In vitro fermentation and hydration properties of commercial dietary fiber-rich supplements

Nuhition Research, Vol. 18. No. 6. pp. 1077-1089.1998 Copyright Q 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0271-5317/98 %19.00+ .CHl ELSEVIER

PI1SO271-5317(98)00090-6

IN VITRO FERMENTATION AND HYDRATION PROPERTIES DIETARY FIBER-RICH SUPPLEMENTS

OF COMMERCIAL

Isabel Got?, Ph.D., Nuria Martin-Car&, BSc Department of Nutrition I, Facultad de Farmacia University Complutense of Madrid, Spain

ABSTRACT

Eight commercial dietary fiber (DF)-rich supplements wsre analyzed in vitro to assess swelling, water retention capacity (WRC) and fermentation. The supplements were fermented with rat cecal as inoculum for 24 h, and short chain fatty acid (SCFA) production, fermentability, dry matter disappearance (DMD) and gas production were determined. Products containing mainly soluble fiber components (Metamucil, Humamil, Fybogel and Fibraplan) showed the highest capacity of retaining water and fermentability. Swelling (mL per g dry matter) ranged from 6.2 for Fibra Leo Apple Prune to 24 for Fibraplan. Total SCFA production was significantly correlated to DMD (r = 0.969, R2 = 0.9385) and to gas production (r = 0.901, R* = 0.8113). Hydration properties and fermentative characteristics were different among the studied supplements, these in vitro techniques could be useful to evaluate their potential physiological effects. 8 1998 Elscvier Sciwcehc. KEY WORDS: Dietary Fiber, Commercial Supplements, Hydration Properties, Fermentation, Short Chain Fatty Acids, Gas Production

INTRODUCTION

The ingestion of dietary fiber (DF) has been shown to have beneficial effects on chronic Western diseases such as obesity, diabetes, coronary heart disease, large bowel cancer and other bowel disorders (1, 2). The dietary guidelines of the Nutrition Advisory Committee on Nutrition Education (NACNE) recommend a DF intake of 30 g per person per day, howwer, DF intakes in European countries are lower, ranging from 16 to 21 g/day (3) and they should be increased. This fact could only be achieved by changes in lifestyles and habits or by supplementing the diet with commercial fiber-rich products. DF is defined as the part of foodstuffs which is not digested by the secretions of the human gastrointestinal tract (2). It comprises a great variety of compounds with different Addres correspondence to: Dr. Isabel Gofli, Departamento de Nutricih y Bromatologia I, Facultad de Farmacia. Universidad Complutense de Madrid. Avda. Complutense. s/n. Ciudad Universitaria, 28040 Madrid, Spain 1077

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I. GOfiI and N. MART/N-CARR6N

structure. The physiological effects of DF depend, in great part, on the chemical and physical properties of its components (2). Thus, soluble dietary fiber (SDF) swells in the stomach and increases the viscosity of the digesta, making the absorption of nutrients from the intestinal mucosa difficult, and lowering the postprandial blood glucose and insulin responses (4, 5). This effect could be beneficial to control non-insulin dependent diabetes mellitus (6). The increase in digesta viscosity has also been related to increased feelings of satiety and may be useful to treat overeating and obesity (7). The effect of SDF on LDLcholesterol level in plasma has also been shown (8). SDF is fermented in the colon. The physiological effects of fiber sources in man are significantly influenced by the degree of fermentation in the colon. Fiber fermentation results in the production of short-chain fatty acids (SCFA), principally acetate, propionate and butyrate, gases (carbon dioxide, methane and hydrogen), microbial cell mass and lactate (9). The 95% of SCFA is absorbed from the colonic lumen and metabolized by various body tissues (10). Butyrate appears to be almost extensively utilized by the colonic epithelial cells (11) and has been found to act as a protective agent against experimental tumourogenesis of these cells (12). Propionate is cleared by the liver and may modulate hepatic carbohydrate and lipid metabolism, that could be related to hypocholesterolemic effects (13, 14). Acetate largely escapes colonic and hepatic metabolism and is utilized by peripheral tissues (15). Insoluble dietary fiber (IDF) has a high water-holding capacity, it increases the fecal bulk and reduces the gastrointestinal transit time (16). This effect may be related to the prevention and treatment of different intestinal disorders, such as constipation, diverticulitis, haemorrhoids and other bowel1 conditions (2). It is difficult to measure the rate of SCFA production in tivo due to the inaccesibility of the human colon. In vitro methods have been developed to predict the physiological effects of DF consumption. These methods try to determine the extent of substrate fermentation, SCFA production and the influence of substrates on fecal bulk. The aim of the present research was to evaluate using in vitro techniques the physiological properties of some commercialized fiber products: fermentability, SCFA profile and hydration properties.

MATERIALS

AND METHODS

Substrates Eight fiber-rich products were analyzed: Agiolax (Madaus Cerafarm S.A., Barcelona, Spain), Cenat (Madaus Cerafarm S.A., Barcelona, Spain), Metamucil (Procter and Gamble S.A., Spain), Fibra Kneipp (Fher S.A, Barcelona, Spain), Humamil (Daker-Fannasimes S.A., Barcelona, Spain), Fibra Leo Apple and Prune (Byk Leo S.L., Madrid, Spain), Fybogel

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(Reckitt and Colman, Brussels, Belgium) and Fibraplan (Laboratory 73-I.P.M., Dietary Register 2602452/M, Spain) Products were purchased in health food shops and pharmacies. Among fiber-rich products, they were chosen as the most frequently used by the consumers to treat constipation, obesity or as a dairy fiber complement. Dietary Fiber (DF) analvsis Total DF was analyzed by the enzymatic-gravimetric method of Prosky et al. (17), slightly modified to solve some methodological problems found due to the nature of the samples analysed. Samples could not be filtered through crucible due to the high viscosity of the enzyme mixture, and DF fractions (insoluble and soluble) could not be separated. Therefore, total DF was precipitated with 80% ethanol and the residues were isolated by centrifugation (3000 x g, 15 min) and gravimetrically quantified. Total DF residues were hydrolyzed with 1M H&O4 (1 OOOC,1.5 h) (18) and the residue after acidic hydrolysis was quantified as insoluble DF. Both total and insoluble DF values were calculated as the residue minus the protein and ash reported on a dry weight basis. Soluble DF was determined by difference between total DF and insoluble DF. Hvdration prooerties Swelling and water retention were assessed following the experimental protocol used in Profibre (European Air Concerted Action, 19). Swelling: The method involves the dispersion of a known weight of dry sample in a volume of water in a measuring cylinder and the measurement of the volume occupied by the hydrated fiber after 18 h. 500 mg of sample were weighed into a graduated measuring cylinder (0.1 mL graduations), and 10 mL of solvent (0.02% w/v sodium azide in deionised water) were added. The sample was dispersed with gentle stirring and left on a level surface overnight at room temperature to allow sample to settle. The volume occupied by the sample was recorded (mL). Swelling was expressed as mL per g of dry sample. Water retention capacity (WRC): The method involves hydrating a known weight of sample, subjecting it to a centrifugal force to allow the excess of supernatant to drain from the pellet, and recording the weight of water retained by the sample. 500 mg of sample were weighed into a 50 mL centrifuge tube and 30 mL deionised water were added. The sample was shaked to aid dispersion and hydration during 1 h at room temperature, finally the sample was centrifuged (3000 x g, 20 minutes), supernatant was discarded and the residue was weighed. Water retention was expressed as g water retained per g dry sample. Suaar extraction The fiber-rich products were milled to a particle size less than 1 mm. 2 g of the powders were extracted with 40 mL ethanol 85% in hot (SOOC)with energic shaking (125 strokes per min) for 60 min. After centrifugation at 2500 x g for 15 min, the supernatants were discarded and the residues were dried at 60°C for 16 h. Finally, the dry sugar-free

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residues were sifted to obtain an uniform particle size of 1 mm. In vitro fermentation Sugar-free samples were fermented in vitro in a batch system under strict anaerobic conditions for 24 h. Fresh rat cecal contents were used as inoculum. Anaerobic conditions were maintained using oxygen-free carbon dioxide. The fermentation medium has been adapted from Goering and Van Soest (20). It contained the following (per L of distilled water): 2.5 g trypticase, 125 JJI micromineral solution, 250 mL buffer solution, 250 mL macromineral solution and 1.25 mL resazurine solution O,l% (w/v). The micromineral solution contained (per L of distilled water): 132 g CaC12.2H20, 100 g MnCl,.4H20, 10 g CoC12.6H20 and 80 g FeC13.6H20. The buffer solution contained (per L of distilled water): 4 g (NH4)HC03 and 35 g NaHC03. The macromineral solution was prepared with 5.7 g NasHPOd, 6.2 g KHz PO4 and 0.6 g MgS04.7H20, per L of distilled water. 33.5 mL of reducing solution, containing 6.25 g cysteine hydrochloride, 6.25 g Na&9H20 and 40 mL NaOH IM per L of distilled water, were added to 1 L of medium and they were sterilized at 1OOOCfor 15 min. The inoculum was prepared from the cecal contents of six male Wistar rats from the breeding centre at the Facultad de Farmacia (Universidad Complutense, Madrid), with an average body weight of 190 g. Rats were anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight) and ceca were removed. Rat cecal contents were scraped, weighed and added to a flask containing sterile and anaerobic medium to give a 10% (w/v) inoculum. The inoculum was mixed for 10 min in a Stomacher 80 Lab Blender (Seward medical, London, UK), and filtered (0.5 mm mesh) before use. 100 mg of sugar-free substrate was weighed in 50 mL serum vials (Supelco), and 8 mL medium added. Substrates were hydrated at 4OC for 16 h. Then, 2 mL inoculum was added to each vial. The concentrations of substrate and inoculum were 1% (w/v) and 2% (w/v), respectively. Vials were placed in a shaking water bath (80 strokes per min) at 37OC for 24 h. Fiber products were fermented in triplicate and twelve controls, six containing no substrate and six containing lactulose (Sigma L-7877) were included in the experiments as zero and as easily fermentable substrate, respectively. At 24 h, gas pressure was measured in the vials to calculate gas production. Fermentation was stopped by adding an excess of 1M NaOH (2.5 mL), samples were centrifuged at 2500 x g for IO min, and 3 mL of supernatant in duplicate were taken for SCFA determinations. Non fermented residue determination The method has been adapted from Guillon et al. (21). After fermentation, the residues were suspended in 50 mL NaCl 0.9% (w/v), stirred in a Stomacher 80 (Lab Blender, Seward Medical, London, UK) for 3 min, and filtered through Dacron cloth (mesh

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size 150 pm). Residues were resuspended in 50 mL NaC10.9°h (w/v) and washed again twice. The residues wsre finally washed with 5 mL 96% ethanol and 5 mL acetone and dried in an oven at 60°C overnight. Non fermented residue was determined gravimetrically.

Results obtained for controls without substrate and time 0 h were sustracted from the samples to correct the SCFA production from the inoculum. Percentage of fermentability of substrates with respect to lactulose was calculated considering total SCFA produced by lactulose as 100% fermentability. SCFA produced were expressed proportions (%) acetate:propionate:butyrate.

as ymoles/mg

dry substrate

and as molar

Non fermented residue = Sample residue weight - Control no substrate residue weight

(Initial dry substrate weight - Non fermented dry substrate weight) DMD (%) = Initial dry substrate weight

SCFA analvsis bv gas chromatonraphy A 400 PL aliquot of supematant with 100 PL internal standard (Cmethyl valeric acid, 50 Fmol mL’) and 50 PL of 850 g L’ phosphoric acid were made up to 1 mL with Milli-Q water and centrifuged (4OC, 7300 x g, 15 min). 2 PL of supematant wre injected into a 5890 Hewlett Packard gas chromatograph equipped with a flame ionisation detector and a fused silica column (Carbowax 20M, IOm x 0.53 mm i.d.). Nitrogen was the carrier gas at a pressure of 17 Wa. injector and detector temperature was 200°C and column temperature 120°C (isothermal). SCFA were identified and quantified by comparison with known fatty acid standards. Statistical analvsis The results are reported as means f SD. Linear regression analysis have been performed to compare SCFA production with percentage of fermentability and with gas production experimental values by using a Statgrafics computer system.

RESULTS AND DISCUSSION

Rich-fiber supplements are commonly used in obesity treatments as satiating agents

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and to regulate the intestinal transit time. Different types of fiber from husks, brans, gums and pectins were found in the composition of the studied products. The principal components in the samples wre ispaghula, wheat and oat bran and apple pectin (Table 1). DF composition was not detailed in the label of the products, only total DF content was indicated in Fibra Kneipp and Fibra Leo, but soluble and insoluble fractions were indicated in none of the supplements. Products labels recommend these supplements mainly for obesity treatments as satiating agents and for prevention and treatment of constipation and haemorrhoids. Two products are also indicated as beneficial for other diseases, Humamil for insulin-dependent diabetes patients and as hypolypidemic agent, and Fibraplan for hepaticdigestive disorders. Labels indicate that non side effects are derived from an habitual use of these products.

Composition,

Indications

TABLE 1 and Dietary Fiber (DF) Content labelled in Commercial Dietary Fiber-rich Supplements Indications

Commercial name

Composition

AGIOLAX

lspaghula seed and husk, cassia fruit

Constipation, diverticulosis,

irritable colon, haemorrhoids

lspaghula

Constipation,

haemorrhoids

CENAT METAMUCIL

seed and husk

lsphaghula

Constipation

husk

FIBRA KNEIPP

Fruit fiber, wheat bran, guar gum

Regulation intestinal transit time, constipation, slendering diets

HUMAMIL

Glucomannan

Slendering and hypolypidemic diets, constipation, insulin-dependent diabetes

FIBRA LEO

Wheat bran, apple pectin, prune pulp

Slendering diets

FYBOGEL

lspaghula husk

FIBRAPLAN

Soluble fiber from algae, seeds, Flours, plants non specified

Regulation

intestinal transit time

Slendering diets, hepaticdigestive disorders. constioation

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Total, soluble and insoluble DF content of the products are shov,n in Table 2. Total DF content was high in all the products (>70% of the dry matter). The main fiber fraction was soluble, more than 60°h of total DF, and some products, such as Metamucil and Fibraplan, were almost exclusively composed of soluble DF.

TABLE 2 Total, Soluble and Insoluble Dietary Fiber Content of Commercial Dietary Fiber-rich Supplements*

Commercial name

Total

insoluble

Soluble

AGIOLAX

71.1+1.1

23.9&l.1

47.2

CENAT

66.Oa.7

22.5ko.9

43.5

METAMUCIL

77.8fl.2

2.6ti.4

75.2

FIBRA KNEIPP

68.3k1.8

15.9*3.1

52.4

HUMAMIL

82.9k1.3

16.4+1.4

66.5

FIBRA LEO

76.4a.4

19.3*1.4

57.1

FYBOGEL

88.5k3.5

17.2M.6

71.3

FIBRAPLAN

86.6a.3

4.lfl.l

82.5

* Mean + SD Hydration properties (swelling and water retention capacity) of fibers may be useful predictors of their effects on small intestine. The gel-forming properties of certain fibers have been suggested to slow the intestinal digestion of carbohydrates and lipids (22). WRC of products is shown in Table 3. WRC was directly correlated with the amount of soluble DF, the products with the highest soluble DF content (Metamucil, Humamil, Fybogel and Fibraplan) showed the highest water retention values, Agiolax and Cenat had an intermediate WRC, and Fibra Kneipp and Fibra Leo retained the lowest amount of water. The water holding capacity of the substrates may be an estimation of the fecal bulking effect of the substrates. Some authors have reported an inverse relationship between original substrate water retention and fecal bulking (23). This can be explained because soluble fiber-rich products, which showed the highest WRC values, ferment in the colon and may have a small contribution to the fecal bulk. /n vitro results of WRC agree with in viva studies of the bulking characteristics of some substrates, several studies indicate that soluble fiber sources such as pectin cause little increase in fecal bulk in humans (24) while insoluble fiber sources such as wheat bran significantly increase fecal bulk in humans (25).

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and N. MART/N-CARR6N

Swslling property did not correlate with WRC or with soluble DF content. Humamil and Fibraplan had higher swelling capacity (18-24 mUg dry matter) than the others (Table 3) Agiolax and Metamucil swelling capacity was intermediate and the rest retained about 6 mL water/g dry matter. Swelling in the stomach gives an idea of the satiating effects of the substrates, when DF swells in the stomach causes an increase in the viscosity of the stomach contents increasing the feeling of satiety and making nutrients absorption difficult (26).

TABLE 3 Hydration Properties: Water Retention Capacity (WRC) and Swelling of Commercial Dietary Fiber-rich Supplements* Commercial name

WRC

Swelling

(g water I g dry matter)

(mL water / g dry matter)

AGIOLAX

6.6*0.1

9.9*0.3

CENAT

9.0*0.1

6.8*0.1

METAMUCIL

19.6kO.6

8.2kO.2

FIBRA KNEIPP

4.8ztO.1

6.7kO.3

HUMAMIL

30.6*0.7

17.7kO.8

FIBRA LEO

3.1*0.1

6.2kO.3

FYBOGEL

23.3kO.6

7.0io.4

FIBRAPLAN l

47.6kl.l

24.Okl.2

Mean f SD

Table 4 shows SCFA production and fermentability of the products. Metamucil, Humamil, Fybogel and Fibraplan were the most fermented substrates, with a percentage of fermentability that ranged from 70 to 90°% Agiolax and Cenat were the least fermented substrates. Fibra Kneipp and Fibra Leo presented an intermediate fermentability value, 48 and 59%, respectively. There was a direct correlation between fermentability and the amount of soluble DF in the products. Similar results of extent of fermentation have been previously reported for different SDF compounds such as pectin (27) and gums (23) and IDF sources such as tieat bran (23) or oat bran (28). Fermentabilities of Agiolax and Cenat were lower than of other ispaghula husk-based supplements, it may be that Agiolax and Cenat contain also ispaghula seeds, that constitute IDF fraction. The differences in

FIBER PRODUCTS: IN VITRO STUDY

1085

fermentability among the products could also be due to the different chemical composition. Fibraplan had the highest soluble fiber content, but it did not show the highest fermentability, it coul be due to the poor fermentability of the seaweeds that composed this product (29).

TABLE 4 Short Chain Fatty Acid (SCFA) Production ( pmoles/mg dry substrate), Fermentability and Dry Matter Disappearance (DMD) in in v&o Fermentation of Commercial Dietary Fiber-rich Supplements* Acetate

Propionate

Butyrate

Total SCFA

Fermenta

DMD’

bility+ (%)

(%)

AGIOLAX

3.4kO.5

2.5*0.2

0.6kO.04

6.5fl.7

38.9*4.2

43.3k2.7

CENAT

2.9*0.2

2.1ztO.l

0.5*0.02

5.5kO.2

32.621.2

39.6k2.4

METAMUCIL

7.1*0.3

5.6k0.1

0.8kO.04

13.5*0.4

80.2k2.3

84.2k0.5

FIBRA KNEIPP

4.8k0.2

2.4k0.1

0.8*0.08

8.0k0.3

47.7*1.9

50.4*1.2

HUMAMIL

9.2*1.3

5.0k0.6

1 .O*O.l

15.2&l .9

90.3*11.3

89.0i6.5

FIBRA LEO

6.0k0.2

2.9*0.1

1.0*0.02

9.9*0.3

58.9*1.5

46.4*1.9

FYBOGEL

7.3io.3

5.4kO.2

0.5*0.04

13.2kO.5

78.4k2.7

79.0*1.2

FIBRAPLAN

6.4k0.3

4.9kO.2

0.4f0.02

11.6kO.5

69.1Q.7

74.7zko.7

Mean f SD ’ Percentage of fermentability * Dry Matter Disappearance l

respect to lactulose = (SCFASUPPLEMEN~/ SCFACACTUL& x 100

Changes in SCFA may explain the variations in the effects of different sources of DF. Products studied showed a wide diversity in their extent of fermentation, and in consequence in the amount and profile of SCFA produced. Molar proportions of SCFA, more than SCFA production, may be indicative of the potential effects of fermentable substrates on health. Molar ratios were very similar for all the studied products (Figure 1). The greater acetate production (60%) was for Fibra Kneipp, Humamil and Fibra Leo; Metamucil, Fybogel and Fibraplan produced the major proportion of propionate(40%) and Agiolax, Cenat, Fibra Kneipp and Fibra Leo produced the highest percentage of butyrate (10%). These results are similar to others obtained for pure substrates in in IMU studies with human fecal bacteria, 58:27:8 for guar gum (21) and 58:35:8 for isphagula (30). The nature of the SCFA produced in the fermentative

process is related to the type

I. GOal and N. MARTiN-CARR6N

1086

of substrate available. Pectin is constituted of uranic acids which seem to be principally involved in the production of acetic acid, whereas the production of propionic acid could be promoted by the fermentation of glucose and, to a lesser extent, by that of xylose and arabinose, and the production of butyrate seems to be related to xylose fermentation (31). Substrates which contained higher amounts of pectins (Fibra Leo) or glucomannans (Humamil) produced higher amounts of acetate.

FIG. 1 Molar Proportions (Ok) Acetate, Propionate and Butyrate in in vitro Fermentation of Commercial Dietary Fiber-rich Supplements

FIBRA KNEIPP!

40

60

Molar Proportions

80

100

(%)

Percentages of dry matter disappearance were very similar to fermentability with respect to SCFA production for all the products (Table 4). In general, percentages of dry matter disappearance were slightly higher than fermentability, but the differences were practically negligible (l-7%). In this experiment, dry matter disappearance seemed to predict end-product formation because the percentage of substrate that had disappeared coincided fairly well with the percentage of fermentabilityscFA (r = 0.969, R2 = 0.9385) but this does not always occurred (32). SCFA production was significantly correlated with gas production (r = 0.901, R2 = 0.8113). Therefore, gas production could be a good indicator of fermentability. In conclusion,

dry matter disappearance

and gas production

were well correlated

FIBER PRODUCTS: IN V/T/70 STUDY

1087

with total SCFA production during the in vtifo fermentation of the studied DF-rich products. Hydration properties and fermentability could be useful to predict the potential physiological effects of the fiber supplements.

ACKNOWLEDGEMENTS

Authors would like to acknowledge the financial support of the Spanish Interministerial Commission of Science and Technology (CICYT ALI 97-0683) and the concession of a scholarship from the Universidad Complutense de Madrid.

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Accepted for

publication

March 23, 1998.

in