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Effect of liquid diets with or without partially hydrolyzed guar gum on intestinal microbial flora and function of rats
Nutrition Research, Vol. 15, No. 4. pp. 527-536, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0271.5317/95 $9.50 + .OO
Pergamon
EFFECT OF LIQUID DIETS WITH OR WITHOUT PARTIALLY HYDROLYZED GUAR GUM ON INTESTINAL MICROBIAL FLORA AND FUNCTION OF RATS
Hidehisa Takahashi, MScf *, Shigehiro Akachi, MSc2, Yoshihiro Ueda, MScf , Shigemitsu Akachi, PhD1, Mujo Kim, PhD1, Kazuyuki Hirano, PhD3 and Takehiko Yamamoto, PhDl 1Central Research Laboratories, Taiyo Kagaku Co. Ltd., Yokkaichi, Mie 510, Japan. 2Kishu Livestock Hygiene Service Center, Kumano, Mie 519-43, Japan 3Gifu College of Pharmacy Gifu, Gifu 502, Japan.
ABSTRACT
The effect of liquid diets with or without partially hydrolyzed guar gum (a water-soluble dietary fiber, PHGG) on intestinal function and microflora of rats was investigated. Male Wistar rats, allocated into five groups’ standard rats chow (MF) ad libitum, low-residue diets (LRD) and elemental diet (ED) with or without 1.5% (W/V) PHGG at the dose of 60-70 kcal dally for 2 weeks. The atrophy of terminal ileum villi was detected in LRD and ED groups. Supplementation with PHGG, however, improved the observed atrophy of terminal ileum(p<0.05). The specific activity of diamine oxidase and alkaline phosphatase in mucosal scrapings was significantly lower in the lower ileum of rats in LRD and ED groups than those in MF group. Those activities of rats supplemented with PHGG, however, significantly increased. The cell number of Bifidobacterium and Lactobacillus per one gram cecal content remarkably decreased in LRD or ED groups, but there was no change in those of rats in LRD+ and ED+PHGG groups compared with MF group. The results of this study suggest that the addition of PHGG to liquid diet improves the gastrointestinal tolerance and bowel control in long-term enterally fed patients. KEY WORDS: Partially hydrolyzed height, Mucosal enzyme.
guar gum, Intestinal
microflora,
Villous
Hidehisa Takahashi, Central *Correspondence should be addressed to: Laboratories, Taiyo Kagaku Ltd., l-3 Takaramachi, Yokkaichi, Mie 510, Japan.
527
Research
H. TAKAHASHI et al.
528
Enteral formula diets are used in pre- and post-operative bowel preparations to provide complete nutrition orally and in treatment of some diseases such as inflammatory bowel disease and irritable bowel syndrome. Constipation and diarrhea are potentially serious problems experienced in chronic care patients who are fed exclusively on liquid formula diets. Several studies have indicated that supplementation of liquid diets with fiber alleviates constipation and diarrhea. Dietary fiber has thus been reported to regulate fecal transit time thereby reducing both constrpation and diarrhea (1). The regulatory effects of fiber containing diets has been ascribed to as; [l] increases transit time in patients experiencing diarrhea to the optimal range by preventing the bacterial overgrowth in the small intestine (2); and [2] shortening transit time in patients experiencing constipation by reducing the extent of water absorption into the gut (3). Several other researchers have speculated that diarrhea caused by liquid formula diet is often due to reduction in the absorptive capacity and area associated with the loss of brush border enzymes and mucosal cells (4,5) It has been reported that the addition of fiber (soluble:insoluble=37:63) to liquid diets improves severe atrophy of villi and the decrease of diamrne oxidase activity in mucosa. Determination of diamine oxidase activity was employed to monitor the structural and functional change in the gut (6). The purpose of this study was to examine the effects of two liquid diets containing partially hydrolyzed guar gum (PHGG) on the structural and functional changes in small intestine and on cecal microflora. Partially hydrolyzed guar gum, a water-soluble dietary fiber, prepared by partial hydrolysis of guar gum with O-1,4Wells endomannanase, show much less viscosity (111,000) than original guar gum (7). et al. have reported that a liquid diet supplemented with PHGG decreased the incidence of bacterial translocation in lipopolysaccharide-treated mice (8).
MATERIALS Animals
AND METHODS
and Diets
A total of 40 male weanling (5-week-old) Wistar rats (SPF strain) were They were housed in wire-mesh purchased from Japan SLC Inc., Shizuoka, Japan. cages in a constant-temperature room (24°C) with a 12 hr (06:00-18:00) light cycle. All After 7 days feeding, the rats were initially given a standard control diet (MF) ad libitum rats were weighed and divided into five groups with approximately equal body weight (130.2k2.3). Rats in MF group were fed conventional rat chow (MF, Oriental Yeast Co. Ltd.. Tokyo); rats in LRD group were fed low-residue diet (Ensure Liquid, Dainabot Co. Ltd., Osaka) which consisted of 3.52 g protein (sodium caseinate, sodium calcium caseinate and soy bean protein), 3.52 g lipid (corn oil, soy bean phospholipid), 13.72 g carbohydrate (dextrin and sucrose), vitamins and minerals in 100 ml; rats in ED group were fed an elemental diet (Elental, Roussel Morishita Co. Ltd., Osaka) which consisted of 4.7 g amino acids, 0.16 g lipid (soy bean oil), 21.2 g carbohydrate (dextrin), vitamins and minerals in 100 ml. Rats in LRD+PHGG and ED+PHGG group were fed LRD and ED diets supplemented with 1.5 % (w/v) PHGG. PHGG used in the present study was a commercial preparatron named “Sunfiber” (Taiyo Kagaku Co. Ltd., Mie). The average molecular weight of PHGG was about 20 KDa which was estimated by HPLC technique
DIETS WITH HYDROLYZED
GUAR GUM
529
(G3dOOPW column, Tosoh Co. Ltd., Tokyo Japan) using pullulan as a marker (Shordex standard P-82, Showa Denko Co.. Tokyo) (7). Experimental
procedure
At the end of the experiment, all rats were sacrificed under anesthesia with diethyl ether. Blood was withdrawn by abdominal aortic puncture, and plasma was separated In a refrigerated centrifuge. The small intestine was removed from pylous to the ileocecal junction. Intestinal length was measured in ice-cold 0.9 % saline The intestine was opened longitudinally and rinsed with ice-cold 0.9 % saline and weighed. Mucosal scraping of proximal Ileum (7-17 cm from ileocecal junction) was homogenized in 10 ml of phosphate buffer (0.1 M, pH7.2) followed by centrifuging at 10,000 xg for 20 min An aliquot of the supernatant was used for enzyme and protein assays. The terminal Ileum (5-7 cm from ileocecal junction) was fixed in IO % formalin and processed in the usual manner for light microscopy. Height of mucosal villi was determined on a tissue cross section, stained with hematoxylin and eosin, under an ocular micrometer. Villous height was measured in five different specimen from each rat. Activities of intestinal and plasma diamine oxidase (DAO) were assayed according to Hosoda et al. (6) using cadaverine (Wako Pure Chemical Co. Osaka) as substrate. One unit of DA0 activity is defined as the amount of enzyme that will produce 1 pmol Hz02 per min. Alkaline phosphatase (ALP) was measured according to Kind and King method (9) usrng disodium phenyl phosphate as substrate. One unit of ALP activity is defined as the amount of enzyme that will produce 1 pmol p-nitrophenol per min. Maltase activrty was assayed as described in previous report (10) and protein was measured by the method of Lowry eT al (11). The cecal contents were collected under aseptic condition. One portion of the sample was bacteriologically analyzed and the other used to measure pH. The pH was directly measured by inserting a glass electrode (Horiba, Tokyo) into cecal contents. Bacteriological analysis was carried out according to the method by Mitsuoka et al. (12). The samples were diluted by IO-fold steps with phosphate buffer (pH7.2) containing 0.05?/, L-cysteine HCI for counting the bacterial cells. The media used were BL, DHL, BS (Erken Chemical Co.. Tokyo), EG, ES (Nissui Seiyaku Co., Tokyo), Neomycin Nagler agars (CW) NBGT, LBS (BBL microbiology Systems, Cockeysville, MD), TATAC, PEES (Difco Laboratories, Detroit, Ml), and trypticase soy agar (TS) supplemented with 5% horse blood Anaerobic cultivation was performed in a steel jar under CO2 atmosphere at 37°C. Bacteria grown were broadly classified, and enumerated. Statistical
Procedure Statistical analysis was done using Student’s test for unpaired data.
RESULTS The intestinal weight and length of rats at sacrifice expressed per 1OOg body weight are shown in Table 1. The weight of small- and large intestine of ED and ED+PHGG group were slightly lower than those of MF group. Cecal weight of rats in LRD+ and ED+PHGG groups significantly increased compared to MF, LRD and ED groups. No difference was observed for the length of small intestine. Large intestinal length of rats in ED and ED+PHGG groups, however, was shorter than that of LRD supplemented groups. Cecal content of the rats in LRD+ and ED+PHGG groups was
H. TAKAHASHI et al.
530
significantly greater than that of LRD and ED groups. Cecal pH of LRD+ and ED+PHGG groups was significantly lower than those of LRD ED and MF groups.
TABLE 1 Body weight gain, intestinal length and organ weight of rats fed liquid diets with or without partially hydrolyzed guar gum. Diets MF’ Body weight gain 51.6+1 .!Ya (g/2weeks) Organ weight (g/lOOg b.w.) Small intestine 2.88kO.07ab Cecum 0.61+0.03a Large intestine 0.45+0.01a Organ length (cm/lOOg b.w ) Small intestine 39.3kO.7 Large intestine 5.0~0. I ab Cecal content (g) 1.99+0.15a Cecal pH 7.11+_0.lOa
LRD*
LRD+PHGGs
ED4
ED+PHGG
30.2+2.7b
30.8+2.3b
39.3&2.1C
42.6+-l .2’
3.08+0.09a 0 56+0.02ab 0.33+0.01b
3.05+0.07a
2.73ko.o5b
2.80-cO.06b
o.92ao51”
0.48+0.04c 0.3O+O.Olc
0.32*0.01 bc
41.9k1.5 5.1*0.1a 1.60&O. 16a 6.75rc_O.O3a
43.7&l .6 5.2+0 2a 7.1 so.9ob 6.09s-0.06b
0.35+0.02b
39 3&l .8 4.620.2bc 0.95*0.06c
6.80*O.lOa
o.99ko.oS’
39.5kO.8 4.8&0.1c 7.74kO.31 b 5.16*0.03c
Means + SEM (n=5) not sharing a common superscript letter within the same row were significantly different at p
Photographs of ileum villi of rats fed liquid diets with or without partially hydrolyzed guar gum are shown in Fig 1. A slight atrophy and shedding of the mucosal cells were observed for the rats of LRD and ED groups. These changes were moderated The villous height of MF, LRD+PHGG and ED+PHGG in LRD+ and ED+PHGG groups. groups were 266.Ok4.5, 259.9k3.6 and 256.lk3.4 pm, respectively, with no significant difference among the three groups. In contrast, the height of rats in LRD group was 228.6k3.8 pm and that of in ED group was 224.2k2.7 pm showing a significant difference from above three groups. A striking reduction in the specific activities of both diamine oxidase (DAO) and alkaline phosphatase (ALP) was observed in the lower ileum of the rats in LRD and ED groups (Table 2). The activities of which enzymes in LRD+ and ED+PHGG groups, remained almost unchanged. Mucosal maltase activity in LRD group was however, significantly lower than that of other experimental groups. Serum DA0 activity of rats in LRD and ED groups was lower than MF, but the enzyme activity in PHGG-contarning A similar trend was observed LRD and ED groups was similar to that in MF group. Serum ALP activities in LRD and between serum and mucosal DA0 activities. LRD+PHGG groups were slightly higher than those of other three groups.
DIETS WITH HYDROLYZED
MF’ 266.0+4.5a
GUAR GUM
531
LRD*
LRD+PHGGs
ED4
ED+PHGG
228.6+3.8b
259.9+3.6a
224.222.7b
256.1*3.4a
FIG. 1 Photographs of ileum vi/Ii of rats fed liquid diets with or without partially hydrolyzed guar gum. The value under each photograph represents the height of viili (pm). Means + SEM (n=8) not sharing a common superscript letter within the same row were significantly different at p
TABLE 2 Diamine oxidase (DAO), alkaline phosphatase (ALP), and Maltase activities of rats fed liquid diets with or without partially hydrolyzed guar gum MF’
LRD*
LRD+PHGGs
ED4
ED+PHGG
U/mg protein M ucosa DA0 ALP Maltase Serum DA0 ALP
0.28*0.03a 0.64*0.13a 0.88&O. 1Oa
0.15+0.02b 0.21+0.02b 0.62+0.05b
0.24+-0.02a
1.05+0.12a
0.59k0.05b 4.82+o.loab
4.72+0.06a
o.l4+o,olb o.35+o.o4b
0.24+0.03a
o.9320.14a
o.92+o.f3ab
o.67+o.o7a”
0.75+0.07ab
0.97-co.09a
0.39+0.04c
4.95-co.o6b
4.79k0.03a
0.96+0.14a 4.83&0.07ab
o.9om4a
Means f SEM (n=8) not sharing a common superscript letter within the same row were significantly different at ~~0.05. 1 MF: conventional rat chow. * LRD: low-residue diet (Ensure liquid). 3 PHGG: partially hydrolyzed guar gum. “ED: elemental diet (Elental).
H. TAKAHASHI et al.
532
10 c‘ 5
8
E
6
8
4
t
(100) (80) (60) (100) (60)
Bifidobacterium
Lactobacillos 12 b
c
bl
12
,
c a
b
10
8
8
6
6
4
4
4
2
2
2
0
0 I I--L (1ClO)(l(
b
b
0
S
b
10
ab
@
bc
Staphylococcus
6
O-
(100)(100)(100)(100)(100)
fnterobacteriaceae
Bacteroidaceae
FIG. 2 Intestinal microflora of the rats fed liquid diets with or without guar gum. Means + SEM (n=5) of log10 bacteria/gram of wet frequency of occurrence (100 x number of content with bacteria/five alphabetical letter was significantly different at ~~0.05. 0; MF group, LRD+PHGG group, 1; ED group, IZl9;ED+PHGG group.
b
c
bc
Total bacteria partially hydrolyzed cecal content, with rats) not sharing an q; Cl; LRD group,
Figure 2 shows the change in cecal microflora of the rats of each group. The cell count of each bacterium per one gram of wet cecal content was expressed as logarithm. The average number of Lactobaci//us srgnificantly decreased in LRD (108.5), specially in ED (104.7) groups compared with those of MF (lO*.a), LRD+ (109.1) and ED+PHGG (I 08.7) groups. The number of 0ifidobacter;um In ED group (105.*) were also signrficantly less than that of the other four groups (10*.2-108.6). As for Staphy/ococcus, the number in cecal contents of rats supplement with PHGG was in a lower level than other experimental groups and their occurrence frequency in LRD+ and ED+PHGG were 60% (3/5). The total count of bacteria (1010.2-1010.5), and those of Ertterobacter\aceae (107.6-l 08 7, and Bacteroidaceae (109.9-l 0104) of liquid diet groups were higher than those of MF group (lC@s,lOs.s and 109.0). However, no significant difference was observed for the cell number of fnterobacteriaceae (10e7I 08.7). Streptococcus (I o 6 3-l 07.‘), Eubacterium (10*.7-l@.*) and Peptococcaceae (1 O*.o-109.1) (data not shown)
DIETS WITH HYDROLYZED GUAR GUM
533
DISCUSSION
It has been documented that diarrhea induced by long-term consumption of a liquid diet was associated with the reduction in absorptive capacity and absorptive area because of the loss of brush border enzymes and mucosal cells (56). The addition of dietary fiber (soluble:insoluble=37:63) to a liquid diet leads to the moderation of the atrophy of mucosa villi and the decrease of enzyme located in mucosa (7). Diamine oxidase (DAO) activity in intestinal mucosa and plasma has been determined as an index of intestinal function (13). In the present experiment, the DA0 activities in intestinal mucosa and serum of rats with fiber-free liquid diets significantly decreased. Those enzyme activities of rats fed on the diets supplemented with PHGG, however, were kept in the same levels as those of MF group. The changes in DA0 activities of mucosa and serum reflected those of the mucosal villi height. This results may be due to the stimulation of the growth of mucosal villi in ileum with PHGG as an indigestible polysaccharide. The activities of other enzymes, such as alkaline phosphatase and maltase located in mucosa also changed similarly to DAO. These data suggested that PHGG in liquid diets helps maintaining the mucosal cell growth and functions. Constipation is also frequently found in chronrc care patients who are fed exclusively on enteral formula or low-residue diets. Several studies indicated that the addition of fiber into the diet alleviates constipation (14,15). Dietary fiber has been reported to regulate fecal transit time by reducing both constipation and diarrhea (1). Fecal transrt time was speculated to be associated with the colonic fermentation (16). Dietary fiber seems to reduce diarrhea by protecting from bacterial overgrowth in the Intestine (17). However, there are few pieces of evrdence about the change of intestinal microflora when liquid diet is administered to laboratory animals or human beings. Our results shows that the administration of LRD or ED decreases the cell number of Lactobacillus and/or Bifidobacterium in cecal content and thus, one rat of ED group lacked Bifrdobacterial cells. On the contrary, PHGG in liquid diets kept the number of It is Lactobacillus and Bifidobacterium in cecal content of rats as same as MF group. and well known that the lactic acid forming bacteria such as Lactobacillus Bifidobacterium exert beneficial effects on our health by increasing defense activity against infection, pathogen inhibition and immunopotentiation (18, 19). Additionally, the admrnistration of Bifidobacterial preparation has been reported to normalize the abnormal microflora and improve pediatric intractable diarrhea (20). PHGG does not directly affect the growth of 5ifidobacterium and Lactobacihs in vitro test (data not shown). Previous studies have revealed that the growth of these bacteria appear to be induced by lowering pH (21,22). On the other hand, the cell number and the occurrence frequency of Staphylococcus in cecal content of the rats with PHGG diets decreased compared with PHGG-free diets. The growth of thts bacteria may be suppressed at lower pH. In our previous investigation, fecal pH was decreased by ingesting PHGG in healthy persons (7). In the present study, the addition of PHGG to liquid diets also resulted in a decrease of pH at cecal content. Based on this results, the growth of cecal Bifidobacterium and Lactobacillus may be due to lowering pH by PHGG metabolites. Our previous study showed that short fatty chain acids (SCFAs) were remarkably increased in cecal content of rats fed with the diet containing PHGG (23). It was revealed that SCFAs in the colon usually facilitate the absorption of Na+ and water (24). In addition, Meier et al. showed that a liquid formula diet supplemented with PHGG prolonged colonic transit time compared to the fiber-free diet (17). This data may elicit that the addition of PHGG to liquid diets reduces diarrhea. Fecal moisture of constipated
534
f-i. TAKAHASHI et al.
women ingested 1 lg of PHGG daily, however, increased, and a reverse correlation between fecal pH and moisture was detected (25). The present Investigation suggests that the water-holding capacity and the adequate growth of several bacteria by PHGG may prevent the formation of hard feces. Otherwise. the stimulation of mucosa in small intestine by PHGG promotes the growth of mucosal cells which result in keeping its structure and enzyme activities unchanged. In addition, the absorption of water by SCFAs resulted from PHGG and formation of feces consisted largely of bacteria mass and PHGG may lead to prolong the transit time. In conclusion, the addition of PHGG to liquid diet can normalize physiological condition of the colon and can lead to moderation of both constipation diarrhea resulting from long-term consumption of a liquid diet.
the and
REFERENCE
1.
Shankardass K. Chuchmach S, Chelswick K, Stefanovich C, Spurr S, Brooks J, Tsai M, Saibil FG. Cohen LB, Edington JD. Bowel function of long-term tube-fed patients consuming formulae with and without dietary fiber. JPEN 1990; 14: 508512.
2.
Eastwood MA. Brydon WG, Tadasse K. Effect of fibre on colon function. In Medical Aspects of Dretary Fibre. Spiller GA, Kay RM (eds). Plenum Press, New York, 1980; 109- 129.
3.
Findlay JM. Smith AN, Mitchell WD, Anderson AJB and Eastwood MA. Effects of unprocessed bran on colon function in normal subjects and in diverticular disease. Lancet 1974: 2: 146-149.
4
Levine GM, Deren JJ, Steiger E and Zinno R Role of oral intake in maintenance gut mass and disaccharide activity. Gastroenterology 1974; 67: 975-982
5.
Johnson LR. Copeland EM, Dudrick SJ, Lichtenberger Structural and hormonal alterations in the gastrointestinal rats. Gastroenterology 1975; 68: 1177-I 183.
6.
Hosoda N, Nishii M, Nakagawa M, Hiramatsu Y, Hioki K and Yamamoto M. Structural and functional alterations in the gut of parenterally or enterally fed rats. J Surg Res 1989; 47: 129-133.
7.
Takahashi H, Yang SI, Hayashi C. Kim M, Yamanaka J and Yamamoto T. Effect Of partially hydrolyzed guar gum on fecal output in human volunteers. Nutr Res 1993; 13: 649-657
a.
Wells CL, Barton RG, Jechorek RP, Gillingam KJ and Cerra FB. Effect of fibre supplementation of liquid diet on cecal bacteria and bacterial translocation in mice. Nutrition 1992; 8: 266-271.
of
LMc and Castro GA. tract of parenterally fed
DIETS WITH HYDROLYZED
GUAR GUM
535
9.
Kind PRN and King EJ. Estimation of plasma phosphatase by determination hydrolysed phenol with amino-antipyrine. J Clin Pathol 1954; 7: 322-326
10.
Sugiura M and Hirano K. A new calorimetric glucose. Clin Chim Acta 1977; 75: 387-391.
11.
Lowry PH, Rosebrough NJ, Farr AL and Randall RJ. Protein measurement Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
12.
Mitsuoka T, Ohno K, Benno Y, Suzuki K and Namba K. The fecal flora of man IV. Communication : Comparison of the Newly developed method with the old conventional method for the analysis of intestinal flora. Hyg I Abt Orig 1976; A234: 219-233.
13.
D’Agostino L. D’Argenio G, Ciacci C, Daniele Diamine oxidase in rat small bowel: Distribution location. Enzyme 1984; 31: 217-220.
14.
Frankenfield DC and Beyer PL. Dietary fiber and bowel patients. J Am Diet Assoc 1991; 91: 590-599.
15.
Hull D, Greco RS and Brooks DL. supplementation. J Am Gerontol Sot
16.
Meier R. Beglinger C, Schneider H, Rowedder A and Gyr K. The effect of a liquid diet with or without soluble fiber supplementation on intestinal transit and cholecystokinin release in volunteers. JPEN 1993; 17: 231-235.
17.
Cummings JH, Southgate DAT, Branch W, Houston H, Jenkins DJA and James WPT. Colonic response to dietary fibre from carrot, cabbage, apple, bran, and guar gum. Lancet 1978; 1: l-9.
18.
Berg RD. Host immune response to antigen of the indigenous intestinal flora. In: KK Rouland. ed. Human Intestinal Microflora in Health and Disease, New York: Academic press, 1983: 101-l 26.
19.
Hentges DJ. Role of the intestinal microflora in host defense against infection. In: KK Rouland, ed. Human Intestinal Microflora in Health and Disease, New York: Academic press, 1983: 311-331.
20.
Hotta M, Sato Y. lwata S, Yamashita N, Sunakawa K, Oikawa T, Tanaka T, Watanabe K. Takayama H, Yajima M, Sekiguchi S, Arai S, Sakurai T and Mutai M. Clinical effects of Biffidobacterium preparation on pediatric intractable diarrhea. Keio J Med 1987; 36: 298-314.
21
Hood SK and Zottola EA. Effect of low pH on the ability of Lactobacj1lu.s acidophilus to survive and adhere to human intestinal cells. J Food Set 1988; 53: 1514-1516.
22.
method for determination
of
of serum
with the
B, Macchia V and Mazzacca G. in different segments and cellular
Alleviation of constipation 1980; 28: 410-414.
function
in tube-fed
in the eldery fibre
Bullen CL. Tearle PV and Willis AT Bifidobacteria in the intestinal tract of infants: an J Med Microbial 1976; 9. 325-333. study
in-viva
536
H. TAKAHASHI et al.
23.
Takahashi H, Yang SI, Kim M and Yamamoto T. Protein and energy utilization of growing rats fed on the diets containing intact or partially hydrolyzed guar gum. Corn Biochem Physiol 1994; 107A: 255260.
24.
Engelhardt WV and Rechkemmer G. Absorption of inorganic irons and short-chain fatty acids In colon of mammals. In: Intestinal transport ed. Baillien MG and Gilles R. Springer-Verlag, 1983; 26-45.
25.
Takahashi H, Wako N, Okubo T. lshihara N, Yamanaka J and Yamamoto T. Influence of partially hydrolyzed guar gum on constipation in women. J Ntur Sci Vitamin01 1994: 40: 251-259.
Accepted for
publication
September 27, 1994.
Pergamon
EFFECT OF LIQUID DIETS WITH OR WITHOUT PARTIALLY HYDROLYZED GUAR GUM ON INTESTINAL MICROBIAL FLORA AND FUNCTION OF RATS
Hidehisa Takahashi, MScf *, Shigehiro Akachi, MSc2, Yoshihiro Ueda, MScf , Shigemitsu Akachi, PhD1, Mujo Kim, PhD1, Kazuyuki Hirano, PhD3 and Takehiko Yamamoto, PhDl 1Central Research Laboratories, Taiyo Kagaku Co. Ltd., Yokkaichi, Mie 510, Japan. 2Kishu Livestock Hygiene Service Center, Kumano, Mie 519-43, Japan 3Gifu College of Pharmacy Gifu, Gifu 502, Japan.
ABSTRACT
The effect of liquid diets with or without partially hydrolyzed guar gum (a water-soluble dietary fiber, PHGG) on intestinal function and microflora of rats was investigated. Male Wistar rats, allocated into five groups’ standard rats chow (MF) ad libitum, low-residue diets (LRD) and elemental diet (ED) with or without 1.5% (W/V) PHGG at the dose of 60-70 kcal dally for 2 weeks. The atrophy of terminal ileum villi was detected in LRD and ED groups. Supplementation with PHGG, however, improved the observed atrophy of terminal ileum(p<0.05). The specific activity of diamine oxidase and alkaline phosphatase in mucosal scrapings was significantly lower in the lower ileum of rats in LRD and ED groups than those in MF group. Those activities of rats supplemented with PHGG, however, significantly increased. The cell number of Bifidobacterium and Lactobacillus per one gram cecal content remarkably decreased in LRD or ED groups, but there was no change in those of rats in LRD+ and ED+PHGG groups compared with MF group. The results of this study suggest that the addition of PHGG to liquid diet improves the gastrointestinal tolerance and bowel control in long-term enterally fed patients. KEY WORDS: Partially hydrolyzed height, Mucosal enzyme.
guar gum, Intestinal
microflora,
Villous
Hidehisa Takahashi, Central *Correspondence should be addressed to: Laboratories, Taiyo Kagaku Ltd., l-3 Takaramachi, Yokkaichi, Mie 510, Japan.
527
Research
H. TAKAHASHI et al.
528
Enteral formula diets are used in pre- and post-operative bowel preparations to provide complete nutrition orally and in treatment of some diseases such as inflammatory bowel disease and irritable bowel syndrome. Constipation and diarrhea are potentially serious problems experienced in chronic care patients who are fed exclusively on liquid formula diets. Several studies have indicated that supplementation of liquid diets with fiber alleviates constipation and diarrhea. Dietary fiber has thus been reported to regulate fecal transit time thereby reducing both constrpation and diarrhea (1). The regulatory effects of fiber containing diets has been ascribed to as; [l] increases transit time in patients experiencing diarrhea to the optimal range by preventing the bacterial overgrowth in the small intestine (2); and [2] shortening transit time in patients experiencing constipation by reducing the extent of water absorption into the gut (3). Several other researchers have speculated that diarrhea caused by liquid formula diet is often due to reduction in the absorptive capacity and area associated with the loss of brush border enzymes and mucosal cells (4,5) It has been reported that the addition of fiber (soluble:insoluble=37:63) to liquid diets improves severe atrophy of villi and the decrease of diamrne oxidase activity in mucosa. Determination of diamine oxidase activity was employed to monitor the structural and functional change in the gut (6). The purpose of this study was to examine the effects of two liquid diets containing partially hydrolyzed guar gum (PHGG) on the structural and functional changes in small intestine and on cecal microflora. Partially hydrolyzed guar gum, a water-soluble dietary fiber, prepared by partial hydrolysis of guar gum with O-1,4Wells endomannanase, show much less viscosity (111,000) than original guar gum (7). et al. have reported that a liquid diet supplemented with PHGG decreased the incidence of bacterial translocation in lipopolysaccharide-treated mice (8).
MATERIALS Animals
AND METHODS
and Diets
A total of 40 male weanling (5-week-old) Wistar rats (SPF strain) were They were housed in wire-mesh purchased from Japan SLC Inc., Shizuoka, Japan. cages in a constant-temperature room (24°C) with a 12 hr (06:00-18:00) light cycle. All After 7 days feeding, the rats were initially given a standard control diet (MF) ad libitum rats were weighed and divided into five groups with approximately equal body weight (130.2k2.3). Rats in MF group were fed conventional rat chow (MF, Oriental Yeast Co. Ltd.. Tokyo); rats in LRD group were fed low-residue diet (Ensure Liquid, Dainabot Co. Ltd., Osaka) which consisted of 3.52 g protein (sodium caseinate, sodium calcium caseinate and soy bean protein), 3.52 g lipid (corn oil, soy bean phospholipid), 13.72 g carbohydrate (dextrin and sucrose), vitamins and minerals in 100 ml; rats in ED group were fed an elemental diet (Elental, Roussel Morishita Co. Ltd., Osaka) which consisted of 4.7 g amino acids, 0.16 g lipid (soy bean oil), 21.2 g carbohydrate (dextrin), vitamins and minerals in 100 ml. Rats in LRD+PHGG and ED+PHGG group were fed LRD and ED diets supplemented with 1.5 % (w/v) PHGG. PHGG used in the present study was a commercial preparatron named “Sunfiber” (Taiyo Kagaku Co. Ltd., Mie). The average molecular weight of PHGG was about 20 KDa which was estimated by HPLC technique
DIETS WITH HYDROLYZED
GUAR GUM
529
(G3dOOPW column, Tosoh Co. Ltd., Tokyo Japan) using pullulan as a marker (Shordex standard P-82, Showa Denko Co.. Tokyo) (7). Experimental
procedure
At the end of the experiment, all rats were sacrificed under anesthesia with diethyl ether. Blood was withdrawn by abdominal aortic puncture, and plasma was separated In a refrigerated centrifuge. The small intestine was removed from pylous to the ileocecal junction. Intestinal length was measured in ice-cold 0.9 % saline The intestine was opened longitudinally and rinsed with ice-cold 0.9 % saline and weighed. Mucosal scraping of proximal Ileum (7-17 cm from ileocecal junction) was homogenized in 10 ml of phosphate buffer (0.1 M, pH7.2) followed by centrifuging at 10,000 xg for 20 min An aliquot of the supernatant was used for enzyme and protein assays. The terminal Ileum (5-7 cm from ileocecal junction) was fixed in IO % formalin and processed in the usual manner for light microscopy. Height of mucosal villi was determined on a tissue cross section, stained with hematoxylin and eosin, under an ocular micrometer. Villous height was measured in five different specimen from each rat. Activities of intestinal and plasma diamine oxidase (DAO) were assayed according to Hosoda et al. (6) using cadaverine (Wako Pure Chemical Co. Osaka) as substrate. One unit of DA0 activity is defined as the amount of enzyme that will produce 1 pmol Hz02 per min. Alkaline phosphatase (ALP) was measured according to Kind and King method (9) usrng disodium phenyl phosphate as substrate. One unit of ALP activity is defined as the amount of enzyme that will produce 1 pmol p-nitrophenol per min. Maltase activrty was assayed as described in previous report (10) and protein was measured by the method of Lowry eT al (11). The cecal contents were collected under aseptic condition. One portion of the sample was bacteriologically analyzed and the other used to measure pH. The pH was directly measured by inserting a glass electrode (Horiba, Tokyo) into cecal contents. Bacteriological analysis was carried out according to the method by Mitsuoka et al. (12). The samples were diluted by IO-fold steps with phosphate buffer (pH7.2) containing 0.05?/, L-cysteine HCI for counting the bacterial cells. The media used were BL, DHL, BS (Erken Chemical Co.. Tokyo), EG, ES (Nissui Seiyaku Co., Tokyo), Neomycin Nagler agars (CW) NBGT, LBS (BBL microbiology Systems, Cockeysville, MD), TATAC, PEES (Difco Laboratories, Detroit, Ml), and trypticase soy agar (TS) supplemented with 5% horse blood Anaerobic cultivation was performed in a steel jar under CO2 atmosphere at 37°C. Bacteria grown were broadly classified, and enumerated. Statistical
Procedure Statistical analysis was done using Student’s test for unpaired data.
RESULTS The intestinal weight and length of rats at sacrifice expressed per 1OOg body weight are shown in Table 1. The weight of small- and large intestine of ED and ED+PHGG group were slightly lower than those of MF group. Cecal weight of rats in LRD+ and ED+PHGG groups significantly increased compared to MF, LRD and ED groups. No difference was observed for the length of small intestine. Large intestinal length of rats in ED and ED+PHGG groups, however, was shorter than that of LRD supplemented groups. Cecal content of the rats in LRD+ and ED+PHGG groups was
H. TAKAHASHI et al.
530
significantly greater than that of LRD and ED groups. Cecal pH of LRD+ and ED+PHGG groups was significantly lower than those of LRD ED and MF groups.
TABLE 1 Body weight gain, intestinal length and organ weight of rats fed liquid diets with or without partially hydrolyzed guar gum. Diets MF’ Body weight gain 51.6+1 .!Ya (g/2weeks) Organ weight (g/lOOg b.w.) Small intestine 2.88kO.07ab Cecum 0.61+0.03a Large intestine 0.45+0.01a Organ length (cm/lOOg b.w ) Small intestine 39.3kO.7 Large intestine 5.0~0. I ab Cecal content (g) 1.99+0.15a Cecal pH 7.11+_0.lOa
LRD*
LRD+PHGGs
ED4
ED+PHGG
30.2+2.7b
30.8+2.3b
39.3&2.1C
42.6+-l .2’
3.08+0.09a 0 56+0.02ab 0.33+0.01b
3.05+0.07a
2.73ko.o5b
2.80-cO.06b
o.92ao51”
0.48+0.04c 0.3O+O.Olc
0.32*0.01 bc
41.9k1.5 5.1*0.1a 1.60&O. 16a 6.75rc_O.O3a
43.7&l .6 5.2+0 2a 7.1 so.9ob 6.09s-0.06b
0.35+0.02b
39 3&l .8 4.620.2bc 0.95*0.06c
6.80*O.lOa
o.99ko.oS’
39.5kO.8 4.8&0.1c 7.74kO.31 b 5.16*0.03c
Means + SEM (n=5) not sharing a common superscript letter within the same row were significantly different at p
Photographs of ileum villi of rats fed liquid diets with or without partially hydrolyzed guar gum are shown in Fig 1. A slight atrophy and shedding of the mucosal cells were observed for the rats of LRD and ED groups. These changes were moderated The villous height of MF, LRD+PHGG and ED+PHGG in LRD+ and ED+PHGG groups. groups were 266.Ok4.5, 259.9k3.6 and 256.lk3.4 pm, respectively, with no significant difference among the three groups. In contrast, the height of rats in LRD group was 228.6k3.8 pm and that of in ED group was 224.2k2.7 pm showing a significant difference from above three groups. A striking reduction in the specific activities of both diamine oxidase (DAO) and alkaline phosphatase (ALP) was observed in the lower ileum of the rats in LRD and ED groups (Table 2). The activities of which enzymes in LRD+ and ED+PHGG groups, remained almost unchanged. Mucosal maltase activity in LRD group was however, significantly lower than that of other experimental groups. Serum DA0 activity of rats in LRD and ED groups was lower than MF, but the enzyme activity in PHGG-contarning A similar trend was observed LRD and ED groups was similar to that in MF group. Serum ALP activities in LRD and between serum and mucosal DA0 activities. LRD+PHGG groups were slightly higher than those of other three groups.
DIETS WITH HYDROLYZED
MF’ 266.0+4.5a
GUAR GUM
531
LRD*
LRD+PHGGs
ED4
ED+PHGG
228.6+3.8b
259.9+3.6a
224.222.7b
256.1*3.4a
FIG. 1 Photographs of ileum vi/Ii of rats fed liquid diets with or without partially hydrolyzed guar gum. The value under each photograph represents the height of viili (pm). Means + SEM (n=8) not sharing a common superscript letter within the same row were significantly different at p
TABLE 2 Diamine oxidase (DAO), alkaline phosphatase (ALP), and Maltase activities of rats fed liquid diets with or without partially hydrolyzed guar gum MF’
LRD*
LRD+PHGGs
ED4
ED+PHGG
U/mg protein M ucosa DA0 ALP Maltase Serum DA0 ALP
0.28*0.03a 0.64*0.13a 0.88&O. 1Oa
0.15+0.02b 0.21+0.02b 0.62+0.05b
0.24+-0.02a
1.05+0.12a
0.59k0.05b 4.82+o.loab
4.72+0.06a
o.l4+o,olb o.35+o.o4b
0.24+0.03a
o.9320.14a
o.92+o.f3ab
o.67+o.o7a”
0.75+0.07ab
0.97-co.09a
0.39+0.04c
4.95-co.o6b
4.79k0.03a
0.96+0.14a 4.83&0.07ab
o.9om4a
Means f SEM (n=8) not sharing a common superscript letter within the same row were significantly different at ~~0.05. 1 MF: conventional rat chow. * LRD: low-residue diet (Ensure liquid). 3 PHGG: partially hydrolyzed guar gum. “ED: elemental diet (Elental).
H. TAKAHASHI et al.
532
10 c‘ 5
8
E
6
8
4
t
(100) (80) (60) (100) (60)
Bifidobacterium
Lactobacillos 12 b
c
bl
12
,
c a
b
10
8
8
6
6
4
4
4
2
2
2
0
0 I I--L (1ClO)(l(
b
b
0
S
b
10
ab
@
bc
Staphylococcus
6
O-
(100)(100)(100)(100)(100)
fnterobacteriaceae
Bacteroidaceae
FIG. 2 Intestinal microflora of the rats fed liquid diets with or without guar gum. Means + SEM (n=5) of log10 bacteria/gram of wet frequency of occurrence (100 x number of content with bacteria/five alphabetical letter was significantly different at ~~0.05. 0; MF group, LRD+PHGG group, 1; ED group, IZl9;ED+PHGG group.
b
c
bc
Total bacteria partially hydrolyzed cecal content, with rats) not sharing an q; Cl; LRD group,
Figure 2 shows the change in cecal microflora of the rats of each group. The cell count of each bacterium per one gram of wet cecal content was expressed as logarithm. The average number of Lactobaci//us srgnificantly decreased in LRD (108.5), specially in ED (104.7) groups compared with those of MF (lO*.a), LRD+ (109.1) and ED+PHGG (I 08.7) groups. The number of 0ifidobacter;um In ED group (105.*) were also signrficantly less than that of the other four groups (10*.2-108.6). As for Staphy/ococcus, the number in cecal contents of rats supplement with PHGG was in a lower level than other experimental groups and their occurrence frequency in LRD+ and ED+PHGG were 60% (3/5). The total count of bacteria (1010.2-1010.5), and those of Ertterobacter\aceae (107.6-l 08 7, and Bacteroidaceae (109.9-l 0104) of liquid diet groups were higher than those of MF group (lC@s,lOs.s and 109.0). However, no significant difference was observed for the cell number of fnterobacteriaceae (10e7I 08.7). Streptococcus (I o 6 3-l 07.‘), Eubacterium (10*.7-l@.*) and Peptococcaceae (1 O*.o-109.1) (data not shown)
DIETS WITH HYDROLYZED GUAR GUM
533
DISCUSSION
It has been documented that diarrhea induced by long-term consumption of a liquid diet was associated with the reduction in absorptive capacity and absorptive area because of the loss of brush border enzymes and mucosal cells (56). The addition of dietary fiber (soluble:insoluble=37:63) to a liquid diet leads to the moderation of the atrophy of mucosa villi and the decrease of enzyme located in mucosa (7). Diamine oxidase (DAO) activity in intestinal mucosa and plasma has been determined as an index of intestinal function (13). In the present experiment, the DA0 activities in intestinal mucosa and serum of rats with fiber-free liquid diets significantly decreased. Those enzyme activities of rats fed on the diets supplemented with PHGG, however, were kept in the same levels as those of MF group. The changes in DA0 activities of mucosa and serum reflected those of the mucosal villi height. This results may be due to the stimulation of the growth of mucosal villi in ileum with PHGG as an indigestible polysaccharide. The activities of other enzymes, such as alkaline phosphatase and maltase located in mucosa also changed similarly to DAO. These data suggested that PHGG in liquid diets helps maintaining the mucosal cell growth and functions. Constipation is also frequently found in chronrc care patients who are fed exclusively on enteral formula or low-residue diets. Several studies indicated that the addition of fiber into the diet alleviates constipation (14,15). Dietary fiber has been reported to regulate fecal transit time by reducing both constipation and diarrhea (1). Fecal transrt time was speculated to be associated with the colonic fermentation (16). Dietary fiber seems to reduce diarrhea by protecting from bacterial overgrowth in the Intestine (17). However, there are few pieces of evrdence about the change of intestinal microflora when liquid diet is administered to laboratory animals or human beings. Our results shows that the administration of LRD or ED decreases the cell number of Lactobacillus and/or Bifidobacterium in cecal content and thus, one rat of ED group lacked Bifrdobacterial cells. On the contrary, PHGG in liquid diets kept the number of It is Lactobacillus and Bifidobacterium in cecal content of rats as same as MF group. and well known that the lactic acid forming bacteria such as Lactobacillus Bifidobacterium exert beneficial effects on our health by increasing defense activity against infection, pathogen inhibition and immunopotentiation (18, 19). Additionally, the admrnistration of Bifidobacterial preparation has been reported to normalize the abnormal microflora and improve pediatric intractable diarrhea (20). PHGG does not directly affect the growth of 5ifidobacterium and Lactobacihs in vitro test (data not shown). Previous studies have revealed that the growth of these bacteria appear to be induced by lowering pH (21,22). On the other hand, the cell number and the occurrence frequency of Staphylococcus in cecal content of the rats with PHGG diets decreased compared with PHGG-free diets. The growth of thts bacteria may be suppressed at lower pH. In our previous investigation, fecal pH was decreased by ingesting PHGG in healthy persons (7). In the present study, the addition of PHGG to liquid diets also resulted in a decrease of pH at cecal content. Based on this results, the growth of cecal Bifidobacterium and Lactobacillus may be due to lowering pH by PHGG metabolites. Our previous study showed that short fatty chain acids (SCFAs) were remarkably increased in cecal content of rats fed with the diet containing PHGG (23). It was revealed that SCFAs in the colon usually facilitate the absorption of Na+ and water (24). In addition, Meier et al. showed that a liquid formula diet supplemented with PHGG prolonged colonic transit time compared to the fiber-free diet (17). This data may elicit that the addition of PHGG to liquid diets reduces diarrhea. Fecal moisture of constipated
534
f-i. TAKAHASHI et al.
women ingested 1 lg of PHGG daily, however, increased, and a reverse correlation between fecal pH and moisture was detected (25). The present Investigation suggests that the water-holding capacity and the adequate growth of several bacteria by PHGG may prevent the formation of hard feces. Otherwise. the stimulation of mucosa in small intestine by PHGG promotes the growth of mucosal cells which result in keeping its structure and enzyme activities unchanged. In addition, the absorption of water by SCFAs resulted from PHGG and formation of feces consisted largely of bacteria mass and PHGG may lead to prolong the transit time. In conclusion, the addition of PHGG to liquid diet can normalize physiological condition of the colon and can lead to moderation of both constipation diarrhea resulting from long-term consumption of a liquid diet.
the and
REFERENCE
1.
Shankardass K. Chuchmach S, Chelswick K, Stefanovich C, Spurr S, Brooks J, Tsai M, Saibil FG. Cohen LB, Edington JD. Bowel function of long-term tube-fed patients consuming formulae with and without dietary fiber. JPEN 1990; 14: 508512.
2.
Eastwood MA. Brydon WG, Tadasse K. Effect of fibre on colon function. In Medical Aspects of Dretary Fibre. Spiller GA, Kay RM (eds). Plenum Press, New York, 1980; 109- 129.
3.
Findlay JM. Smith AN, Mitchell WD, Anderson AJB and Eastwood MA. Effects of unprocessed bran on colon function in normal subjects and in diverticular disease. Lancet 1974: 2: 146-149.
4
Levine GM, Deren JJ, Steiger E and Zinno R Role of oral intake in maintenance gut mass and disaccharide activity. Gastroenterology 1974; 67: 975-982
5.
Johnson LR. Copeland EM, Dudrick SJ, Lichtenberger Structural and hormonal alterations in the gastrointestinal rats. Gastroenterology 1975; 68: 1177-I 183.
6.
Hosoda N, Nishii M, Nakagawa M, Hiramatsu Y, Hioki K and Yamamoto M. Structural and functional alterations in the gut of parenterally or enterally fed rats. J Surg Res 1989; 47: 129-133.
7.
Takahashi H, Yang SI, Hayashi C. Kim M, Yamanaka J and Yamamoto T. Effect Of partially hydrolyzed guar gum on fecal output in human volunteers. Nutr Res 1993; 13: 649-657
a.
Wells CL, Barton RG, Jechorek RP, Gillingam KJ and Cerra FB. Effect of fibre supplementation of liquid diet on cecal bacteria and bacterial translocation in mice. Nutrition 1992; 8: 266-271.
of
LMc and Castro GA. tract of parenterally fed
DIETS WITH HYDROLYZED
GUAR GUM
535
9.
Kind PRN and King EJ. Estimation of plasma phosphatase by determination hydrolysed phenol with amino-antipyrine. J Clin Pathol 1954; 7: 322-326
10.
Sugiura M and Hirano K. A new calorimetric glucose. Clin Chim Acta 1977; 75: 387-391.
11.
Lowry PH, Rosebrough NJ, Farr AL and Randall RJ. Protein measurement Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
12.
Mitsuoka T, Ohno K, Benno Y, Suzuki K and Namba K. The fecal flora of man IV. Communication : Comparison of the Newly developed method with the old conventional method for the analysis of intestinal flora. Hyg I Abt Orig 1976; A234: 219-233.
13.
D’Agostino L. D’Argenio G, Ciacci C, Daniele Diamine oxidase in rat small bowel: Distribution location. Enzyme 1984; 31: 217-220.
14.
Frankenfield DC and Beyer PL. Dietary fiber and bowel patients. J Am Diet Assoc 1991; 91: 590-599.
15.
Hull D, Greco RS and Brooks DL. supplementation. J Am Gerontol Sot
16.
Meier R. Beglinger C, Schneider H, Rowedder A and Gyr K. The effect of a liquid diet with or without soluble fiber supplementation on intestinal transit and cholecystokinin release in volunteers. JPEN 1993; 17: 231-235.
17.
Cummings JH, Southgate DAT, Branch W, Houston H, Jenkins DJA and James WPT. Colonic response to dietary fibre from carrot, cabbage, apple, bran, and guar gum. Lancet 1978; 1: l-9.
18.
Berg RD. Host immune response to antigen of the indigenous intestinal flora. In: KK Rouland. ed. Human Intestinal Microflora in Health and Disease, New York: Academic press, 1983: 101-l 26.
19.
Hentges DJ. Role of the intestinal microflora in host defense against infection. In: KK Rouland, ed. Human Intestinal Microflora in Health and Disease, New York: Academic press, 1983: 311-331.
20.
Hotta M, Sato Y. lwata S, Yamashita N, Sunakawa K, Oikawa T, Tanaka T, Watanabe K. Takayama H, Yajima M, Sekiguchi S, Arai S, Sakurai T and Mutai M. Clinical effects of Biffidobacterium preparation on pediatric intractable diarrhea. Keio J Med 1987; 36: 298-314.
21
Hood SK and Zottola EA. Effect of low pH on the ability of Lactobacj1lu.s acidophilus to survive and adhere to human intestinal cells. J Food Set 1988; 53: 1514-1516.
22.
method for determination
of
of serum
with the
B, Macchia V and Mazzacca G. in different segments and cellular
Alleviation of constipation 1980; 28: 410-414.
function
in tube-fed
in the eldery fibre
Bullen CL. Tearle PV and Willis AT Bifidobacteria in the intestinal tract of infants: an J Med Microbial 1976; 9. 325-333. study
in-viva
536
H. TAKAHASHI et al.
23.
Takahashi H, Yang SI, Kim M and Yamamoto T. Protein and energy utilization of growing rats fed on the diets containing intact or partially hydrolyzed guar gum. Corn Biochem Physiol 1994; 107A: 255260.
24.
Engelhardt WV and Rechkemmer G. Absorption of inorganic irons and short-chain fatty acids In colon of mammals. In: Intestinal transport ed. Baillien MG and Gilles R. Springer-Verlag, 1983; 26-45.
25.
Takahashi H, Wako N, Okubo T. lshihara N, Yamanaka J and Yamamoto T. Influence of partially hydrolyzed guar gum on constipation in women. J Ntur Sci Vitamin01 1994: 40: 251-259.
Accepted for
publication
September 27, 1994.