.26 on the GE rate of the lipid test meal is due to coincidence and should be explained by a type I statistical error. In conclusion, this study showed that the addition of a low dose of AXOS-15-0.26 to a meal did not influence the supply of lipids and proteins to the colon. As a consequence, it can be assumed that the possible effects of AXOS-15-0.26 on colon fermentation processes will effectively be due to colonic events and not to changes in the proximal gastrointestinal tract.

Acknowledgements LC, VD and KV have designed the study, KS, WFB and CMC have prepared AXOS; LC was responsible for data acquisition and analysis; LC, VD and KV contributed to data interpretation. All authors read and approved the final manuscript.

Conflict of interest All authors have stated that there are no conflicts of interest.

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