Chicory inulin does not increase stool weight or speed up intestinal transit time in healthy male subjects

Abstract

Inulin is a non-digestible oligosaccharide classified as a prebiotic, a substrate that promotes the growth of certain beneficial microorganisms in the gut. We examined the effect of a 20 g day⁻¹ supplement of chicory inulin on stool weight, intestinal transit time, stool frequency and consistency, selected intestinal microorganisms and enzymes, fecal pH, short chain fatty acids and ammonia produced as by-products of bacterial fermentation. Twelve healthy male volunteers consumed a well-defined, controlled diet with and without a 20 g day⁻¹ supplement of chicory inulin (degree of polymerization (DP) ranging for 2–60), with each treatment lasting for 3 weeks in a randomized, double-blind crossover trial. Inulin was consumed in a low fat ice cream. No differences were found in flavor or appeal between the control and inulin-containing ice creams. Inulin consumption resulted in a significant increase in total anaerobes and Lactobacillus species and a significant decrease in ammonia levels and β-glucuronidase activity. Flatulence increased significantly with the inulin treatment. No other significant differences were found in bowel function with the addition of inulin to the diet. Thus, inulin is easily incorporated into a food product and has no negative effects on food acceptability. Twenty grams of inulin was well tolerated, but had minimal effects on measures of laxation in healthy, human subjects.

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Introduction

Dietary fiber escapes digestion and absorption in the upper digestive tract, but is fermented in the colon. Intake of dietary fiber is linked to improved health outcomes, including less cardiovascular disease, improved bowel function, and stabilization of blood glucose levels.¹ Additionally, fermentable fiber may provide a number of health benefits by altering the composition of the intestinal flora.

Prebiotics are non-digestible substances that provide a beneficial physiological effect to the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria.² This generally refers to the ability of a fiber to increase the growth of bifidobacteria and lactobacilli, which are considered beneficial to human health. Benefits of prebiotics include improvement in gut barrier function and host immunity, reduction of potentially pathogenic bacteria subpopulations (e.g. clostridia), and enhanced short chain fatty acid (SCFA) production.³ Inulin, oligofructose, and fructooligosaccharides (FOS) have been extensively studied as prebiotics, and have been shown to significantly increase fecal bifidobacteria at fairly low levels of consumption (5–8 grams per day). Roberfroid⁴ points out the specificity of Bifidobacteria and Lactobacillus for inulin-type compounds. These species possess intracellular 2,1-β-D-fructan-fructanohydrolase (EC 3.2.1.7) to hydrolyse the type β(2–1) fructans of inulin, fructooligosaccharides (FOS) and oligofructose.

Fermentable fibers that don't meet the definition for prebiotics still provide health benefits via production of SCFAs. The three most abundant SCFAs are acetate, propionate, and butyrate, each of which exerts unique physiological effects.⁵ Of these, butyrate is considered the most beneficial in terms of colonic health. Butyrate is the preferred energy source for colonic epithelial cells, and promotes normal cell differentiation and proliferation. SCFAs also help regulate sodium and water absorption, and can enhance absorption of calcium and other minerals. In addition, SCFAs act to lower colonic pH, which can inhibit growth of potential pathogens and promote the growth of beneficial bacteria such as bifidobacteria and lactobacilli. Different fibers vary in the amounts and ratio of SCFA produced, as well as in the rate of production.⁶ Fibers that are fermented quickly may lead to excessive gas production and bloating, so the dose of fiber consumed is an important consideration. The fermentation pattern may be related to the molecular weight, chain length, and structure of the fiber. Short chain molecules, such as FOS, are generally fermented more rapidly than larger, longer chain molecules such as acacia gum.⁷

The objective of this study was to examine the effects of a 20 g day⁻¹ supplement of chicory inulin (average degree of polymerization (DP) = 9) on several gastrointestinal parameters, including stool weight, defecation frequency and intestinal transit time, fecal microbes, fecal microbial enzyme activity, fecal SCFA and fecal ammonia. Information on the sensory qualities of inulin added to ice cream and subjective measures of gastrointestinal tolerance to the inulin were also measured.

Methods

Subjects

All aspects of this research were approved by the University of Minnesota Institutional Review Board Human Subjects Committee. We recruited male subjects from the surrounding university community. Subjects were screened for their ability to adhere to a controlled diet and collect fecal samples on specific days. All subjects were non-smokers and had not been on antibiotics for 6 months prior to beginning the study. Other exclusion criteria included body mass index > 32, any pre-existing medical condition, alcohol or drug use, extreme diet, or extreme exercise pattern. We screened 54 subjects for possible inclusion in the study and 12 male subjects, ages 27 to 49 years, were chosen to participate. Individual written consent was obtained from each subject.

Subjects were asked to pick up meals and consume all the food they were given each day, recording any food not consumed. Compliance to the diet was assessed by daily food check-off sheets, which were collected and monitored throughout the study. All 12 subjects completed the study. However, only 10 of the 12 subjects successfully completed the anaerobic fecal collection so data on microflora represent n = 10.

Assessment of subjects' habitual diets

Prior to the study, each subject was asked to fill out detailed diet records for 3 consecutive days. Nutrient calculations were performed using the Nutrition Data System for Research (NDS-R) software version 4.0, developed by the Nutrition Coordinating Center (NCC), University of Minnesota, Minneapolis, MN, Food and Nutrient Database 28. If an analytical value is not available for a nutrient in a food, NCC calculates the value based on the nutrient content of other nutrients in the same food or on a product ingredient list, or estimates the value based on the nutrient content of similar foods.

Study design

This study used a randomized, double-blind crossover design, with no washout period between treatments. Subjects were randomly assigned to either a basal, low fiber control diet, or to the basal diet with the addition of 20 g of chicory inulin (Frutafit® supplied by Imperial Suiker-Unie, Sugar Land, TX; produced by Sensus, Roosendaal, Netherlands) incorporated into low fat vanilla ice cream. Each treatment period lasted 21 days, after which subjects were crossed over to the other diet. Thus, the entire study lasted for 42 days.

Controlled diet composition

A 4-day cycle menu was used for this 42-day study. This ensured that on day 21 and day 42, the meals consumed would be the same prior to fecal collections. Each day subjects were asked to come to the University of Minnesota McNeal Hall test kitchen to pick up meals for the next 24 hour period. On Fridays, subjects were given food for the weekend through to breakfast on Monday. Energy needs were calculated for each subject and modified if necessary to prevent weight fluctuations >5% during the study by providing unit muffins as the source of extra calories. No caffeine-containing beverages were allowed during the study since caffeine alters intestinal transit, one parameter of interest in this study. Caffeine-free cola was provided in two of the test meals. Additionally, subjects filled out sensory evaluations of the ice cream products as well as symptom evaluation of flatulence, stool frequency and consistency.

Inulin supplement

Long-chain inulin binds water and produces a fat-like particle gel, providing a means to work well in fat replacer systems.³ We incorporated inulin into low-fat (3.5%) vanilla ice cream in the University of Minnesota Department of Food Science and Nutrition dairy pilot plant. Each pint of test ice cream contained 20 g of inulin. A control ice cream was also made, using corn syrup instead of inulin. Sensory evaluations using trained sensory judges indicated that there was no difference between test and control ice creams with respect to sweetness, mouth feel, off-flavor, chewiness and ice content. Some judges indicated a full-fat mouth feel in the inulin-containing ice cream compared to the control.

Subjective measurement of gastrointestinal tolerance

Subjects were asked about stool frequency, stool consistency, and flatulence at the end of each diet. Subjects completed a subjective assessment of gastrointestinal symptoms with a self-scoring line measuring from 0 to 15.3 cm long at the end of the each treatment.

Sample collection

On days 16 and 37 of the study, each subject swallowed 20 plastic radio opaque pellets to mark intestinal transit time. All feces were subsequently collected into individual containers, defecation times were recorded and samples were weighed and frozen immediately at −20 °C until analyzed. Fecal samples were X-rayed and pellets per stool were counted. A sample containing >80% of the 20 pellets was used as the transit time sample. For the SCFA analyses, a 5 day fecal collection was prepared from the samples that were first X-rayed for transit time measurement.

A fresh fecal sample was obtained from each subject at the conclusion of the transit time collection. Subjects were asked to defecate into sterile bags and add an anaero-pouch sachet (Mitsubishi Gas Chemical Company, Inc., New York, NY) and seal with a clip to keep the atmosphere reduced until analyzed. Subjects delivered the fresh fecal samples to the University of Minnesota microbiology laboratory within 1 h of defecation. All data for microbiology and fecal enzymes were made from this fresh fecal sample collected at the end of each feeding period.

Microbiology

11 g of fresh fecal sample was obtained from the center of each stool and homogenized in 99 mL of pre-reduced 0.1% peptone water to provide a 10% (wt/vol) fecal slurry. One ml of slurry was diluted serially in peptone water and duplicate spread plates were made using 0.1 ml of each dilution. Total anaerobes were counted using Wilkins-Chalgren agar (Difco Laboratories, Detroit, MI) and enterobacteria were counted using MacConkey agar (Difco). Total lactic acid bacteria were counted using Lactobacilli MRS medium (Difco) supplemented with 0.05% L-cysteine·HCl (Sigma Chemical, St. Louis, MO), 0.075% Bacto agar (Difco), 0.02% Na₂CO₃ and 0.01% CaCl₂·2H₂O.⁸Bifidobacterium spp. were counted on a X-β-Gal based medium as described by Chevalier and colleagues using a final concentration of 100 mM 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside dissolved in 0.2% N,N-dimethylformamide.⁹Clostridium spp. were isolated on sulfite-polymyxin-milk agar containing 15 g of tryptone (Difco), 10 g of yeast extract (Difco), 0.5 g of ferric citrate, and 18 g of Bacto-Agar (Difco) per 930 ml of distilled water. Following sterilization, 5 ml of filter-sterilized 5% Na₂CO₃, 10 ml of 0.1% colistin sulfate, 4 ml of neutral red, and 50 ml whole cow's milk were added to the medium prior to pouring plates.¹⁰ Plates were incubated at 37 °C in the AnaeroPack™ (Mitsubishi Gas Company) containing 20% CO₂ and read after 48 h. Stool slurry pH was determined in each sample with a glass pH electrode.

Enzyme assays

Samples (40 ml) of 1 : 10 diluted stool from microbial enumeration studies were placed in 50 ml tubes. 4 ml of Oxyrase® for Broth (Oxyrase, Inc., Mansfield, OH) was added to each sample to maintain an anaerobic environment. Samples were stored at −20 °C until analyzed. Samples were thawed, sonicated on ice for 3 min and centrifuged for 5 min at 12,000 × g to pellet particulate matter. Samples were transferred to capped microfuge tubes for individual enzyme assays.

β-Glucosidase

β-Glucosidase activity was assayed at 37 °C under atmospheric conditions by following the hydrolysis of 3 mM p-nitrophenyl-β-D-glucopyranoside after 1 h and comparing the p-nitrophenol liberated to a standard curve at an absorbance of 405 nm. The pH of the 1 ml samples were adjusted with the addition of 100 μl 1.0 M potassium phosphate, 1.5 M NaCl, pH 5.5. The reaction was stopped with the addition of 100 μl 1M Na₂CO₃.

β-Glucuronidase

β-Glucuronidase activity was assayed at 37 °C in atmospheric conditions using 3 mM p-nitrophenyl-β-glucuronide as the substrate. The pH of each 1 ml sample was adjusted with the addition of 100 μl 1.0 M potassium phosphate in 1.5 M NaCl, pH 7.0. The reactions were carried out for 60 min and stopped with the addition of 100 μl 1M Na₂CO₃. Samples were centrifuged at 5000 × g for 5 min, room temperature and concentrations of p-nitrophenol were compared to a p-nitrophenol standard curve at an absorbance of 405 nm.

Ammonia assay

Fecal ammonia levels were assayed using the CHEMets^® Ammonia-Nitrogen Kit (CHEMetrics, Calverton, VA). 1 ml fecal supernatant samples were diluted with 24 ml distilled, deionized water. Glass ampoules containing Nessler's reagent, an alkaline solution comprised of mercuric iodide and sodium hydroxide were inserted into diluted fecal samples and filled. Ampoules were mixed, allowed to react for 1 min and quantified by comparing to a set of colored standards. A yellow color developed in the presence of ammonia.

Short chain fatty acids

After transit time calculations, 5-day fecal collections were homogenized in a blender and stored at −20 °C for SCFA analysis. Samples were thawed and 5 g aliquots were placed in Centriprep fluid concentrators, MWCO 30,000 kDa (Amicon Inc., Beverly, MA). Samples were centrifuged for 30 min at 1000 × g, room temperature and supernatants (total volume 0.75–1.0 ml) were placed in 15 ml polypropylene tubes. 0.3 ml of 25% m-phosphoric acid was added to each tube, samples were vortexed and incubated at room temperature for 25 min. Samples were centrifuged at 5000 × g for 15 min at room temperature. Supernatants were decanted and frozen overnight. The following day, samples were thawed and the pH of each sample was measured with a pH electrode. pH was adjusted to 6.5 using 4 N KOH. Oxalic acid was added at a final concentration of 0.03% and SCFA concentrations were determined by gas chromatography with use of a Hewlett-Packard 5880A gas chromatograph (Hewlet Packard, Palo Alto, CA) containing an 80/120 Carbopack B-DA/4% Carbowax 20M column (Supelco, Inc., Bellefonte, PA).¹¹

Statistical analysis

Values in tables represent mean and standard deviation. p values reported are for a one-tailed students paired t-test for means. Data were analyzed using Microsoft Excel for Windows 95.

Results

General observations

Test meals were well tolerated by all subjects. There were no significant fluctuations in body weight (97.54 ± 6.39 and 97.36 ± 6.45 kg, control and inulin phases, respectively). All subjects completed the study without complication.

Large-bowel function

Fecal composite weight and 24-hour defecation volume, intestinal transit time, and self-scored stool frequency, consistency and degree of flatus are presented in Table 1. No statistically significant differences between diets were seen in stool weight, transit time, stool frequency, and stool consistency. Flatulence increased significantly (p = 0.046) during the inulin phase of the study (Table 1). No differences were reported for stool consistency or stool frequency in the subjective assessment completed by the subjects.

Table 1 Large bowel function and symptom evaluation before and after inulin administration

Parameter	Control phase	Inulin phase	p value
	Mean ± SD	Mean ± SD
a Grams. b Hours. c Self-scored line length: 0 = constipated, 15.3 = urgent. d Self-scored line length: 0 = diarrhea, 15.3 = hard pellets. e Self-scored line length: 0 = minimal, 15.3 = excessive.
5-Day fecal composite weight^a	751.38 ± 269.91	821.45 ± 284.20	0.20
Mean 24-hour fecal weight^a	150.28 ± 53.98	164.29 ± 56.84	0.20
Intestinal transit^b	32.5 ± 25.30	30.50 ± 16.10	0.33
Stool frequency^c	7.80 ± 2.59	9.80 ± 1.70	0.14
Stool consistency^d	6.36 ± 2.88	4.56 ± 2.42	0.10
Flatulence^e	4.52 ± 3.16	10.06 ± 6.39	0.04

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Intestinal microflora

There were significant differences in levels of total anaerobes and Lactobacillus spp. after the inulin phase of the study (Table 2). Levels of fecal Bifidobacterium and Clostridium spp. did not differ significantly between the control and inulin phases. Levels of Enterobacteriaceae tended (p = 0.067) to decrease after inulin consumption, although this change was not statistically significant.

Table 2 Select Fecal Microbial Endpoints Before and After Inulin Administration

Parameter	Control phase	Inulin phase	p value
	Mean ± SD	Mean ± SD
a Activity expressed as μmol L^−1 g^−1 h^−1. b Ammonia expressed as parts per million (ppm).
Total Aanaerobes	1.98 E10 ± 1.65 E10	2.82 E10 ± 1.79 E10	0.03
Lactobacillus spp.	1.55 E09 ± 2.44 E09	2.85 E09 ± 1.79 E10	0.05
Bifidobacterium spp.	1.75 E09 ± 2.44 E09	2.15 E09 ± 3.29 E09	0.33
Clostridium spp.	2.26 E09 ± 2.51 E09	1.64 E09 ± 3.07 E09	0.17
Enterobacteriaceae	2.11 E08 ± 1.11 E08	3.42 E06 ± 1.81 E06	0.07
β-Glucosidase^a	2.57 ± 1.87	2.62 ± 1.95	0.45
β-Glucuronidase^a	14.06 ± 5.24	10.54 ± 5.33	0.008
Ammonia^b	87.50 ± 42.90	51.50 ± 28.68	0.001
Acetate : propionate	3.26 ± 0.66	3.81 ± 0.91	0.0005
Acetate : butyrate	4.00 ± 1.12	3.80 ± 0.79	0.30
Propionate : butyrate	1.27 ± 0.47	1.02 ± 0.31	0.07

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Bacterial enzyme activity, fecal pH, ammonia, and SCFA ratios

We observed significant decreases in both fecal β-glucuronidase and ammonia levels after administration of inulin (Table 2). β-Glucosidase and fecal pH did not significantly differ between treatments. None of the individual SCFAs analyzed changed significantly after inulin administration. However, we noted a significant change in the acetate : propionate ratio after the inulin phase (Table 2).

Discussion

It is well recognized that fiber is important for normal laxation. This is due primarily to the ability of fiber to increase stool weight.¹ The increased weight is due to the physical presence of the fiber itself, water held by the fiber, and increased bacterial mass from fermentation. Larger and softer stools increase the ease of defecation and reduce transit time through the intestinal tract, which may help to prevent or relieve constipation. In general, cereal fibers are the most effective at increasing stool weight.¹

It is not only insoluble fibers that have an effect on laxation. SCFAs produced from fermentation of soluble fibers contribute to fecal bulk and increase the water content of feces. Fermentable fibers may provide a number of health benefits by altering the composition of the intestinal flora. Inulin, the fiber fed in our study, is best known as a prebiotic. Prebiotics are non-digestible substances that provide a beneficial physiological effect to the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria.³ This generally refers to the ability of a fiber to increase the growth of bifidobacteria and lactobacilli, which are considered beneficial to human health. Benefits of prebiotics include improvement in gut barrier function and host immunity, reduction of potentially pathogenic bacteria subpopulations (e.g. clostridia), and enhanced SCFA production. Since inulin is readily fermented and increases the bacterial content of the stool, it might be expected to increase stool weight.

Our subjects entered the study with normal stool weights and transit times so we might not expect a readily fermentable carbohydrate source to alter these parameters. Different fibers have different effects on laxation. Wheat bran increases stool weight 5 g for each gram of wheat bran fiber fed, while other extensively fermented fibers such as pectin have minimal effects on stool weight, and often only increases stool weight 1.5 g for each gram of pectin fed.¹ In our controlled study, inulin had even less effect on stool weight with less than a gram increase in stool weight with each gram of inulin consumed. Other studies with inulin and shorter chain fructans suggest that inulin can soften and enhance weight and bulk of stools. Both short and long chain fructans significantly increased fecal wet and dry matter, nitrogen, and energy excretion¹² in normal subjects. Inulin has been shown to have a more desirable laxative effect in elderly constipated persons as compared to lactose.¹³ When inulin was added to beverages and consumed by institutionalized adults, perceived stool output increased.¹⁴

Van Dokkum¹⁵ fed 15 g of inulin to male subjects and also reported no changes in measures of bowel function. While changes in fecal 24-hour output, stool frequency and consistency in our study were not statistically significant, defecation frequency tended to increase and stool consistency tended to be softer on a self-scoring symptom evaluation. It is likely that inulin is similar to other soluble fibers like pectin and has little effect on stool weight or gastrointestinal transit time.¹

An increase in gas formation would be expected since inulin provides a highly fermentable substrate to the resident microflora for fermentation.^16–19 Subjects in our study reported a lessening of flatulence over time, indicative of an adaptive response to inulin consumption. In a recent study we compared 5 and 10 grams of short and long chain inulin on gastrointestinal tolerance.²⁰ Only the shorter chain inulin at the 10 g dose significantly increased reports of gastrointestinal intolerance. A review of tolerance of low digestible carbohydrates concluded that 40 grams of inulin may cause diarrhea, but tolerance of inulin is generally fine up to 15 g day⁻¹.²¹

Fecal slurries were assayed for β-glucosidase and β-glucuronidase, two microbial enzymes capable of converting metabolites to potential precarcinogens. Levels of β-glucosidase did not change during the study, while we found a significant decrease in β-glucuronidase activity and ammonia levels after the inulin phase of the study. We also measured fecal SCFA and computed ratios and found a significant increase in the ratio of acetate to propionate (A : P). No other individual SCFA or ratios differed significantly in our study. Implications of an increase in A : P are not clear. The concentration of SCFA produced in humans after dietary fiber interventions are difficult to measure. Fecal SFCA are not necessarily indicative of the concentrations present in the luminal contents since approximately 90% of SCFA are rapidly absorbed on generation.

Total anaerobes and Lactobacillus spp. significantly increased after inulin consumption. Buddington et al.²² also noted a significant increase in total anaerobes after feeding 4 g day⁻¹ fructooligosaccharide (FOS) to twelve healthy humans for 6 weeks. Other studies in humans²³ report a significant increase in Bifidobacterium spp. after inulin or FOS administration. In the present study, we did not find a significant increase in Bifidobacterium spp. This may have been due to subjectivity in scoring Bifidus-positive colonies, nutrient competition between Lactobacillus spp. and Bifidobacterium spp. and/or large individual variation between individuals.

Interest in the ability of fermentable carbohydrates to change the gut microbiota has increased with the finding that the gut microflora may be linked to obesity. The involvement of gut microbiota in the regulation of host energy homeostasis was suggested by studies reporting that obese people have lower Bacteroidetes and more Firmicutes in their distal gut than lean control individuals, alterations that were abolished after 52 weeks of diet-induced weight loss.²⁴ Changing gut microflora may be more difficult in free-living individuals and the long term consequences of changes in gut microflora are unknown.²⁵

It is known that fiber intake is linked to satiety.²⁶ The gastrointestinal tract is also important in obesity prevention because of the role of gut hormones in eating.²⁷ Prebiotics and gut microbiota are also proposed to play a role in a broad range of gastrointestinal diseases.²⁸ Additionally, a review of the effect of prebiotics on immune function, infection, and inflammation was conducted.²⁹ The results from human trials are mixed. Ten prebiotics trials involving infants and children reported beneficial effects on infectious outcomes, while 15 adult trials showed little effect. The relationship of the gut microflora and functional food components such as prebiotics holds much promise.³⁰

Thus, consumption of 20 g day⁻¹ inulin in the controlled diets of healthy adults had minimal effects on bowel function measures. Inulin, as consumed in ice cream, was well tolerated in male subjects, although flatulence measures by self report were significantly higher when on the inulin treatment. Inulin is easily incorporated into well liked food products and should be a useful vehicle to increase the fiber content of the diet.

Acknowledgements

The authors wish to thank Ms. Lisa Schaller-Povolny, Dr David Smith, and Rey Miller for producing the test ice creams, Ms. Jennifer Causey for conducting the clinical study, Ms. Lynda Enright for help with menu selection, diet records and food preparation, Ms. Ying Xin-Chu and Mr. Richard Flores for microbial enumeration and processing of fecal samples.

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Footnote

† This work was supported in part by grant MO1-RR00400 from the National Center for Research Resources and funding from Imperial Sensus USA and Sensus, Netherlands.

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