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Influence of long-term feeding of different purified dietary fibers on the volatile fatty acid (VFA) profile, pH and fiber-degrading activity of the cecal contents in rats
NUTRITION RESEARCH, Vol. 9, pp. 761-772, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Maxwell Pergamon Macmillan plc. All rights reserved.
INFLUENCE OF LONG-TERM FEEDING OF DIFFERENT PURIFIED DIETARY FIBERS ON THE VOLATILE FATTY ACID (VFA) PROFILE, pH AND FIBER-DEGRADING ACTIVITY OF THE CECAL CONTENTS IN RATS. Furio Brighenti, Giulio Testolin, Enrica Canzi*, Annamaria Ferrari*, Thomas M.S. Wolever **, Salvatore Ciappellano, Marisa Porrini, and Paolo Simonetti Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, division of Nutrition; *division of Microbiology, University of Milan, Milan, Italy and ** Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
ABSTEAC~ The aim of this study was to investigate the effect of four weeks ingestion of 10% fiber diets (cellulose, lignocellulose, pectin or guar g ~ ) and fiber free diets on volatile fatty acid (VFA) concentration, pH, and ~ fiber-degrading activity o f the cecal contents of female Sprague Dawley rats. The pH was significantly lower (p
intestinal
INTRODUCTION Volatile fatty acids (VFAs), mainly acetate (Ac), propionate (Pr) and butyrate (Bu), are known to be produced in large amounts in the colon of monogastric animals and man by the anaerobic saccharolytic bacteria of the large bowel (I-2). Small amounts of branched-chain VFAs (isobutyrate, isovalerate, isocaproate) have also been found in the fecal fluids, probably produced from the breakdown of the branched-chain amino acids valine, leucine and isoleucine (3). The substrates for the colonic fermentation include complex endogenous polysaccharides, such as mucin and glycoproteins produced by epithelial cells of the gastrointestinal tract, and carbohydrates of exogenous origin escaping digestion in the small intestine, such as amylase-resistent starch, saccharidase-resistent oligosaccharides and some dietary fibers (fermentable DF).
Address Correspondence to:
Furio Brighenti, Dipartimento Scienze Alimentari e Nicrobiol., University of G.Celoria 20188 - Milan, ITALY
761
e Tecnologie Milan, 2, Via
762
F. BRIGHENTI et al.
Recently, the presence and fate of VFAs in the large intestine have been suggested to be related to some of the important clinical effects observed in patients consuming diets rich in dietary fiber. Reduction of serum cholesterol (4), development of ulcerative colitis (5) and irritable bowel syndrome (6), mutagenic activity of bile salts and nitroso compounds (7), and fecal trapping of nitrogen with consequent reduction of serum urea (8) have been hypothesized to be due to an increase in the luminal VFAs, or to an increase of the colonic microbial activity, of which the VFA concentration is a marker. Thus, efforts have been made to clarify the relationships between fermentable DF, VFA production, and the colonic environment in general. However, the results have been contradictory. Some studies have failed to demonstrate any direct effect of the SOLace of fiber on fecal microflora (9-10), concentration of VFAs (11), or pH (12), while other studies have reported significant relationships (13-17). The difference in fermentability of the sources of fiber used can generally explain the variability of these results. However, differences in the experimental models of intestinal fermentation of fiber can also play a role, particularly: i) time of adaptation of the colonic microflora to the fibers fed, ii) type of specimen, (i.e. feces or cecal content) since VFAs absorption tends to reduce the concentration gradient between the cecum and the rectum and iii) composition of the experimental diets, especially the starch intake. The purpose of this study was to investigate the effect of chronic ingestion of cellulose, lignocellulose, pectin, guar gum and fiber free diets on the cecal volatile fatty acids (VFAs) and I>}{ in rats, as well as to assess the fiber-degrading activities of the cecal contents by means of an in vitro fermentation system. MATERIALS AND METHODS Female Sprague-Dawley rats (Nossan, Milan, ItalY) 80 days old, average weight 89• g, were divided into five groups of 8 animals. Each group was housed in two cages of four rats in a temperature controlled room kept on a 12h light-dark cycle. The rats were trained to meal-eat over a 4-h period from 9 a.m. to 1 p.m.; each cage was provided with 80 g of basal diet, AIN 76; water was provided ad libitum. The weight of diet consumed daily was taken as the difference between the weight provided and the residue. After a 15-day period of training, four groups were provided with experimental diets containing 18.0% albumin, 4.5% corn oil, 56.2% sucrose, 6.3% starch, 10% fiber, 4.1% AIN 78 minerals mix, 0.9% AIN 76 vitamins mix (table I). The dietary fibers (DF) used were: guar gum (diet G) (Sigma, St Louis, MO, USA), citrus pectin (diet P) (Sigma), cellulose solka-flock (diet C) (Laboratori Piccioni, Milan, Italy), lignocellulose from hazel-nut shells (diet H) (Also, Zelbio, Italy). The composition of the hazel-nut fiber was: total dietary fiber 98%; lignin 24%; cellulose 68%; and hemicellulose 6% (98% xylose). The fifth group of rats was fed a fiber free diet (diet FF) with an identical relative composition of macronutrients, vitamins and minerals but with no fiber. The proximate analysis of the diets after pelletting is presented in table 2. After consL~ing the diets for 4 weeks, 20-h fasting rats were anaesthetized and killed by bleeding, the whole intestine was excised and divided into different s e c t i o n s b y d o u b l e ligature with surgical suture. The cecum, assumed to be the first 2 cm of the large intestine measured from the ileo-cecal valve, was resected and immediately placed into a sterile bag flushed with CO~. The ceca from five rats of each group were then weighed and opened in the fundus with a 5mm cut. A pHmicroprobe (Radiometer, Copenhagen, DM) was inserted twice, at different depths, into the cecum and the average of the pH readings recorded. From 0.5 to I g of the cecal contents was weighed and diluted 1:2 with ice-cold saline into 5 ml preweighed plastic vials.
FERMENTATION OF DIETARY FIBERS TABLE 1
TABLE 2
Diet Composition (g/lO0 g of diet)
Proximate Analysis (g/lO0 g of diet)
~t6AL IOZ FIBI~R FIBII~I~|E (AIE 76) DIET DI~
Protein ~ Fat Carbohydrate | Fiber ~inerale Vitains Integrators $
Y,O.O 5.0 65.0 5.0 3.5 1.0 0.5
16.0 4.5 62.5 10.0 4.1 0.9 0.0
763
20.0 5.0 70.0 0.0 4.0 1.0 0.0
DIITG DIITP DIETH DIITC DIgTFF Moisture Protein rat Av. Curb. Fikr Ash
12.7 16.0 3.0 54.5 10.0 3.8
11.3 16.4 2.4 56.4 9.9 3.6
12.5 14.7 2.7 56.7 10.0 3.4
13.7 15.1 2.6 55.1 9.9 3.4
10.4 18.1 3.5 63.9 0.1 4.0
* Albtmin in the experimental diets, casein in AII 76; S sucrose 90Z corn starch IOZ ; $ ebo]ine, zetionine Vitains and zineraIe as in AI| 76 The remaining cecal content was washed out with saline, the cecum was gently dried by blotting on a filter paper and then weighed again. The whole wet weight of the cecal content wa~ calculated from the difference between the weight of the full and empty cecum. Five hundred ~l of suspension were used for VFA analysis as described below; vials were then freeze-drled after addition of 500 ~i of 110 mM NaOH, weighed and the resulting weights used to calculate the dry weight of cecal content. In vitro bacterial degradation. The cecal content from the remaining three rats of each group were used for the in vitro studies of the DF degradation. For these experiments all the media used were pre-reduced, and all the manipulations were done into an anaerobic cabinet (Anaerobic Glove Box model 1029, Forma Scientific, Marietta, OH, USA) filled with a mixture of N2:H2:C02 85:10:5 % V/V under strict anaerobic conditions (02<20 ppm). The samples of cecal contents (0.5-1 g) were diluted with 70 ml of Dilution Blank (18). Ten ml of suspension were added to i00 ml of Medium i0 broth (19) prepared without VFA mix and with 0.3 % W/V of DF as the only source of carbohydrates. The fiber used in each culture was the same as that in the diet of the rat from which the cecal contents were obtained. In addition, the cecal contents of the rats on diet FF were added to media containing all four different DFs. The residual DF in the media was estimated on I0 ml of culture suspension after O, 6, 24 and 48 h for the guar and pectin groups, and after O, 48 and 72 h for the lignocellulose and cellulose groups. The results were expressed as the percent of DF undigested. Chromatography The analysis of the VFAs was performed b y g a s liquid chromatography (GLC) on the cecal samples prepared according to Varel et al (20). In brief, samples were spiked with internal standard, centrifuged 45.000 x g for 20", acidified to pH 2.0 using HsP04, and directly injected into the chromatograph. The chromatographic apparatus included a chromatograph model GC 6000 MEGA Series 2 fitted with a FID detector, and a MEGA Series SP 4290 integrator (Carlo Erba, Milan, Italy). A coiled glass column (6" lenght X 1/4" I.D.) packed with 15% SP - 1220/1% H3P04 on 100/200 mesh Chromosorb (Supelco, Bellefonte, PA, USA), was used. The gas flows were H2 and N2 30 ml/min and air 340 ml/min. The carrier gas was saturated with formic acid to minimize the tailing and ghosting peaks
764
F. BRIGHENTI et al.
effect that have been reported to occur in the GLC analysis of VFAs (21). The analysis was performed isothermally at 165 ~ with injector and detector temperatures of 185 and 195 ~ respectively. Before use the column was conditioned 72 h at 190 ~ under carrier gas flow. The quantification of the peaks was made by the internal standard procedure, using heptanoic acid (Merck, Darmstadt, FRG) as the internal standard and a calibration mixture of CI-C6 VFAs I0 mM each (Supelco Inc., Bellefonte, PENN.). The method is routinely used in our Department for the detection of VFAs in culture media and fecal specimens, and achieves a mean recovery of C2-C6 VFAs of 98% . Statistics The comparisons between results were conducted by analysis of variance with the fibers as determinant, using the Fisher's F test. When a level of significance p<=O.05 was found, the means were compared by the Tukey's Studentized range test. Regression analyses were performed and correlation coefficents were determined when appropriate. RESULTS After 4 weeks of experimental feeding the weight gain of the rats was not significantly different among groups, ranging from 81• for the P group to 90• for the C group. The feed-efficency of the diets (body weight increment / Kcalories consumed x I00) was also equivalent, ranging from 5.7 for the P and G groups to 5.1 for the FF group (table 3). The total wet weight of the cecal contents was significantly higher in the P group compared with the FF and H groups. This difference was principally related to the water content, since the dry weights did not differ significantly among groups (table 4). There was significant difference in the cecal pH between diets (table 4). Diets G and P significantly increased the acidity of the luminal contents by approximately one pH unit, with respect to the FF, H and C diets. TABLE 3 Body Weight Increment, Food Consumption and Feed Efficency of Diets ~OgP
Body wei~ht (g)
weight s increment (l)
food Z feed consuaed efficeney (g) I/[cal (xlO0)
Guar gm~
aean S~f
188.3 7.3
83.3 5.2
476
5.7 0.3
Pectin
aean Sell
191.7 7.3
81.7 5.3
462
5.7 0.3
Hazel nut
mean 8~
199.2 7.0
89.2 5.0
532
5.4 0.3
Cellulose
mean 8m
204.6 4.9
90.4 2.5
532
5.6 0.2
199.1 7.8
90.2 5.5
504
5.1 0.3
Fiber free u u Sire
S calculated fron the beginning of feedi~ eith the experilental diets.
| =8
FERMENTATION OF DIETARY FIBERS
765
TABLE 4
Wet and Dry Weights and pH of the Cecal Contents OI~P
Net Nei~ht (g} of DU HeiSt (g) of cecal contents cecal contents 0.69 0.10 25.50
6.09 ~C 0.35 11.54
2.90 ABb 0.45 31.00
0.72 a 0.15 31.00
6.38 DE a 0.27 8.59
8uel nut lean $~ CVl
1.71 A 0.24 32.06
0.60 0.09 20.38
7.23 A 0.16 5.04
Cellulose lean 6m C~
1.79 0.21 23.24
0.62 0.09 18.40
7.53 BD 0.08 1.48
Xiber free mean Sm CVZ
1.45 B ac 0.19 26.17
0.50 a 0.09 22.00
7.55 CX 0.06 1.48
~ar ~m
Pectin
6E C~
2.48 0.33 26.25
mean S~ CVZ
lean
a
I~
he
a
Groups in colasnmarhedwith the ease superscript are eilnifieantl7
different (p(O.05) to the Yinher's (nail letter) and the 1~lkey's (capital letter) analysis of the variance. |: 5; Hazel nut |: 8. TABLE 5
Concentration (~mol/g W.W.) of Volatile Fatty Acids in Cecal Contents GI~P
Acetic ~opionic Isobotyrtc Butyric inovaleric Valeric ineeaproic Caproic Total V~aa
Gu~ ~m
~n SBi CVl
46.34 9.35 40.38
27.31 nhe 1.36 6.97 0.37 51.03 54.50
7.65 1.79 46.76
2.07 0.51 49.28
2.21 0.37 33.70
0.44 0.20 89.57
0.52 a 0.20 76.80
87.89 17.09 38.89
Pectin
lean 69 C~
54.78 9.65 35.24
21.02 2.93 27.88
1.49 0.22 29.11
11.36 0.94 16.60
1.87 0.27 28.00
2.12 0.25 23.79
0.60 0.20 65.89
0.55 0.20 72.63
93.80 11.93 25.45
Hazel nut mean 36.24 SI~ 8.50 CVI 53.47
14.90 a 3.03 45.23
1.90 0.58 67.64
11.89 3.91 73.54
2.40 0.64 59.96
2.43 0.74 68.11
1.06 0.65 ]36.97
1.34 0.70 116.39
72.56 14.24 43.88
Cellulose Man 6~ Ct~
S0.71 5.45 35.51
12.60 b 2.88 45.70
2.33 0.56 47.72
7.14 1.33 37.30
2.90 0.75 51.55
2.78 0.77 55.15
1.78 0.62 69.06
2.26 a 0.94 83.33
02.50 12.25 39.20
Fiber free u u Sin CVl
49.14 7.09 28.80
15.23 c 1.91 25.05
1.89 0.14 14.91
8.70 0.12 21.49
2.60 0.29 22.56
2.56 0.33 26.65
0.75 0.24 63.34
0.78 0.37 95.11
79.65 9.81 24.64
~ou~ in cotton mk~l with the m e superscript are ntpificastly different (1~0.05) to the Yinher's analysis of the variance. |= 5; lane] nut I.--6
766
F. BRIGHENTI et al.
There were no significant differences of VFA concentration in cecal contents among the different diets except that Pr was significantly higher (p
Acetic
I~0pionic Butyric
Guar g',m mean 119.71 SR 34.46 CV% 57.58 Pectin
72.30 A ab 20.34 24.60 6.21 68.05 60.97
Iso-VFAs Hin0rVFAs Total VBs 9.78 3.23 66.01
7.03 1.89 53.81
229.16 64.48 56.28
mean 162.13A ab 82.67 cd 33.51 sb 11.19 S{H 44.43 15.29 6.31 2.22 CVt 54.80 48.79 37.68 39.68
8.15 2.35 57.75
277,64 abc 64.24 46.28
9azel nut mean S~ CV%
62.90 a 13.80 49.06
27.32 a 7.38 60.42
23.51 9.75 92.80
9.86 3.97 89.91
6.77 2.80 92.67
130.35 u 32.07 54.77
Cellulose teas 8~ CV%
51.29 A 6.14 23.95
20.86 A c 4.25 40.80
11.76 s 1.06 18.01
11.08 1.77 31.98
7.74 1.76 45.43
102.71 a 12.27 23.90
Fiberfree seas
72.57
22.45
Ixl 10.10
8.02
5.31
1t8.45 c
S~
15.01
b
4.59
2,43
b
2.01
1.79
24.90
CV~
41.36
40.90
48.15
50.24
67.33
42.04
Groups in column Barked eith the sane superscript are significantly different (p
FERMENTATION OF DIETARY FIBERS
767
TABLE 7 VFA Profile G~P
acetic
Guarg~
(% of total VFA)
prepionic
mean 52.9
betyric
61~ Or%
3.0
29.2 ~IBCD 2.7
II.3
18.5
Pectin
mean SE CVZ
57.2 2.7 9.6
Hazel nut
mean 6E CVl
Cellulose
8.8 a 1.2
iso-u
minor TWAs
26.7
5.5 a 2.0 71.7
3.6 a 1.0
57.9
22.3 a 0.6 5.4
12.7 1.6 24.9
4.6 b 0.9 38.4
3.1 0.8 48.0
51.5 5.3 23.1
20.7 B 0.6 8.3
15.1 ab 7.5 3.2 1.8 47.7 55.1
5.1 1.5 61.6
mean SE CV%
50.1 a 2.4 9.5
19.7 C 2.0 20.2
11.7 0.7 11.4
10.6 ab 1.2 21.2
7.7 ab 1.6 40.4
Fiber free mean Sl~ C~
61.2 a 2.1 6.9
19.2 D 1.2 12.5
8.7 b 0.9 20.4
6.7 0.7 21.8
4.2 0.8 30.7
Groups in column marked sith the same superscript are significantly different p
Cellulose and lignocellulose were only slightly degraded in vitro with about 90-95% remaining after 72 hours. Moreover, the inocula from the cecal slurries of the fiber-free adapted rats showed the same fermentative performance as those from the fiber adapted rats (Fig. I). Fig. 1 In vitro indigestibility of the fibre snbstrates incubated sith cecal slurries frot long-tern fiber fed rats. Close figures represant the inocola frol fiber-edapted rats, open fifuren represent the ioocula f r ~ fiber-free adapted rats. hch point is the average of three aniaalo. - - - - - Gear ~ ; --0-- Pectin; --~-- Lipocellulose; - - i - - Cellulose
100
25 ~
60
~
4o "r
0
I
I
I
,
6
24
48
72
INCUBATION t i m e ( h )
768
F. BRIGHENTI et al.
Pectin and guar gum were degraded to the same extent (20%) when incubated for 48 hours with cecal contents from rats on the fiber-free diet. However, the fermentability of guar gum and pectin increased to 60% and 37% respectively, when incubated with cecal contents of rats adapted to diets containing guar and pectin. DISCUSSION Many factors have been thought to affect the rate of production and the concentration of VFAs in the large intestine of both animals and man: the type and the amount of dietary fiber and starch intake and processing (22), transit time (23), and food restriction (24). In an attempt to isolate the dietary fiber as the only experimental variable we fed the rats identical semisinthetic diets containing 10% purified fiber. The available carbohydrates in the experimental diets consisted mainly of sucrose since a number of studies have shown that 5-10% of dietary starch escapes digestion in the upper intestinal tract and reaches the colon (22). The protein source (albumin) was chosen since its fermentation is known to produce preferentially branched VFAs rather than Ac, Pr and Bu (3). The period of feeding was kept constant and the analyses were made after a period of time (20 h) presumed to be sufficent for the fiber to reach the colon and undergo fermentation. In this experimental model, diets C and H showed approximately the same cecal content and profile of VFAs as diet FF. This suggests that, with C, H, and FF, colonic VFAs were derived from the fermentation of endogenous carbohydrates. The almost complete indigestibility of cellulose and lignocellulose over a 20-h period, demostrated by the in vitro experiment, also supports this hypothesis. In contrast, diets P and G resulted in significantly higher amounts of VFAs than the other diets, although not as high as expected by the rate of fermentation observed in vitro, perhaps due to colonic absorption of VFA. The rate of VFAs absorption from the colon has been found to be directly related to VFAs concentration (25). An indirect relationship between VFA absorption and pH has been also demonstrated in the rumen, and hypothesized to occour in other gastrointestinal tracts (26). Thus, an increased amount of fermentable substrate entering the colon would produce a higher concentration of bacterial acidic metabolites and a lower pH, both resulting in an increased rate of uptake of VFAs and excretion of bicarbonate, until the initial conditions of equilibrium are restabilished. This can explain the close similarity in the molar ratio of fecal VFA that invariably has been found with different diets and ~n various species (27-29). In our study, the profile of the major VFA (Ac, Pr, Bu) in the cecum of the rats on diets C and H was almost exactly the same found by Remesy and Demigne (28) in the cecum of rats fed 10% cellulose. Diet P resulted in a higher but not significant percentage of acetate, with propionate and butyrate percentages falling in the same range of values of diets C and H. Hovewer, a marked increase in the percentage of Pr was observed on guar diet, suggesting that this fiber is possibly more metabolized via the succinate than the acetate pathways (30). This could reflect a difference in the species of bacteria present in the colon of guar-adapted rats, or in metabolism of the monosaccharides (mannose and galactose) components of guar g ~ . Mortensen et el., testing the in vitro VFA production from mono- and disaccharides incubated with human fecal inocula, found that glucuronic and galacturonic acids (components of pectin), but not galactose, increased Pr (31). They did not test mannose. Thus, if the rise in Pr we observed on guar was related to its sugar composition, this would be due to the mannose rather than the galactose. The
relationship
between "colonic
pH
and
VFA
concentration
is
still
FERMENTATION OF DIETARY FIBERS
769
controversial. Thomsen et al. (32) found a significant effect of pectin in lowering the pH of the cecal content of rats fed high fat diets. Topping et al. (16) similarly, found a direct relationship between decrease in cecal pH and the amount of gum arabic fed to rats, either alone or mixed with cellulose. In humans, when the non-absorbed but readily fermented disaccharide lactulose was added to the diet, the pH in the right colon decreased significantly from 6.0 to 4.5, whereas in the left colon and rectum no difference from the placebofed controls was observed, both being in a range of 6.3-6.8 pH (33). However, no mechanism bywhich lactulose p r o ~ e d the observed increase of acidity was demonstrated. Other workers have reported a lack of correlation between fecal and intestinal pH and VFAs concentrations in pigs (I) and humans (12) fed high fiber diets. Ruppin et al. (25) even reported an increase in pH with increasing propionate concentration in the intestinal lumen of humans. From their data, the stability of the colonic pH could be attributed to the accumulation of bicarbonate into the lumen , as result of the nonionic transport of VFA across the mucosa. This study shows that the pH of the cecal contents of rats adapted to a controlled fiber diet depends upon the kind of fiber f e d . The readily fermentable fibers, pectin and guar, produced a significant reduction in the cecal pH whereas the non-fermentable fibers, cellulose and ligno-cellulose, had no effect. Moreover, the effect on pH was significantly correlated to the concentrations of acetate, propionate and tota] VFAs. However, it is likely that the concentration of VFAs is not the only factor determining the lower pH observed in the G and P diet groups. The lowest pH was found in the G group, which had a lower concentration of acetic and total VFAs than the P group. In addition the cecal pH correlated better with the Pr than the Ac concentration, although Pr accounted for only 19-25% of total VFA compared to 50-61% for Ac. Therefore, we hypothesize that the reduction in colonic pH is at least partly due to an increase in the utilization of bases present in the lumen, (e.g. ammonia and urea) for bacterial protein synthesis. This has been demostrated by some authors as consequence of fermentable fiber ingestion (34). The fermentative pattern found for Guar gum and Pectin after in vitrQ incubation with cecal inocula from fiber-adapted or fiber-free rats is consistent both with the hypothesis of inducibility of the polysaccharidedegrading enzymes from the intestinal bacteria or with a change of the speciesprofile of the microflora itself. Many studies have shown no changes of the fecal flora in humans after chronic ingestion of fermentable fibers (9,10). However, Slade et al. recently found a reproducible modification of the bacterial profile of fecal bacteria after in vitro incL~ation with purified fibers (35).
CmNCUUSIONS The long term feeding of 10% readily fermentable dietary fibers (guar gum and pectin) had a significant influence on the colonic environment in rats. These fibers lead to an increase in the total amoLmt of VFAs in cecal contents, reflecting an increased metabolic activity of the intestinal bacteria. The pH was significantly lowered in presence of fermentable substrate, partly due to the higher concentration of acetic and propionic acids in the cecal fluid. The amounts of the minor VFAs (iso-acids, valeric and caproic acids.) were influenced neither by the presence nor by the type of fiber, supporting the hypothesis that these acids are mainly produced by bacterial metabolism of undigested proteins. Of the fibers studied, g11ar g ~ resulted in the lowest pH and the highest concentration, amount, and molar ratio of Pr. In addition the increase in fermentability of fibers in vitro by colonic bacteria from rats adapted to a high fiber diet was most marked for guar gum. In light of the suggested metabolic effects of the propionate in the control of cholesterol synthesis and in the stimulation of insulin release (36), its production by the
770
F. BRIGHENTI et al.
colonic fermentation of selected dietary fibers should warrant further on the effect of the colonic fermentation in metabolic disorders.
1
Argenzio RA, Southworth M. absorption in gastrointestinal 228:454-460
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2
Cummings JH. Fermentation in the human large intestine: implication for health. Lancet 1983; i:1206-1209
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and
Zarling EJ, Ruchim MA. Protein origin of the volatile fatty acids isobutyrate and isovalerate in human stool. J Lab Clin Med 1987; 109:566570 4
Jenkins DJA, Leeds AR, Gassul MA, Huston H, Golf DV, Hill MJ. cholesterol lowering properties of g m r and pectin. Clin Sci Mol 1976; 51:8P-gP
The Med
Onderdonk AB, Bartlett JG. 1979. Bacteriological studies of experimental ulcerative colitis. Am J Clin Nutr 1979; 32:258-265 Goy JAE, Eastwood MA, Mitchell WD, Pritchard JL, Smith AN. Fecal characteristics contracted in the irritable bowel syndrome and diverticular disease. Am J Clin Nutr 1976; 29:1480-1486. 7
Lederman M, Van Tassel R, West SEH, Ehrich MF, Wilkins TD. production of hL~an fecal mutagens. Murat Res 1980; 79:115-120
In
vitro
Demigne" C, Remesy C. Stimulation of absorption of volatile fatty acids and mineral in the cecum of rats adapted to a very high fiber diet. J Nutr 1985; 115:53-60 9
McLean-Baird I, Walters RL, Davies PS, Hill MJ, Drasar BS, Southgate DAT. The effect of two dietary fiber supplements on gastrointestinal transit, stool weight, and frequency, and bacterial flora, and fecal bile acids in normal subjects. Metabolism 1977; 26:117-124
i0
Savage DC. Effects of food and fibre on the intestinal luminal environment. In: Fibre in Human and Animal Nutrition. Wallace G & Bell L eds. 1982; pp. 125-134. Wellington: Royal Society of New Zealand.
11
Rasmussen HS, Holtug K, Andersen JR, Krag E, Mortensen PB. The influence of ispaghula husk and lactulose on the in vivo and ~n vitro production capacity of short-chain fatty acids in humans. Scand J Gastroenterol 1987; 22:406-410
12
function and Fleming SE, Marthinsen D., Kuhnlein H. Colonic 1983; 113:2535fermentation in men consuming high fiber diets. J Nutr 2544
13
Wyatt GM, Bayliss CE, Holcroft JD. A change in human faecal flora in response to inclusion of gum arabic in the diet. Brit J Nutr 1986; 55:261-266
14
Spiller GA, Chernoff MC, Hill RA, Gates JE, Nassar JJ, Shipley EA. Effect of purified cellulose, pectin and a low residue diet on fecal volatile fatty acids, transit time, and fecal weight in humans. Am J Clin Nutr 1980; 33:754-759
FERMENTATION OF DIETARY FIBERS
771
15
Walker ARP, Walker BF, Segal I. Fecal pH value and its modification by dietary means in South Africans black and white schoolchildren. S Afr Med J 1979; 55:495-498
16
Topping DL, lllman RJ, Trimble RP. Volatile fatty acid concentration in rats fed diets containing gum arabic and cellulose separately and as a mixture. Nutr Rep Int 1985; 32:809-814
17
Tomlin J. Which fiber is best for the colon? Scand J Gastroenterol 22 (Suppl.129):lO0-104
18
Holdeman LV, Moore WEC. Anaerobic laboratory manual, 4 zh ed. Virginia Laboratory. Polytechnic Institute ~d State University Anaerobe Blacksburg, VA. 1973. p. 145
19
Caldwell DR, Bryant MP. Medit~ without rt~en fluid for non selective em~eration and isolation of rt~en bacteria. Appl Microbiol 1966; 14: 794801
20
Varel VH, Hashimoto AG, Chen YR. Effect of temperature and retention time on methane production from beef cattle waste. Appl Environ Microbiol 1980; 40:217-222
21
Dankert J, Zijlstra JB, Wolthers BG. Volati]e fatty acids in human perpheral ~id portal blood: quantitative determination by vac~a~ distillation and gas chromatography. Clin Chim Acta 1981; 110:301-307
22
CLnnmings JH, Englyst HN. 1987. Fermentation in the htman large intestine and the available substrates. Am J Clin Nutr 1987; 45:1243-]255
23
Mortensen PB, A~dersen JR, Arffmann S, Krag E. Short-chain fatty acids and the irritable bowel syndrome: the effect of wheat bran. Scand J Gastroenterol 1987; 22:185-192
24
Illman RJ, Topping DL, Trimble RP. Effects of food restriction and starvation-refeeding on volatile fatty acid concentrations in the rat. J Nutr 1986; 116:1694-1700
25
Ruppin H, Bar-Melt S, Soergel KH, Wood CM, Schmitt Jr. MG. Absorption of Short-chain fatty acids by the colon. Gastroenterology 1980; 78:1500-1507
26
Stevens CE, Stettler BK. Factors affecting the transport of volatile fatty acids across rumen epithelium. Am J Phisiol 1966; 210:365-372
27
Imoto S, Namioka S. Volatile fatty acid metabolism in intestine. J Anim Sci 1978; 47:467-478
28
Remesy C, Demigne C. Partition and absorption of volatile fatty acids the alimentary canal of the rat. Ann Rech Vet 1976; 7:39-55
29
Cummings JH. Short chain fatty acids in the human colon. Progress report. Gut 1981; 22:763-779
30
Miller TL, Wolin MJ. Fermentation by saccharolytic intestinal Am J Clin Nutr 1979; 32:164-172
31
Mortensen PB, Holtug K, Rasmussen HS. Short-chain fatty acid production from mono- and disaccharides in a fecal incubation system: implications for colonic fermentation of dietary fiber in humans. J Nutr 1987; 118:321-325
the
pig
1987;
large
in
bacteria.
772
F. BRIGHENTI et al.
32
Thomsen LL, Tasman-Jones C, Lee SP, Roberton AM. Dietary factors in the control of pH and volatile fatty acid production in the rat caecum. Falk Symp 1982; 32:47-53
33
Bown RL, Gibson JA, Sladen GE, Hicks B, Dawson AM. Effects of lactulose and other laxatives on ileal and colonic pH as measured by a radiotelemetry device. Gut 1974; 15:999-1004
34
Demigne C, Remesey C. Urea recycling and ammonia absorption in vivo in the digestive tract of the rat. Ann Biol Anim Biochim Biophys 1979; 19:929-935
35
Slade AP, Wyatt GM, Bayliss CE, Waites WM. Comparison of populations of hi,man faecal bacteria before and after "in vitro" incubation with plant cell wall substrates. J Appl Bacteriol 1987; 62:231-240
36
Anderson JW, Bridges SR. Plant fiber metabolites alter and lipid metabolism. Diabetes 1981; 30 (Supp. l):133A
Accepted for publication March 29, 1989.
hepatic
glucose
INFLUENCE OF LONG-TERM FEEDING OF DIFFERENT PURIFIED DIETARY FIBERS ON THE VOLATILE FATTY ACID (VFA) PROFILE, pH AND FIBER-DEGRADING ACTIVITY OF THE CECAL CONTENTS IN RATS. Furio Brighenti, Giulio Testolin, Enrica Canzi*, Annamaria Ferrari*, Thomas M.S. Wolever **, Salvatore Ciappellano, Marisa Porrini, and Paolo Simonetti Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, division of Nutrition; *division of Microbiology, University of Milan, Milan, Italy and ** Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada
ABSTEAC~ The aim of this study was to investigate the effect of four weeks ingestion of 10% fiber diets (cellulose, lignocellulose, pectin or guar g ~ ) and fiber free diets on volatile fatty acid (VFA) concentration, pH, and ~ fiber-degrading activity o f the cecal contents of female Sprague Dawley rats. The pH was significantly lower (p
intestinal
INTRODUCTION Volatile fatty acids (VFAs), mainly acetate (Ac), propionate (Pr) and butyrate (Bu), are known to be produced in large amounts in the colon of monogastric animals and man by the anaerobic saccharolytic bacteria of the large bowel (I-2). Small amounts of branched-chain VFAs (isobutyrate, isovalerate, isocaproate) have also been found in the fecal fluids, probably produced from the breakdown of the branched-chain amino acids valine, leucine and isoleucine (3). The substrates for the colonic fermentation include complex endogenous polysaccharides, such as mucin and glycoproteins produced by epithelial cells of the gastrointestinal tract, and carbohydrates of exogenous origin escaping digestion in the small intestine, such as amylase-resistent starch, saccharidase-resistent oligosaccharides and some dietary fibers (fermentable DF).
Address Correspondence to:
Furio Brighenti, Dipartimento Scienze Alimentari e Nicrobiol., University of G.Celoria 20188 - Milan, ITALY
761
e Tecnologie Milan, 2, Via
762
F. BRIGHENTI et al.
Recently, the presence and fate of VFAs in the large intestine have been suggested to be related to some of the important clinical effects observed in patients consuming diets rich in dietary fiber. Reduction of serum cholesterol (4), development of ulcerative colitis (5) and irritable bowel syndrome (6), mutagenic activity of bile salts and nitroso compounds (7), and fecal trapping of nitrogen with consequent reduction of serum urea (8) have been hypothesized to be due to an increase in the luminal VFAs, or to an increase of the colonic microbial activity, of which the VFA concentration is a marker. Thus, efforts have been made to clarify the relationships between fermentable DF, VFA production, and the colonic environment in general. However, the results have been contradictory. Some studies have failed to demonstrate any direct effect of the SOLace of fiber on fecal microflora (9-10), concentration of VFAs (11), or pH (12), while other studies have reported significant relationships (13-17). The difference in fermentability of the sources of fiber used can generally explain the variability of these results. However, differences in the experimental models of intestinal fermentation of fiber can also play a role, particularly: i) time of adaptation of the colonic microflora to the fibers fed, ii) type of specimen, (i.e. feces or cecal content) since VFAs absorption tends to reduce the concentration gradient between the cecum and the rectum and iii) composition of the experimental diets, especially the starch intake. The purpose of this study was to investigate the effect of chronic ingestion of cellulose, lignocellulose, pectin, guar gum and fiber free diets on the cecal volatile fatty acids (VFAs) and I>}{ in rats, as well as to assess the fiber-degrading activities of the cecal contents by means of an in vitro fermentation system. MATERIALS AND METHODS Female Sprague-Dawley rats (Nossan, Milan, ItalY) 80 days old, average weight 89• g, were divided into five groups of 8 animals. Each group was housed in two cages of four rats in a temperature controlled room kept on a 12h light-dark cycle. The rats were trained to meal-eat over a 4-h period from 9 a.m. to 1 p.m.; each cage was provided with 80 g of basal diet, AIN 76; water was provided ad libitum. The weight of diet consumed daily was taken as the difference between the weight provided and the residue. After a 15-day period of training, four groups were provided with experimental diets containing 18.0% albumin, 4.5% corn oil, 56.2% sucrose, 6.3% starch, 10% fiber, 4.1% AIN 78 minerals mix, 0.9% AIN 76 vitamins mix (table I). The dietary fibers (DF) used were: guar gum (diet G) (Sigma, St Louis, MO, USA), citrus pectin (diet P) (Sigma), cellulose solka-flock (diet C) (Laboratori Piccioni, Milan, Italy), lignocellulose from hazel-nut shells (diet H) (Also, Zelbio, Italy). The composition of the hazel-nut fiber was: total dietary fiber 98%; lignin 24%; cellulose 68%; and hemicellulose 6% (98% xylose). The fifth group of rats was fed a fiber free diet (diet FF) with an identical relative composition of macronutrients, vitamins and minerals but with no fiber. The proximate analysis of the diets after pelletting is presented in table 2. After consL~ing the diets for 4 weeks, 20-h fasting rats were anaesthetized and killed by bleeding, the whole intestine was excised and divided into different s e c t i o n s b y d o u b l e ligature with surgical suture. The cecum, assumed to be the first 2 cm of the large intestine measured from the ileo-cecal valve, was resected and immediately placed into a sterile bag flushed with CO~. The ceca from five rats of each group were then weighed and opened in the fundus with a 5mm cut. A pHmicroprobe (Radiometer, Copenhagen, DM) was inserted twice, at different depths, into the cecum and the average of the pH readings recorded. From 0.5 to I g of the cecal contents was weighed and diluted 1:2 with ice-cold saline into 5 ml preweighed plastic vials.
FERMENTATION OF DIETARY FIBERS TABLE 1
TABLE 2
Diet Composition (g/lO0 g of diet)
Proximate Analysis (g/lO0 g of diet)
~t6AL IOZ FIBI~R FIBII~I~|E (AIE 76) DIET DI~
Protein ~ Fat Carbohydrate | Fiber ~inerale Vitains Integrators $
Y,O.O 5.0 65.0 5.0 3.5 1.0 0.5
16.0 4.5 62.5 10.0 4.1 0.9 0.0
763
20.0 5.0 70.0 0.0 4.0 1.0 0.0
DIITG DIITP DIETH DIITC DIgTFF Moisture Protein rat Av. Curb. Fikr Ash
12.7 16.0 3.0 54.5 10.0 3.8
11.3 16.4 2.4 56.4 9.9 3.6
12.5 14.7 2.7 56.7 10.0 3.4
13.7 15.1 2.6 55.1 9.9 3.4
10.4 18.1 3.5 63.9 0.1 4.0
* Albtmin in the experimental diets, casein in AII 76; S sucrose 90Z corn starch IOZ ; $ ebo]ine, zetionine Vitains and zineraIe as in AI| 76 The remaining cecal content was washed out with saline, the cecum was gently dried by blotting on a filter paper and then weighed again. The whole wet weight of the cecal content wa~ calculated from the difference between the weight of the full and empty cecum. Five hundred ~l of suspension were used for VFA analysis as described below; vials were then freeze-drled after addition of 500 ~i of 110 mM NaOH, weighed and the resulting weights used to calculate the dry weight of cecal content. In vitro bacterial degradation. The cecal content from the remaining three rats of each group were used for the in vitro studies of the DF degradation. For these experiments all the media used were pre-reduced, and all the manipulations were done into an anaerobic cabinet (Anaerobic Glove Box model 1029, Forma Scientific, Marietta, OH, USA) filled with a mixture of N2:H2:C02 85:10:5 % V/V under strict anaerobic conditions (02<20 ppm). The samples of cecal contents (0.5-1 g) were diluted with 70 ml of Dilution Blank (18). Ten ml of suspension were added to i00 ml of Medium i0 broth (19) prepared without VFA mix and with 0.3 % W/V of DF as the only source of carbohydrates. The fiber used in each culture was the same as that in the diet of the rat from which the cecal contents were obtained. In addition, the cecal contents of the rats on diet FF were added to media containing all four different DFs. The residual DF in the media was estimated on I0 ml of culture suspension after O, 6, 24 and 48 h for the guar and pectin groups, and after O, 48 and 72 h for the lignocellulose and cellulose groups. The results were expressed as the percent of DF undigested. Chromatography The analysis of the VFAs was performed b y g a s liquid chromatography (GLC) on the cecal samples prepared according to Varel et al (20). In brief, samples were spiked with internal standard, centrifuged 45.000 x g for 20", acidified to pH 2.0 using HsP04, and directly injected into the chromatograph. The chromatographic apparatus included a chromatograph model GC 6000 MEGA Series 2 fitted with a FID detector, and a MEGA Series SP 4290 integrator (Carlo Erba, Milan, Italy). A coiled glass column (6" lenght X 1/4" I.D.) packed with 15% SP - 1220/1% H3P04 on 100/200 mesh Chromosorb (Supelco, Bellefonte, PA, USA), was used. The gas flows were H2 and N2 30 ml/min and air 340 ml/min. The carrier gas was saturated with formic acid to minimize the tailing and ghosting peaks
764
F. BRIGHENTI et al.
effect that have been reported to occur in the GLC analysis of VFAs (21). The analysis was performed isothermally at 165 ~ with injector and detector temperatures of 185 and 195 ~ respectively. Before use the column was conditioned 72 h at 190 ~ under carrier gas flow. The quantification of the peaks was made by the internal standard procedure, using heptanoic acid (Merck, Darmstadt, FRG) as the internal standard and a calibration mixture of CI-C6 VFAs I0 mM each (Supelco Inc., Bellefonte, PENN.). The method is routinely used in our Department for the detection of VFAs in culture media and fecal specimens, and achieves a mean recovery of C2-C6 VFAs of 98% . Statistics The comparisons between results were conducted by analysis of variance with the fibers as determinant, using the Fisher's F test. When a level of significance p<=O.05 was found, the means were compared by the Tukey's Studentized range test. Regression analyses were performed and correlation coefficents were determined when appropriate. RESULTS After 4 weeks of experimental feeding the weight gain of the rats was not significantly different among groups, ranging from 81• for the P group to 90• for the C group. The feed-efficency of the diets (body weight increment / Kcalories consumed x I00) was also equivalent, ranging from 5.7 for the P and G groups to 5.1 for the FF group (table 3). The total wet weight of the cecal contents was significantly higher in the P group compared with the FF and H groups. This difference was principally related to the water content, since the dry weights did not differ significantly among groups (table 4). There was significant difference in the cecal pH between diets (table 4). Diets G and P significantly increased the acidity of the luminal contents by approximately one pH unit, with respect to the FF, H and C diets. TABLE 3 Body Weight Increment, Food Consumption and Feed Efficency of Diets ~OgP
Body wei~ht (g)
weight s increment (l)
food Z feed consuaed efficeney (g) I/[cal (xlO0)
Guar gm~
aean S~f
188.3 7.3
83.3 5.2
476
5.7 0.3
Pectin
aean Sell
191.7 7.3
81.7 5.3
462
5.7 0.3
Hazel nut
mean 8~
199.2 7.0
89.2 5.0
532
5.4 0.3
Cellulose
mean 8m
204.6 4.9
90.4 2.5
532
5.6 0.2
199.1 7.8
90.2 5.5
504
5.1 0.3
Fiber free u u Sire
S calculated fron the beginning of feedi~ eith the experilental diets.
| =8
FERMENTATION OF DIETARY FIBERS
765
TABLE 4
Wet and Dry Weights and pH of the Cecal Contents OI~P
Net Nei~ht (g} of DU HeiSt (g) of cecal contents cecal contents 0.69 0.10 25.50
6.09 ~C 0.35 11.54
2.90 ABb 0.45 31.00
0.72 a 0.15 31.00
6.38 DE a 0.27 8.59
8uel nut lean $~ CVl
1.71 A 0.24 32.06
0.60 0.09 20.38
7.23 A 0.16 5.04
Cellulose lean 6m C~
1.79 0.21 23.24
0.62 0.09 18.40
7.53 BD 0.08 1.48
Xiber free mean Sm CVZ
1.45 B ac 0.19 26.17
0.50 a 0.09 22.00
7.55 CX 0.06 1.48
~ar ~m
Pectin
6E C~
2.48 0.33 26.25
mean S~ CVZ
lean
a
I~
he
a
Groups in colasnmarhedwith the ease superscript are eilnifieantl7
different (p(O.05) to the Yinher's (nail letter) and the 1~lkey's (capital letter) analysis of the variance. |: 5; Hazel nut |: 8. TABLE 5
Concentration (~mol/g W.W.) of Volatile Fatty Acids in Cecal Contents GI~P
Acetic ~opionic Isobotyrtc Butyric inovaleric Valeric ineeaproic Caproic Total V~aa
Gu~ ~m
~n SBi CVl
46.34 9.35 40.38
27.31 nhe 1.36 6.97 0.37 51.03 54.50
7.65 1.79 46.76
2.07 0.51 49.28
2.21 0.37 33.70
0.44 0.20 89.57
0.52 a 0.20 76.80
87.89 17.09 38.89
Pectin
lean 69 C~
54.78 9.65 35.24
21.02 2.93 27.88
1.49 0.22 29.11
11.36 0.94 16.60
1.87 0.27 28.00
2.12 0.25 23.79
0.60 0.20 65.89
0.55 0.20 72.63
93.80 11.93 25.45
Hazel nut mean 36.24 SI~ 8.50 CVI 53.47
14.90 a 3.03 45.23
1.90 0.58 67.64
11.89 3.91 73.54
2.40 0.64 59.96
2.43 0.74 68.11
1.06 0.65 ]36.97
1.34 0.70 116.39
72.56 14.24 43.88
Cellulose Man 6~ Ct~
S0.71 5.45 35.51
12.60 b 2.88 45.70
2.33 0.56 47.72
7.14 1.33 37.30
2.90 0.75 51.55
2.78 0.77 55.15
1.78 0.62 69.06
2.26 a 0.94 83.33
02.50 12.25 39.20
Fiber free u u Sin CVl
49.14 7.09 28.80
15.23 c 1.91 25.05
1.89 0.14 14.91
8.70 0.12 21.49
2.60 0.29 22.56
2.56 0.33 26.65
0.75 0.24 63.34
0.78 0.37 95.11
79.65 9.81 24.64
~ou~ in cotton mk~l with the m e superscript are ntpificastly different (1~0.05) to the Yinher's analysis of the variance. |= 5; lane] nut I.--6
766
F. BRIGHENTI et al.
There were no significant differences of VFA concentration in cecal contents among the different diets except that Pr was significantly higher (p
Acetic
I~0pionic Butyric
Guar g',m mean 119.71 SR 34.46 CV% 57.58 Pectin
72.30 A ab 20.34 24.60 6.21 68.05 60.97
Iso-VFAs Hin0rVFAs Total VBs 9.78 3.23 66.01
7.03 1.89 53.81
229.16 64.48 56.28
mean 162.13A ab 82.67 cd 33.51 sb 11.19 S{H 44.43 15.29 6.31 2.22 CVt 54.80 48.79 37.68 39.68
8.15 2.35 57.75
277,64 abc 64.24 46.28
9azel nut mean S~ CV%
62.90 a 13.80 49.06
27.32 a 7.38 60.42
23.51 9.75 92.80
9.86 3.97 89.91
6.77 2.80 92.67
130.35 u 32.07 54.77
Cellulose teas 8~ CV%
51.29 A 6.14 23.95
20.86 A c 4.25 40.80
11.76 s 1.06 18.01
11.08 1.77 31.98
7.74 1.76 45.43
102.71 a 12.27 23.90
Fiberfree seas
72.57
22.45
Ixl 10.10
8.02
5.31
1t8.45 c
S~
15.01
b
4.59
2,43
b
2.01
1.79
24.90
CV~
41.36
40.90
48.15
50.24
67.33
42.04
Groups in column Barked eith the sane superscript are significantly different (p
FERMENTATION OF DIETARY FIBERS
767
TABLE 7 VFA Profile G~P
acetic
Guarg~
(% of total VFA)
prepionic
mean 52.9
betyric
61~ Or%
3.0
29.2 ~IBCD 2.7
II.3
18.5
Pectin
mean SE CVZ
57.2 2.7 9.6
Hazel nut
mean 6E CVl
Cellulose
8.8 a 1.2
iso-u
minor TWAs
26.7
5.5 a 2.0 71.7
3.6 a 1.0
57.9
22.3 a 0.6 5.4
12.7 1.6 24.9
4.6 b 0.9 38.4
3.1 0.8 48.0
51.5 5.3 23.1
20.7 B 0.6 8.3
15.1 ab 7.5 3.2 1.8 47.7 55.1
5.1 1.5 61.6
mean SE CV%
50.1 a 2.4 9.5
19.7 C 2.0 20.2
11.7 0.7 11.4
10.6 ab 1.2 21.2
7.7 ab 1.6 40.4
Fiber free mean Sl~ C~
61.2 a 2.1 6.9
19.2 D 1.2 12.5
8.7 b 0.9 20.4
6.7 0.7 21.8
4.2 0.8 30.7
Groups in column marked sith the same superscript are significantly different p
Cellulose and lignocellulose were only slightly degraded in vitro with about 90-95% remaining after 72 hours. Moreover, the inocula from the cecal slurries of the fiber-free adapted rats showed the same fermentative performance as those from the fiber adapted rats (Fig. I). Fig. 1 In vitro indigestibility of the fibre snbstrates incubated sith cecal slurries frot long-tern fiber fed rats. Close figures represant the inocola frol fiber-edapted rats, open fifuren represent the ioocula f r ~ fiber-free adapted rats. hch point is the average of three aniaalo. - - - - - Gear ~ ; --0-- Pectin; --~-- Lipocellulose; - - i - - Cellulose
100
25 ~
60
~
4o "r
0
I
I
I
,
6
24
48
72
INCUBATION t i m e ( h )
768
F. BRIGHENTI et al.
Pectin and guar gum were degraded to the same extent (20%) when incubated for 48 hours with cecal contents from rats on the fiber-free diet. However, the fermentability of guar gum and pectin increased to 60% and 37% respectively, when incubated with cecal contents of rats adapted to diets containing guar and pectin. DISCUSSION Many factors have been thought to affect the rate of production and the concentration of VFAs in the large intestine of both animals and man: the type and the amount of dietary fiber and starch intake and processing (22), transit time (23), and food restriction (24). In an attempt to isolate the dietary fiber as the only experimental variable we fed the rats identical semisinthetic diets containing 10% purified fiber. The available carbohydrates in the experimental diets consisted mainly of sucrose since a number of studies have shown that 5-10% of dietary starch escapes digestion in the upper intestinal tract and reaches the colon (22). The protein source (albumin) was chosen since its fermentation is known to produce preferentially branched VFAs rather than Ac, Pr and Bu (3). The period of feeding was kept constant and the analyses were made after a period of time (20 h) presumed to be sufficent for the fiber to reach the colon and undergo fermentation. In this experimental model, diets C and H showed approximately the same cecal content and profile of VFAs as diet FF. This suggests that, with C, H, and FF, colonic VFAs were derived from the fermentation of endogenous carbohydrates. The almost complete indigestibility of cellulose and lignocellulose over a 20-h period, demostrated by the in vitro experiment, also supports this hypothesis. In contrast, diets P and G resulted in significantly higher amounts of VFAs than the other diets, although not as high as expected by the rate of fermentation observed in vitro, perhaps due to colonic absorption of VFA. The rate of VFAs absorption from the colon has been found to be directly related to VFAs concentration (25). An indirect relationship between VFA absorption and pH has been also demonstrated in the rumen, and hypothesized to occour in other gastrointestinal tracts (26). Thus, an increased amount of fermentable substrate entering the colon would produce a higher concentration of bacterial acidic metabolites and a lower pH, both resulting in an increased rate of uptake of VFAs and excretion of bicarbonate, until the initial conditions of equilibrium are restabilished. This can explain the close similarity in the molar ratio of fecal VFA that invariably has been found with different diets and ~n various species (27-29). In our study, the profile of the major VFA (Ac, Pr, Bu) in the cecum of the rats on diets C and H was almost exactly the same found by Remesy and Demigne (28) in the cecum of rats fed 10% cellulose. Diet P resulted in a higher but not significant percentage of acetate, with propionate and butyrate percentages falling in the same range of values of diets C and H. Hovewer, a marked increase in the percentage of Pr was observed on guar diet, suggesting that this fiber is possibly more metabolized via the succinate than the acetate pathways (30). This could reflect a difference in the species of bacteria present in the colon of guar-adapted rats, or in metabolism of the monosaccharides (mannose and galactose) components of guar g ~ . Mortensen et el., testing the in vitro VFA production from mono- and disaccharides incubated with human fecal inocula, found that glucuronic and galacturonic acids (components of pectin), but not galactose, increased Pr (31). They did not test mannose. Thus, if the rise in Pr we observed on guar was related to its sugar composition, this would be due to the mannose rather than the galactose. The
relationship
between "colonic
pH
and
VFA
concentration
is
still
FERMENTATION OF DIETARY FIBERS
769
controversial. Thomsen et al. (32) found a significant effect of pectin in lowering the pH of the cecal content of rats fed high fat diets. Topping et al. (16) similarly, found a direct relationship between decrease in cecal pH and the amount of gum arabic fed to rats, either alone or mixed with cellulose. In humans, when the non-absorbed but readily fermented disaccharide lactulose was added to the diet, the pH in the right colon decreased significantly from 6.0 to 4.5, whereas in the left colon and rectum no difference from the placebofed controls was observed, both being in a range of 6.3-6.8 pH (33). However, no mechanism bywhich lactulose p r o ~ e d the observed increase of acidity was demonstrated. Other workers have reported a lack of correlation between fecal and intestinal pH and VFAs concentrations in pigs (I) and humans (12) fed high fiber diets. Ruppin et al. (25) even reported an increase in pH with increasing propionate concentration in the intestinal lumen of humans. From their data, the stability of the colonic pH could be attributed to the accumulation of bicarbonate into the lumen , as result of the nonionic transport of VFA across the mucosa. This study shows that the pH of the cecal contents of rats adapted to a controlled fiber diet depends upon the kind of fiber f e d . The readily fermentable fibers, pectin and guar, produced a significant reduction in the cecal pH whereas the non-fermentable fibers, cellulose and ligno-cellulose, had no effect. Moreover, the effect on pH was significantly correlated to the concentrations of acetate, propionate and tota] VFAs. However, it is likely that the concentration of VFAs is not the only factor determining the lower pH observed in the G and P diet groups. The lowest pH was found in the G group, which had a lower concentration of acetic and total VFAs than the P group. In addition the cecal pH correlated better with the Pr than the Ac concentration, although Pr accounted for only 19-25% of total VFA compared to 50-61% for Ac. Therefore, we hypothesize that the reduction in colonic pH is at least partly due to an increase in the utilization of bases present in the lumen, (e.g. ammonia and urea) for bacterial protein synthesis. This has been demostrated by some authors as consequence of fermentable fiber ingestion (34). The fermentative pattern found for Guar gum and Pectin after in vitrQ incubation with cecal inocula from fiber-adapted or fiber-free rats is consistent both with the hypothesis of inducibility of the polysaccharidedegrading enzymes from the intestinal bacteria or with a change of the speciesprofile of the microflora itself. Many studies have shown no changes of the fecal flora in humans after chronic ingestion of fermentable fibers (9,10). However, Slade et al. recently found a reproducible modification of the bacterial profile of fecal bacteria after in vitro incL~ation with purified fibers (35).
CmNCUUSIONS The long term feeding of 10% readily fermentable dietary fibers (guar gum and pectin) had a significant influence on the colonic environment in rats. These fibers lead to an increase in the total amoLmt of VFAs in cecal contents, reflecting an increased metabolic activity of the intestinal bacteria. The pH was significantly lowered in presence of fermentable substrate, partly due to the higher concentration of acetic and propionic acids in the cecal fluid. The amounts of the minor VFAs (iso-acids, valeric and caproic acids.) were influenced neither by the presence nor by the type of fiber, supporting the hypothesis that these acids are mainly produced by bacterial metabolism of undigested proteins. Of the fibers studied, g11ar g ~ resulted in the lowest pH and the highest concentration, amount, and molar ratio of Pr. In addition the increase in fermentability of fibers in vitro by colonic bacteria from rats adapted to a high fiber diet was most marked for guar gum. In light of the suggested metabolic effects of the propionate in the control of cholesterol synthesis and in the stimulation of insulin release (36), its production by the
770
F. BRIGHENTI et al.
colonic fermentation of selected dietary fibers should warrant further on the effect of the colonic fermentation in metabolic disorders.
1
Argenzio RA, Southworth M. absorption in gastrointestinal 228:454-460
Sites of organic tract of the pig.
2
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Accepted for publication March 29, 1989.
hepatic
glucose