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Cellobiose is extensively digested in the small intestine by β-galactosidase in rats
Nutrition 24 (2008) 1199 –1204 www.elsevier.com/locate/nut
Basic nutritional investigation
Cellobiose is extensively digested in the small intestine by -galactosidase in rats Tatsuya Morita, Ph.D.a,*, Mayumi Ozawa, M.S.a, Hiroyuki Ito, M.S.b, Sugiyama Kimio, Ph.D.a, and Shuhachi Kiriyama, Ph.D.c a
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan b Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan c Faculty of Nutritional Sciences, University of Shizuoka, Shizuoka, Japan Manuscript received April 8, 2008; accepted June 25, 2008.
Abstract
Objective: The bioavailability of cellobiose (CEB) was investigated with respect to small intestinal digestibility and cecal fermentation in rats. Further, whether small intestinal -galactosidase is responsible for the hydrolysis of CEB was examined. Methods: Ileorectostomized rats were fed diets including 6% CEB or fructo-oligosaccharide with or without 0.1% neomycin in drinking water for 7 d. The fecal excretion of the respective oligosaccharides was determined. In vitro digestion of CEB and lactose was characterized using mucosal enzymes of the small intestine from suckling and adult rats. Cecal fermentation in normal rats fed a control diet or a diet including 3% or 6% CEB for 14 d was examined. Results: The small intestinal digestibility of CEB was 64%, irrespective of the presence of neomycin in drinking water, whereas the digestibility of fructo-oligosaccharide differed significantly between groups administered (26%) or not administered (35%) neomycin. The in vitro digestibility of lactose (62%) and CEB (36%) was three times greater with the enzymes from the suckling rats than with those from the adult rats. Michaelis constant (Km) and maximum velocity (Vmax) values for CEB were 25 and 7 times lower, respectively, than those for lactose. Normal rats fed the 6% CEB diet showed a greater cecal organic acid than those fed the control diet, but no differences were observed between those fed the control and 3% CEB diets. Conclusion: Our results indicate that dietary CEB was extensively digested in the small intestine by -galactosidase in rats, leading to complete digestion of CEB when dietary supplementation was limited. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Cellobiose; Fructo-oligosaccharide; Small intestinal digestibility; -Galactosidase; Ileorectostomized rats; Cecal fermentation
Introduction The physiologic functions of indigestible oligosaccharides have been extensively studied, and it is now well recognized that some of these compounds have beneficial effects on large bowel health through bacterial fermentation. Because no -glucosidase has been reported in the intestinal mucosa, cellobiose (CEB) could be a good candidate for a functional indigestible oligosaccharide. Watanabe [1] and Satouchi et al. [2] reported that in vitro * Corresponding author. Tel./fax: ⫹81-54-238-5132. E-mail address: [email protected] (T. Morita). 0899-9007/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2008.06.029
digestibility of CEB measured using a homogenate of rat intestinal mucosa was 6.3%, and that after oral administration of CEB to rats, the blood glucose concentration showed a gradual increase up to 120 min after administration, implying that CEB was slowly hydrolyzed in the lower part of the small intestine, possibly through bacterial activity [1]. In contrast to the results obtained in rats, Nakamura et al. [3] showed that, when 25 g of CEB was orally administered to human subjects, there were no increases in blood glucose or insulin secretion, whereas the excretion of breath hydrogen was remarkable, indicating that a large part of the administered CEB escaped digestion and fermented in the large bowel. However,
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precise information about the small intestinal digestibility of CEB in experimental animals and humans is scarce. In addition, it remains unclear whether CEB digestion in the small intestine is due to an enzyme on the mucosal membrane or one of bacterial origin. The purpose of the present study was to directly measure the small intestinal digestibility of CEB using ileorectostomized rats whose ileal terminal was directly anastomosed to the rectum. In these rats, the amount of fecal excretion of CEB was expected to correspond to that of undigested CEB coming from the small intestine into the large bowel in normal rats. Glucose and galactose moieties at non-reducing terminals in CEB and lactose (LAC) molecules are epimers at the C4 position, and the chemical structures of these molecules are highly similar. Accordingly, we also examined the in vitro digestibility of CEB with respect to small intestinal -galactosidase (lactase), considering that this enzyme may cleave the -1,4 glucosidic bond. If lactase is responsible for CEB digestion, one might expect that the findings obtained from Japanese subjects [3] could be different from those in Caucasians, many of whom have persistent high lactase activity even as adults [4,5].
Materials and methods Materials Cellobiose was provided by Nippon Paper Chemicals Co. (Tokyo, Japan) and was composed of 96.6% cellobiose (1– 4), 1.9% cello-oligosaccharide, and 1.5% glucose. Fructooligosaccharide (FOS), composed of 44% 1-kestose, 46% nystose, and 10% 1-f--fructofuranosyl nystose, was purchased from Meiji Seika (Tokyo, Japan; Meioligo P). Gentiobiose (1– 6) was provided by Nihon Shokuhin Kako Co. (Shizuoka, Japan). Neomycin sulfate (NM) was purchased from Sigma (St. Louis, MO, USA). Care of animals Wistar rats (purchased from Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were housed in individual stainless steel cages with wire screen bottoms in a room of controlled temperature (23 ⫾ 2°C) and lighting (lights on 0800 –2000 h). For adaptation, rats were fed a control diet for at least 3 d. This diet [6] was formulated from 250 g of casein, 652.25 g of cornstarch, and 50 g of corn oil per kilogram. The remainder of the diet consisted of vitamins and minerals [7]. The rats were then divided into groups on the basis of body weight and allowed free access to the experimental diets and water. Body weight and food intake were recorded every morning before replenishing the diet. The study was approved by the animal use committee of Shizuoka University, and animals were maintained in accordance with the guidelines of Shizuoka University for the care and use of laboratory animals.
In vivo digestibility of CEB (experiment 1) After overnight fasting, 28 rats weighing 90 –115 g were subjected to an ileorectostomy in which the terminal ileum was connected to the rectum according to the method of Lambert [8], with some modifications. To shorten the recovery period, we did not dissect the cecum and colon, but the ileocecal valve was ligatured (closed) and then the colonic terminal was anastomosed to the stoma in the abdominal wall to allow the cecal and colonic contents to be excreted naturally. The surgery was performed for 3 consecutive days (8 –10 rats/d were operated on). Postoperatively, the rats were not allowed food and water for the first 24 h and then were fed the control diet for 14 d. The rats received a daily intramuscular injection of antibiotics [9] at surgery and for 5 d thereafter. The rats lost about 13 g of body weight during immediate postoperative recovery. However, they then gained weight, and constant growth rates (3– 4 g of body weight gain/d) were achieved 5 d after surgery. Rats weighing 120 –170 g were divided into four groups (seven rats each) and were freely fed diets containing 60 g of CEB or FOS per kilogram of diet for 7 d with or without 0.1% NM in drinking water. There were thus four groups: CEB, CEB ⫹ NM, FOS, and FOS ⫹ NM (initial body weights 141 ⫾ 5, 143 ⫾ 5, 137 ⫾ 6, and 139 ⫾ 5 g, respectively). To promote fecal consistency, cellulose powder was also added to each diet at 100 g/kg of diet. Supplementation of cellulose and CEB or FOS was performed by replacement of an equal amount of cornstarch in the control diet. Feces (ileal excreta) were collected for the last 3 d of the experimental period, freeze-dried, and stored at ⫺20°C. During the fecal collection, feces were separated from urine by a separatory funnel and a narrow stainless sieve (20 mesh) that was provided in the individual stainless steel metabolic cage. After the digestibility study was performed, to measure the small intestinal transit time (h) in the ileorectostomized rats, all rats further consumed the control diet including cellulose powder at 100 g/kg of diet at 0800 – 0900 and 1900 –2000 h for 7 d. After adaptation to meal feeding, rats weighing 150 –204 g were fed 3 g of the same diet including 5% carmine (a water-insoluble and unabsorbable dye) at 1900 h. The feces were monitored every 15 min for the first appearance of the red dye. In vitro digestibility of CEB and LAC (experiment 2) Small intestinal mucosa was prepared from 6-mo-old adult rats and 15-d-old suckling rats. After a rat was decapitated, the small intestine except the duodenum was excised, rinsed with ice-cold NaCl solution (0.154 mol/L), and placed on an ice-cold glass dish. The intestine was cut open and the mucosa was scraped off with a coverglass and homogenized with 4 vol of ice-cold NaCl solution in a Teflon homogenizer (five strokes, 800 rpm). The homogenate was centrifuged at 2300 ⫻ g for 30 min at 4°C to obtain supernatant as an enzyme source. The digestibilities of
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hipak NH2P-50 4E column (4.6 mm ⫻ 25 cm; Showa Denko), acetonitrile:water (70:30, v/v) for the mobile phase, a flow rate of 1.0 mL/min, a temperature of 25°C, and an SE-61 refractive index detector (Showa Denko) [11]. When an appropriate amount of CEB or FOS was added to feces, the recovery ratio was higher than 98% for both sugars.
CEB, gentiobiose, and LAC were measured by the method of Dahlqvist [10], with 30 mmol/L of substrate as the final concentration. Digestibility assays contained 0.5 mg of enzyme protein/mL, buffered with 50 mM of sodium acetate (pH 6.0), and were carried out at 37°C for 16 h. In Lineweaver-Burk plot analysis, the assay contained 2.5 mg of enzyme protein from suckling rats per milliliter, with final concentrations of CEB or LAC ranging from 0.5 to 30 mmol/L, and was conducted at 37°C for 60 min within the range of linearity of enzymatic reaction. The reaction was terminated by boiling the assay mixture for 1 min. After cooling, the reaction mixture was centrifuged at 9000 ⫻ g at 4°C for 5 min to obtain supernatant. Glucose released into the supernatant was determined with a commercially available kit (Glucose B test Wako, Wako Pure Chemical Industries, Tokyo, Japan).
After the cecal contents were homogenized, a portion of homogenate was diluted with the same weight of distilled water, and the cecal pH was measured with a compact pH meter (model C-1, Horiba, Tokyo, Japan) [9]. Measurement of cecal organic acids (formate, acetate, propionate, isobutyrate, n-butyrate, iso-valerate, n-valerate, succinate, and lactate) was described previously [9].
Cecal fermentation of CEB (experiment 3)
Statistical analysis
Forty rats weighing 159 –183 g were divided into five groups of eight rats each after acclimation and were allowed free access to the control diet or a diet containing 30 or 60 g of CEB or 30 or 60 g of FOS per kilogram. Dietary addition was performed by the replacement of cornstarch in the control diet with an equal amount of each oligosaccharide. After the respective diets were fed for 14 d, the rats were lightly anesthetized with diethyl ether and sacrificed by decapitation. The cecum was removed and weighed. The cecal contents were homogenized and used for measurements of pH and organic acids. The cecal wall was flushed clean with 0.15 mol/L of ice-cold NaCl, blotted dry on filter paper, and weighed.
Data were analyzed by one-way or two-way analysis of variance, followed by the Tukey-Kramer test. Results were expressed as mean ⫾ standard error, and a 5% level of probability was considered significant. When variances were not homogenous by Bartlett’s test [12], data were logarithmically transformed before analysis. The statistical calculations were carried out using StatView 5.0 (SAS Institute, Tokyo, Japan). Regression analysis was performed using the Stat Cel 2 (Tokyo Shoseki, Tokyo, Japan).
Cecal pH and organic acids
Results In vivo digestibility of CEB (experiment 1)
Analyses of CEB and FOS in feces Amounts of CEB and FOS in feces were quantified by high-performance liquid chromatographic analysis. A freeze-dried sample (1000 mg) was defatted with diethyl ether (10 mL ⫻ 3). After the solvent was removed by evaporation, distilled water (20 mL ⫻ 2) was added, and the mixture was shaken vigorously for 30 min and centrifuged at 1500 ⫻ g for 20 min to remove the supernatant. One milliliter of 363 mmol/L of maltitol as an internal standard was added to the supernatant, which was then filled to 50 mL with distilled water. A portion of the solution was filtered (0.45-m pore size) and passed through anion (Strata SAX, Phenomenex, CA, USA) and cation (Strata SCX) exchange columns according to the manufacturer’s instructions. The solution was filtered (0.22-m pore size), and 10 L (CEB) or 20 L (FOS) of the filtrate was applied to high-performance liquid chromatographic analysis. The CEB analysis consisted of a SUGAR SP0810 column (4.6 mm ⫻ 25 cm; Showa Denko, Tokyo, Japan), distilled water for the mobile phase, a flow rate of 0.5 mL/min, a temperature of 80°C, and an SE-61 refractive index detector (Showa Denko) [1]. The FOS analysis consisted of an Asa-
Fluctuations in daily food intake and body weight gain were observed until day 3, but an improved food intake and growth rate was achieved from days 4 to 7 in all dietary groups (Fig. 1A,B). Final body weights were 154 ⫾ 8 g (CEB), 153 ⫾ 7 g (CEB ⫹ NM), 144 ⫾ 7 g (FOS), and 145 ⫾ 5 g (FOS ⫹ NM), respectively. Total food intakes for 7 d did not differ among the dietary groups. Body weight gains in the FOS diet groups tended to be lower (P ⫽ 0.076) than those in the CEB diet groups, but differences were not statistically significant due to large variations (Table 1). For the last 3 d of the experimental period, intakes of the respective oligosaccharides did not differ among the dietary groups. The results of two-way analysis of variance showed that fecal dry matter excretion was significantly greater in the FOS-fed groups than in the CEB-fed groups. Fecal excretion of FOS was significantly greater than that of CEB irrespective of NM in the drinking water. This translates to fecal recoveries of 36.2%, 35.9%, 65.3%, and 73.5% for the CEB, CEB ⫹ NEM, FOS, and FOS ⫹ NEM groups, respectively. There were significant differences in fecal recovery between CEB and FOS. In this case, the result of two-way analysis of variance showed a significant interac-
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Fig. 1. Changes in body weight (A) and daily food intake (B) in ileorectostomized rats fed diets containing 6% CEB or FOS with or without 0.1% NM in drinking water. Changes in body weight are presented as percentages of the initial body weight. Values are mean ⫾ SE of seven rats per group. CEB, cellobiose; FOS, fructo-oligosaccharide; NM, neomycin.
tion between oligosaccharide and NM, and the fecal recovery of FOS in the presence of NM was greater than in the absence of NM. The homogeneity of intestinal movement after surgery was verified by the measurement of small intestinal transit time. The first appearance of red dye in the feces was distributed between 3 and 4 h in all groups, and no significant differences were observed among groups. In vitro digestibility of CEB and LAC (experiment 2) When the supernatant of the mucosal homogenate from suckling rats was used as the enzyme source, the digestibility of CEB (35.9%) and LAC (62.0%) was triple that obtained in the mucosal homogenate from adult rats (Fig. 2). The digestibility of LAC was always two-fold greater than that of CEB in enzyme preparations from adult and suckling rats. In contrast, the digestibility of gentiobiose was almost negligible in both enzyme preparations. Lineweaver-Burk analyses showed that the Michaelis constant (Km) value of CEB (0.9 mmol/L) was 25 times lower than that of LAC (24.8 mmol/L), whereas the maximum velocity (Vmax) of
CEB (0.006 mol · mL⫺1 · min⫺1) was 7 times lower than that of LAC (0.042 mol · mL⫺1 · min⫺1; Fig. 3). Cecal fermentation of CEB (experiment 3) Body weight gain and food intake in rats fed the 6% FOS diet were significantly lower than in those fed the other diets, but there were no differences among the other dietary groups (Table 2). Dietary treatment, except for the 3% CEB diet, significantly affected cecal variables. The wet weight of the cecal contents was lowest in rats fed the control and 3% CEB diets and highest in those fed the 6% FOS diet, with intermediate values in those fed the 6% CEB and 3% FOS diets, whereas cecal pH was highest in rats fed the control and 3% CEB diets and lowest in those fed the 6% FOS diet, with intermediate values in those fed the 6% CEB and 3% FOS diets. A tendency similar to that of the cecal contents was also observed in the cecal pool size of the respective organic acids. These acids were highest in rats fed the 6% FOS diet and lowest in those fed the control and 3% CEB diets, with intermediate values in those fed the 6% CEB and 3% FOS diets (Table 2).
Table 1 Food intake, body weight gain, oligosaccharide intake, fecal dry matter, and fecal recovery of oligosaccharide in ileorectostomized rats fed diets containing 6% CEB or 6% FOS for 7 d with or without NM in drinking water* CEB
Total food intake (g/7 d) Body weight gain (g/7 d) Oligosaccharide intake (g/3 d)† Fecal dry matter (g/3 d)‡ Fecal oligosaccharide (g/3 d) Fecal recovery (%)
92.1 ⫾ 6.1 12.5 ⫾ 3.3 2.8 ⫾ 0.2 7.7 ⫾ 0.4 1.0 ⫾ 0.1a 36.2 ⫾ 1.0a
CEB ⫹ NM
90.5 ⫾ 3.7 10.3 ⫾ 3.2 2.5 ⫾ 0.1 7.0 ⫾ 0.3 0.9 ⫾ 0.0a 35.9 ⫾ 0.5a
FOS
89.3 ⫾ 2.1 6.2 ⫾ 1.3 2.6 ⫾ 0.1 8.3 ⫾ 0.3 1.7 ⫾ 0.1b 65.3 ⫾ 2.0b
FOS ⫹ NM
87.7 ⫾ 4.5 5.4 ⫾ 3.6 2.5 ⫾ 0.1 8.1 ⫾ 0.4 1.8 ⫾ 0.1b 73.5 ⫾ 1.9c
ANOVA P
0.522 0.076 0.598 0.034 ⬍0.001 ⬍0.001
Oligosaccharide NM
Interaction
0.762 0.632 0.201 0.341 0.634 0.031
0.967 0.829 0.573 0.582 0.120 0.026
ANOVA, analysis of variance; CEB, cellobiose; FOS, fructo-oligosaccharide; NM, neomycin * Data are expressed as mean ⫾ SE (n ⫽ 7); values in a row not sharing a common superscript are significantly different (P ⬍ 0.05) when analyzed using two-way ANOVA, followed by the Tukey-Kramer test. † Calculated from dietary intakes for the last 3 d of the experimental period and then averaged. ‡ Feces were collected for the last 3 d of the experimental period.
Digestibility (%)
T. Morita et al. / Nutrition 24 (2008) 1199 –1204
70 Suckling rat
60
Adult rat
50 40 30 20 10 0 Lactose
Cellobiose
Gentiobiose
Fig. 2. In vitro digestibility of lactose, cellobiose, and gentiobiose when mucosal homogenates of the small intestine from suckling and adult rats were used as enzyme sources. Values are means from duplicate determinations.
Discussion In the present study, we directly measured the small intestinal digestibility of CEB in ileorectostomized rats in the presence or absence of NM in drinking water. The digestibility of CEB (approximately 65%) was much greater than that of FOS (approximately 25–35%) and was not affected by the NM treatment. This differed from FOS, for which the digestibility was reduced by 10% in the presence of NM (Table 1). Previous studies had shown that oral administration of CEB to rats gradually increased the blood glucose concentration up to 120 min after administration, implying that CEB was slowly hydrolyzed in the lower part of the small intestine through bacterial activity [1]. However, the NM used in the present study is an unabsorbable, broad-spectrum antibiotic, and 0.1% NM in drinking water was expected to largely suppress bacterial activity in the small intestine [13]. Accordingly, the present findings indicated that the hydrolysis of CEB was due to a mucosal enzyme in the intestine, not due to an enzyme of bacterial origin. In a human study, the digestibility of FOS in the terminal ileum was estimated to be 11% by an intestinal aspiration method [14]; this value was low compared with the present results obtained with ileorectostomized rats. Because the ileorectostomized rats retain the rectum and preserve the defecation reflex, the transit time of chyme in these rats (3– 4 h) could be longer than is usual in normal rats. This may allow a longer period of time to expose FOS to bacterial degradation, which could partly explain the different digestibility of FOS in the previous [14] and present results. Nevertheless, the present results clearly showed that CEB was extensively digested by a mucosal enzyme in the rat small intestine. In rats, lactase activity is high at birth and declines at about 4 wk of age to a plateau that is maintained throughout
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the life of the animals [15]. This pattern is similar to adultonset lactase decline in humans, which is characterized by a decline in lactase activity at ages 5–7 y to ⬃10% of early childhood levels [16]. In the present study, in vitro LAC digestibility measured at near-neutral pH (6.0) was three times higher in the supernatant of mucosal homogenate prepared from suckling rats (15 d of age) than in that from adult rats (6 mo old). Interestingly, the same tendency was also observed for the in vitro digestibility of CEB (Fig. 2). There are two types of -galactosidases in the small intestine, one having a more neutral pH optimum (approximately 6.0) and the other an acidic pH optimum (approximately 4.0) [17,18], but the latter is lysosomal in origin and is therefore presumably not involved in digesting CEB on the intestinal brush border membrane. From our results considered together, it is reasonable to assume that the rat intestinal lactase should have the potential to cleave the -glucosidic bond as described by Lau [19], indicating that a purified lactase of the human intestine has -glucosidase activities against different chromogenic substrates, which vary between 13% and 18% of the LAC activity. In addition, a profound gap was apparent in the digestibility of CEB between in vitro (10%) and in vivo (65%) assays, indicating that it is impossible to predict the small intestinal digestibility of CEB by extrapolating from the in vitro value. As indicated in Table 2, the cecal variables including fermentation products were virtually the same in the control and 3% CEB groups. This simply means that the amount of CEB entering the cecum was small in rats fed the 3% CEB diet, although the small intestinal digestibility of CEB was approximately 65% (Table 1). Lineweaver-Burk analyses indicated that CEB showed higher affinity with the membrane enzyme than LAC did, but the velocity of CEB hydrolysis was very slow compared with LAC hydrolysis (Fig. 3). These properties of CEB as a substrate imply that a lower dietary intake of CEB may result in higher digestibility of CEB in the small intestine. This may explain why the cecal variables in rats fed the 3%
Fig. 3. Lineweaver-Burk analyses of cellobiose and lactose when the mucosal homogenate of the small intestine from suckling rats was used as the enzyme source.
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Table 2 Food intake, body weight gain, and cecal variables in rats fed a control diet or a diet containing CEB or FOS at a dietary level of 3% or 6% for 14 d* Control Food intake (g/14 d) Body weight gain (g/14 d) Cecal content (g) Cecal pH Cecal organic acids (mol/cecum) Acetate Propionate n-Butyrate Succinate Lactate
3% CEB
6% CEB
3% FOS
6% FOS
228 ⫾ 7 68 ⫾ 4b 1.9 ⫾ 0.0a 7.6 ⫾ 0.0c
230 ⫾ 3 66 ⫾ 3b 1.9 ⫾ 0.1a 7.6 ⫾ 0.0c
227 ⫾ 5 68 ⫾ 3b 2.8 ⫾ 0.2b 7.3 ⫾ 0.1b
228 ⫾ 5 63 ⫾ 2b 3.0 ⫾ 0.2b 7.2 ⫾ 0.0b
199 ⫾ 4a 50 ⫾ 2a 5.2 ⫾ 0.4c 5.9 ⫾ 0.1a
84.4 ⫾ 4.7ab 36.1 ⫾ 2.1a 10.7 ⫾ 1.1a 3.6 ⫾ 1.6a 1.1 ⫾ 0.7
74.3 ⫾ 5.1a 31.4 ⫾ 2.5a 11.9 ⫾ 1.1a 5.1 ⫾ 1.1a 2.1 ⫾ 2.0
112.2 ⫾ 7.9bc 56.7 ⫾ 6.6b 45.8 ⫾ 4.3b 19.0 ⫾ 4.3b 4.9 ⫾ 3.3
141.2 ⫾ 11.5c 68.5 ⫾ 4.9b 33.7 ⫾ 2.8b 36.6 ⫾ 5.0b ND
83.4 ⫾ 17.0abc 100.6 ⫾ 8.3c 23.1 ⫾ 9.1ab 19.3 ⫾ 3.0b 347.9 ⫾ 78.9
b
b
b
b
CEB, cellobiose; FOS, fructo-oligosaccharide; ND, not detected. * Data are expressed as mean ⫾ SE (n ⫽ 8); values in a row not sharing a common superscript are significantly different (P ⬍ 0.05) when analyzed using one-way analysis of variance, followed by the Tukey-Kramer test.
CEB diet were almost the same as in those fed the control diet. Because the administration of 25 g CEB to Japanese subjects induced a remarkable excretion of breath hydrogen without any glycemic response, Nakamura et al. [3] concluded that little CEB was hydrolyzed, and therefore CEB was readily fermented by intestinal microbes. However, two distinct lactase phenotypes exist in humans. One is persistent high lactase activity throughout life, dominating in Caucasians, particularly those with ancestry in Northern and Western Europe [5], and the other is adult-onset lactase decline, which is prevalent in many human populations, such as Asians and Blacks [4,20]. Accordingly, the lactaserelated hydrolysis of CEB in rats, if extrapolated to humans, may have important implications for the bioavailability of CEB in European populations. Whether the findings obtained from Japanese subjects [3] can be extrapolated to Caucasians with persistent high lactase activity requires further investigation. Conclusion Our results indicate that dietary CEB was extensively digested in the small intestine by -galactosidase (lactase) in rats, leading to complete digestion of CEB when dietary supplementation was limited. References [1] Watanabe T. Development of physiological functions of cellooligosaccharides. Cellulose Commun 1998;5:91–7. [2] Satouchi M, Watanabe T, Wakabayashi S, Ohokuna K, Koshijima T, Kuwahara M. Digestibility, absorptivity, and physiological effects of cellooligosaccharides in human and rat (in Japanese). J Jpn Soc Nutr Food Sci 1996;49:143– 8. [3] Nakamura S, Oku T, Ichinose M. Bioavailability of cellobiose by tolerance test and breath hydrogen excretion in humans. Nutrition 2004;20:979 – 83. [4] Bayless TM, Rosensweig NS. A racial difference in incidence of lactase deficiency: a survey of milk intolerance and lactase deficiency in healthy adult males. J Am Med Assoc 1966;197:138 – 42.
[5] Rao DR, Bello H, Warren AP, Brown GE. Prevalence of lactose maldigestion: influence and interaction of age, race, and sex. Dig Dis Sci 1994;39:1519 –24. [6] Morita T, Tanabe H, Ito H, Yuto S, Matsubara T, Matsuda T, et al. Increased luminal mucin does not disturb glucose or ovalbumin absorption in rats fed insoluble dietary fiber. J Nutr 2006;136: 2486 –91. [7] American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J Nutr 1977;107:1340 – 8. [8] Lambert R. Operative technique in intestinal surgery. In: Surgery of the digestive system in the rat. Springfield, IL: Thomas CC, ed. 1965, p. 413–33. [9] Morita T, Kasaoka S, Oh-hashi A, Ikai M, Numasaki Y, Kiriyama S. Resistant proteins alter cecal short-chain fatty acid profiles in rats fed high amylose cornstarch. J Nutr 1998;128:1156 – 64. [10] Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18 –25. [11] Yoshikawa J, Amachi S, Shinoyama H, Fujii T. Purification and some properties of -fructofuranosidase I formed by Aureobasidium pullulans DSM 2404. J Biosci Bioeng 2007;103:491–3. [12] Zar JH. Biostatistical analysis. 2nd ed. Englewood Cliffs, NJ: Prentice-Hall; 1984. [13] Miyasaka H, Sanada H, Ayano Y. Relation between colonic products from corn bran hemicellulose and the lipid metabolism in rats fed on diets supplemented with orotic acid. Biosci Biotech Biochem 1992; 56:157– 8. [14] Molis C, Flourie B, Ouarne F, Gailing MF, Lartigue S, Guilbert A, et al. Digestion, excretion, and energy value of fructooligosaccharides in healthy humans. Am J Clin Nutr 1996;64:324 – 8. [15] Doell RG, Kretchmer N. Studies of small intestine during development. I. Distribution and activity of -galactosidase. Biochim Biophys Acta 1962;62:353– 62. [16] Buller HA, Grand RJ. Lactose intolerance. Annu Rev Med 1990;41: 141– 8. [17] Asp NG. Human small-intestinal-galactosidases. Separation and characterization of three forms of an acid-galactosidase. Biochem J 1971; 121:299 –308. [18] Asp NG, Dahlqvist A, Koldovsky O. Human small-intestinal betagalactosidases. Separation and characterization of one lactase and one hetero beta-galactosidase. Biochem J 1969;114:351–9. [19] Lau HFK. Physicochemical characterization of human intestinal lactase. Biochem J 1987;241:567–72. [20] Savilahti E, Launiala K, Kuitunen P. Congenital lactase deficiency: a clinical study on 16 patients. Arch Dis Child 1983;58:246 –52.
Basic nutritional investigation
Cellobiose is extensively digested in the small intestine by -galactosidase in rats Tatsuya Morita, Ph.D.a,*, Mayumi Ozawa, M.S.a, Hiroyuki Ito, M.S.b, Sugiyama Kimio, Ph.D.a, and Shuhachi Kiriyama, Ph.D.c a
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan b Graduate School of Science and Technology, Shizuoka University, Shizuoka, Japan c Faculty of Nutritional Sciences, University of Shizuoka, Shizuoka, Japan Manuscript received April 8, 2008; accepted June 25, 2008.
Abstract
Objective: The bioavailability of cellobiose (CEB) was investigated with respect to small intestinal digestibility and cecal fermentation in rats. Further, whether small intestinal -galactosidase is responsible for the hydrolysis of CEB was examined. Methods: Ileorectostomized rats were fed diets including 6% CEB or fructo-oligosaccharide with or without 0.1% neomycin in drinking water for 7 d. The fecal excretion of the respective oligosaccharides was determined. In vitro digestion of CEB and lactose was characterized using mucosal enzymes of the small intestine from suckling and adult rats. Cecal fermentation in normal rats fed a control diet or a diet including 3% or 6% CEB for 14 d was examined. Results: The small intestinal digestibility of CEB was 64%, irrespective of the presence of neomycin in drinking water, whereas the digestibility of fructo-oligosaccharide differed significantly between groups administered (26%) or not administered (35%) neomycin. The in vitro digestibility of lactose (62%) and CEB (36%) was three times greater with the enzymes from the suckling rats than with those from the adult rats. Michaelis constant (Km) and maximum velocity (Vmax) values for CEB were 25 and 7 times lower, respectively, than those for lactose. Normal rats fed the 6% CEB diet showed a greater cecal organic acid than those fed the control diet, but no differences were observed between those fed the control and 3% CEB diets. Conclusion: Our results indicate that dietary CEB was extensively digested in the small intestine by -galactosidase in rats, leading to complete digestion of CEB when dietary supplementation was limited. © 2008 Elsevier Inc. All rights reserved.
Keywords:
Cellobiose; Fructo-oligosaccharide; Small intestinal digestibility; -Galactosidase; Ileorectostomized rats; Cecal fermentation
Introduction The physiologic functions of indigestible oligosaccharides have been extensively studied, and it is now well recognized that some of these compounds have beneficial effects on large bowel health through bacterial fermentation. Because no -glucosidase has been reported in the intestinal mucosa, cellobiose (CEB) could be a good candidate for a functional indigestible oligosaccharide. Watanabe [1] and Satouchi et al. [2] reported that in vitro * Corresponding author. Tel./fax: ⫹81-54-238-5132. E-mail address: [email protected] (T. Morita). 0899-9007/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2008.06.029
digestibility of CEB measured using a homogenate of rat intestinal mucosa was 6.3%, and that after oral administration of CEB to rats, the blood glucose concentration showed a gradual increase up to 120 min after administration, implying that CEB was slowly hydrolyzed in the lower part of the small intestine, possibly through bacterial activity [1]. In contrast to the results obtained in rats, Nakamura et al. [3] showed that, when 25 g of CEB was orally administered to human subjects, there were no increases in blood glucose or insulin secretion, whereas the excretion of breath hydrogen was remarkable, indicating that a large part of the administered CEB escaped digestion and fermented in the large bowel. However,
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precise information about the small intestinal digestibility of CEB in experimental animals and humans is scarce. In addition, it remains unclear whether CEB digestion in the small intestine is due to an enzyme on the mucosal membrane or one of bacterial origin. The purpose of the present study was to directly measure the small intestinal digestibility of CEB using ileorectostomized rats whose ileal terminal was directly anastomosed to the rectum. In these rats, the amount of fecal excretion of CEB was expected to correspond to that of undigested CEB coming from the small intestine into the large bowel in normal rats. Glucose and galactose moieties at non-reducing terminals in CEB and lactose (LAC) molecules are epimers at the C4 position, and the chemical structures of these molecules are highly similar. Accordingly, we also examined the in vitro digestibility of CEB with respect to small intestinal -galactosidase (lactase), considering that this enzyme may cleave the -1,4 glucosidic bond. If lactase is responsible for CEB digestion, one might expect that the findings obtained from Japanese subjects [3] could be different from those in Caucasians, many of whom have persistent high lactase activity even as adults [4,5].
Materials and methods Materials Cellobiose was provided by Nippon Paper Chemicals Co. (Tokyo, Japan) and was composed of 96.6% cellobiose (1– 4), 1.9% cello-oligosaccharide, and 1.5% glucose. Fructooligosaccharide (FOS), composed of 44% 1-kestose, 46% nystose, and 10% 1-f--fructofuranosyl nystose, was purchased from Meiji Seika (Tokyo, Japan; Meioligo P). Gentiobiose (1– 6) was provided by Nihon Shokuhin Kako Co. (Shizuoka, Japan). Neomycin sulfate (NM) was purchased from Sigma (St. Louis, MO, USA). Care of animals Wistar rats (purchased from Shizuoka Laboratory Animal Center, Hamamatsu, Japan) were housed in individual stainless steel cages with wire screen bottoms in a room of controlled temperature (23 ⫾ 2°C) and lighting (lights on 0800 –2000 h). For adaptation, rats were fed a control diet for at least 3 d. This diet [6] was formulated from 250 g of casein, 652.25 g of cornstarch, and 50 g of corn oil per kilogram. The remainder of the diet consisted of vitamins and minerals [7]. The rats were then divided into groups on the basis of body weight and allowed free access to the experimental diets and water. Body weight and food intake were recorded every morning before replenishing the diet. The study was approved by the animal use committee of Shizuoka University, and animals were maintained in accordance with the guidelines of Shizuoka University for the care and use of laboratory animals.
In vivo digestibility of CEB (experiment 1) After overnight fasting, 28 rats weighing 90 –115 g were subjected to an ileorectostomy in which the terminal ileum was connected to the rectum according to the method of Lambert [8], with some modifications. To shorten the recovery period, we did not dissect the cecum and colon, but the ileocecal valve was ligatured (closed) and then the colonic terminal was anastomosed to the stoma in the abdominal wall to allow the cecal and colonic contents to be excreted naturally. The surgery was performed for 3 consecutive days (8 –10 rats/d were operated on). Postoperatively, the rats were not allowed food and water for the first 24 h and then were fed the control diet for 14 d. The rats received a daily intramuscular injection of antibiotics [9] at surgery and for 5 d thereafter. The rats lost about 13 g of body weight during immediate postoperative recovery. However, they then gained weight, and constant growth rates (3– 4 g of body weight gain/d) were achieved 5 d after surgery. Rats weighing 120 –170 g were divided into four groups (seven rats each) and were freely fed diets containing 60 g of CEB or FOS per kilogram of diet for 7 d with or without 0.1% NM in drinking water. There were thus four groups: CEB, CEB ⫹ NM, FOS, and FOS ⫹ NM (initial body weights 141 ⫾ 5, 143 ⫾ 5, 137 ⫾ 6, and 139 ⫾ 5 g, respectively). To promote fecal consistency, cellulose powder was also added to each diet at 100 g/kg of diet. Supplementation of cellulose and CEB or FOS was performed by replacement of an equal amount of cornstarch in the control diet. Feces (ileal excreta) were collected for the last 3 d of the experimental period, freeze-dried, and stored at ⫺20°C. During the fecal collection, feces were separated from urine by a separatory funnel and a narrow stainless sieve (20 mesh) that was provided in the individual stainless steel metabolic cage. After the digestibility study was performed, to measure the small intestinal transit time (h) in the ileorectostomized rats, all rats further consumed the control diet including cellulose powder at 100 g/kg of diet at 0800 – 0900 and 1900 –2000 h for 7 d. After adaptation to meal feeding, rats weighing 150 –204 g were fed 3 g of the same diet including 5% carmine (a water-insoluble and unabsorbable dye) at 1900 h. The feces were monitored every 15 min for the first appearance of the red dye. In vitro digestibility of CEB and LAC (experiment 2) Small intestinal mucosa was prepared from 6-mo-old adult rats and 15-d-old suckling rats. After a rat was decapitated, the small intestine except the duodenum was excised, rinsed with ice-cold NaCl solution (0.154 mol/L), and placed on an ice-cold glass dish. The intestine was cut open and the mucosa was scraped off with a coverglass and homogenized with 4 vol of ice-cold NaCl solution in a Teflon homogenizer (five strokes, 800 rpm). The homogenate was centrifuged at 2300 ⫻ g for 30 min at 4°C to obtain supernatant as an enzyme source. The digestibilities of
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hipak NH2P-50 4E column (4.6 mm ⫻ 25 cm; Showa Denko), acetonitrile:water (70:30, v/v) for the mobile phase, a flow rate of 1.0 mL/min, a temperature of 25°C, and an SE-61 refractive index detector (Showa Denko) [11]. When an appropriate amount of CEB or FOS was added to feces, the recovery ratio was higher than 98% for both sugars.
CEB, gentiobiose, and LAC were measured by the method of Dahlqvist [10], with 30 mmol/L of substrate as the final concentration. Digestibility assays contained 0.5 mg of enzyme protein/mL, buffered with 50 mM of sodium acetate (pH 6.0), and were carried out at 37°C for 16 h. In Lineweaver-Burk plot analysis, the assay contained 2.5 mg of enzyme protein from suckling rats per milliliter, with final concentrations of CEB or LAC ranging from 0.5 to 30 mmol/L, and was conducted at 37°C for 60 min within the range of linearity of enzymatic reaction. The reaction was terminated by boiling the assay mixture for 1 min. After cooling, the reaction mixture was centrifuged at 9000 ⫻ g at 4°C for 5 min to obtain supernatant. Glucose released into the supernatant was determined with a commercially available kit (Glucose B test Wako, Wako Pure Chemical Industries, Tokyo, Japan).
After the cecal contents were homogenized, a portion of homogenate was diluted with the same weight of distilled water, and the cecal pH was measured with a compact pH meter (model C-1, Horiba, Tokyo, Japan) [9]. Measurement of cecal organic acids (formate, acetate, propionate, isobutyrate, n-butyrate, iso-valerate, n-valerate, succinate, and lactate) was described previously [9].
Cecal fermentation of CEB (experiment 3)
Statistical analysis
Forty rats weighing 159 –183 g were divided into five groups of eight rats each after acclimation and were allowed free access to the control diet or a diet containing 30 or 60 g of CEB or 30 or 60 g of FOS per kilogram. Dietary addition was performed by the replacement of cornstarch in the control diet with an equal amount of each oligosaccharide. After the respective diets were fed for 14 d, the rats were lightly anesthetized with diethyl ether and sacrificed by decapitation. The cecum was removed and weighed. The cecal contents were homogenized and used for measurements of pH and organic acids. The cecal wall was flushed clean with 0.15 mol/L of ice-cold NaCl, blotted dry on filter paper, and weighed.
Data were analyzed by one-way or two-way analysis of variance, followed by the Tukey-Kramer test. Results were expressed as mean ⫾ standard error, and a 5% level of probability was considered significant. When variances were not homogenous by Bartlett’s test [12], data were logarithmically transformed before analysis. The statistical calculations were carried out using StatView 5.0 (SAS Institute, Tokyo, Japan). Regression analysis was performed using the Stat Cel 2 (Tokyo Shoseki, Tokyo, Japan).
Cecal pH and organic acids
Results In vivo digestibility of CEB (experiment 1)
Analyses of CEB and FOS in feces Amounts of CEB and FOS in feces were quantified by high-performance liquid chromatographic analysis. A freeze-dried sample (1000 mg) was defatted with diethyl ether (10 mL ⫻ 3). After the solvent was removed by evaporation, distilled water (20 mL ⫻ 2) was added, and the mixture was shaken vigorously for 30 min and centrifuged at 1500 ⫻ g for 20 min to remove the supernatant. One milliliter of 363 mmol/L of maltitol as an internal standard was added to the supernatant, which was then filled to 50 mL with distilled water. A portion of the solution was filtered (0.45-m pore size) and passed through anion (Strata SAX, Phenomenex, CA, USA) and cation (Strata SCX) exchange columns according to the manufacturer’s instructions. The solution was filtered (0.22-m pore size), and 10 L (CEB) or 20 L (FOS) of the filtrate was applied to high-performance liquid chromatographic analysis. The CEB analysis consisted of a SUGAR SP0810 column (4.6 mm ⫻ 25 cm; Showa Denko, Tokyo, Japan), distilled water for the mobile phase, a flow rate of 0.5 mL/min, a temperature of 80°C, and an SE-61 refractive index detector (Showa Denko) [1]. The FOS analysis consisted of an Asa-
Fluctuations in daily food intake and body weight gain were observed until day 3, but an improved food intake and growth rate was achieved from days 4 to 7 in all dietary groups (Fig. 1A,B). Final body weights were 154 ⫾ 8 g (CEB), 153 ⫾ 7 g (CEB ⫹ NM), 144 ⫾ 7 g (FOS), and 145 ⫾ 5 g (FOS ⫹ NM), respectively. Total food intakes for 7 d did not differ among the dietary groups. Body weight gains in the FOS diet groups tended to be lower (P ⫽ 0.076) than those in the CEB diet groups, but differences were not statistically significant due to large variations (Table 1). For the last 3 d of the experimental period, intakes of the respective oligosaccharides did not differ among the dietary groups. The results of two-way analysis of variance showed that fecal dry matter excretion was significantly greater in the FOS-fed groups than in the CEB-fed groups. Fecal excretion of FOS was significantly greater than that of CEB irrespective of NM in the drinking water. This translates to fecal recoveries of 36.2%, 35.9%, 65.3%, and 73.5% for the CEB, CEB ⫹ NEM, FOS, and FOS ⫹ NEM groups, respectively. There were significant differences in fecal recovery between CEB and FOS. In this case, the result of two-way analysis of variance showed a significant interac-
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Fig. 1. Changes in body weight (A) and daily food intake (B) in ileorectostomized rats fed diets containing 6% CEB or FOS with or without 0.1% NM in drinking water. Changes in body weight are presented as percentages of the initial body weight. Values are mean ⫾ SE of seven rats per group. CEB, cellobiose; FOS, fructo-oligosaccharide; NM, neomycin.
tion between oligosaccharide and NM, and the fecal recovery of FOS in the presence of NM was greater than in the absence of NM. The homogeneity of intestinal movement after surgery was verified by the measurement of small intestinal transit time. The first appearance of red dye in the feces was distributed between 3 and 4 h in all groups, and no significant differences were observed among groups. In vitro digestibility of CEB and LAC (experiment 2) When the supernatant of the mucosal homogenate from suckling rats was used as the enzyme source, the digestibility of CEB (35.9%) and LAC (62.0%) was triple that obtained in the mucosal homogenate from adult rats (Fig. 2). The digestibility of LAC was always two-fold greater than that of CEB in enzyme preparations from adult and suckling rats. In contrast, the digestibility of gentiobiose was almost negligible in both enzyme preparations. Lineweaver-Burk analyses showed that the Michaelis constant (Km) value of CEB (0.9 mmol/L) was 25 times lower than that of LAC (24.8 mmol/L), whereas the maximum velocity (Vmax) of
CEB (0.006 mol · mL⫺1 · min⫺1) was 7 times lower than that of LAC (0.042 mol · mL⫺1 · min⫺1; Fig. 3). Cecal fermentation of CEB (experiment 3) Body weight gain and food intake in rats fed the 6% FOS diet were significantly lower than in those fed the other diets, but there were no differences among the other dietary groups (Table 2). Dietary treatment, except for the 3% CEB diet, significantly affected cecal variables. The wet weight of the cecal contents was lowest in rats fed the control and 3% CEB diets and highest in those fed the 6% FOS diet, with intermediate values in those fed the 6% CEB and 3% FOS diets, whereas cecal pH was highest in rats fed the control and 3% CEB diets and lowest in those fed the 6% FOS diet, with intermediate values in those fed the 6% CEB and 3% FOS diets. A tendency similar to that of the cecal contents was also observed in the cecal pool size of the respective organic acids. These acids were highest in rats fed the 6% FOS diet and lowest in those fed the control and 3% CEB diets, with intermediate values in those fed the 6% CEB and 3% FOS diets (Table 2).
Table 1 Food intake, body weight gain, oligosaccharide intake, fecal dry matter, and fecal recovery of oligosaccharide in ileorectostomized rats fed diets containing 6% CEB or 6% FOS for 7 d with or without NM in drinking water* CEB
Total food intake (g/7 d) Body weight gain (g/7 d) Oligosaccharide intake (g/3 d)† Fecal dry matter (g/3 d)‡ Fecal oligosaccharide (g/3 d) Fecal recovery (%)
92.1 ⫾ 6.1 12.5 ⫾ 3.3 2.8 ⫾ 0.2 7.7 ⫾ 0.4 1.0 ⫾ 0.1a 36.2 ⫾ 1.0a
CEB ⫹ NM
90.5 ⫾ 3.7 10.3 ⫾ 3.2 2.5 ⫾ 0.1 7.0 ⫾ 0.3 0.9 ⫾ 0.0a 35.9 ⫾ 0.5a
FOS
89.3 ⫾ 2.1 6.2 ⫾ 1.3 2.6 ⫾ 0.1 8.3 ⫾ 0.3 1.7 ⫾ 0.1b 65.3 ⫾ 2.0b
FOS ⫹ NM
87.7 ⫾ 4.5 5.4 ⫾ 3.6 2.5 ⫾ 0.1 8.1 ⫾ 0.4 1.8 ⫾ 0.1b 73.5 ⫾ 1.9c
ANOVA P
0.522 0.076 0.598 0.034 ⬍0.001 ⬍0.001
Oligosaccharide NM
Interaction
0.762 0.632 0.201 0.341 0.634 0.031
0.967 0.829 0.573 0.582 0.120 0.026
ANOVA, analysis of variance; CEB, cellobiose; FOS, fructo-oligosaccharide; NM, neomycin * Data are expressed as mean ⫾ SE (n ⫽ 7); values in a row not sharing a common superscript are significantly different (P ⬍ 0.05) when analyzed using two-way ANOVA, followed by the Tukey-Kramer test. † Calculated from dietary intakes for the last 3 d of the experimental period and then averaged. ‡ Feces were collected for the last 3 d of the experimental period.
Digestibility (%)
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70 Suckling rat
60
Adult rat
50 40 30 20 10 0 Lactose
Cellobiose
Gentiobiose
Fig. 2. In vitro digestibility of lactose, cellobiose, and gentiobiose when mucosal homogenates of the small intestine from suckling and adult rats were used as enzyme sources. Values are means from duplicate determinations.
Discussion In the present study, we directly measured the small intestinal digestibility of CEB in ileorectostomized rats in the presence or absence of NM in drinking water. The digestibility of CEB (approximately 65%) was much greater than that of FOS (approximately 25–35%) and was not affected by the NM treatment. This differed from FOS, for which the digestibility was reduced by 10% in the presence of NM (Table 1). Previous studies had shown that oral administration of CEB to rats gradually increased the blood glucose concentration up to 120 min after administration, implying that CEB was slowly hydrolyzed in the lower part of the small intestine through bacterial activity [1]. However, the NM used in the present study is an unabsorbable, broad-spectrum antibiotic, and 0.1% NM in drinking water was expected to largely suppress bacterial activity in the small intestine [13]. Accordingly, the present findings indicated that the hydrolysis of CEB was due to a mucosal enzyme in the intestine, not due to an enzyme of bacterial origin. In a human study, the digestibility of FOS in the terminal ileum was estimated to be 11% by an intestinal aspiration method [14]; this value was low compared with the present results obtained with ileorectostomized rats. Because the ileorectostomized rats retain the rectum and preserve the defecation reflex, the transit time of chyme in these rats (3– 4 h) could be longer than is usual in normal rats. This may allow a longer period of time to expose FOS to bacterial degradation, which could partly explain the different digestibility of FOS in the previous [14] and present results. Nevertheless, the present results clearly showed that CEB was extensively digested by a mucosal enzyme in the rat small intestine. In rats, lactase activity is high at birth and declines at about 4 wk of age to a plateau that is maintained throughout
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the life of the animals [15]. This pattern is similar to adultonset lactase decline in humans, which is characterized by a decline in lactase activity at ages 5–7 y to ⬃10% of early childhood levels [16]. In the present study, in vitro LAC digestibility measured at near-neutral pH (6.0) was three times higher in the supernatant of mucosal homogenate prepared from suckling rats (15 d of age) than in that from adult rats (6 mo old). Interestingly, the same tendency was also observed for the in vitro digestibility of CEB (Fig. 2). There are two types of -galactosidases in the small intestine, one having a more neutral pH optimum (approximately 6.0) and the other an acidic pH optimum (approximately 4.0) [17,18], but the latter is lysosomal in origin and is therefore presumably not involved in digesting CEB on the intestinal brush border membrane. From our results considered together, it is reasonable to assume that the rat intestinal lactase should have the potential to cleave the -glucosidic bond as described by Lau [19], indicating that a purified lactase of the human intestine has -glucosidase activities against different chromogenic substrates, which vary between 13% and 18% of the LAC activity. In addition, a profound gap was apparent in the digestibility of CEB between in vitro (10%) and in vivo (65%) assays, indicating that it is impossible to predict the small intestinal digestibility of CEB by extrapolating from the in vitro value. As indicated in Table 2, the cecal variables including fermentation products were virtually the same in the control and 3% CEB groups. This simply means that the amount of CEB entering the cecum was small in rats fed the 3% CEB diet, although the small intestinal digestibility of CEB was approximately 65% (Table 1). Lineweaver-Burk analyses indicated that CEB showed higher affinity with the membrane enzyme than LAC did, but the velocity of CEB hydrolysis was very slow compared with LAC hydrolysis (Fig. 3). These properties of CEB as a substrate imply that a lower dietary intake of CEB may result in higher digestibility of CEB in the small intestine. This may explain why the cecal variables in rats fed the 3%
Fig. 3. Lineweaver-Burk analyses of cellobiose and lactose when the mucosal homogenate of the small intestine from suckling rats was used as the enzyme source.
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Table 2 Food intake, body weight gain, and cecal variables in rats fed a control diet or a diet containing CEB or FOS at a dietary level of 3% or 6% for 14 d* Control Food intake (g/14 d) Body weight gain (g/14 d) Cecal content (g) Cecal pH Cecal organic acids (mol/cecum) Acetate Propionate n-Butyrate Succinate Lactate
3% CEB
6% CEB
3% FOS
6% FOS
228 ⫾ 7 68 ⫾ 4b 1.9 ⫾ 0.0a 7.6 ⫾ 0.0c
230 ⫾ 3 66 ⫾ 3b 1.9 ⫾ 0.1a 7.6 ⫾ 0.0c
227 ⫾ 5 68 ⫾ 3b 2.8 ⫾ 0.2b 7.3 ⫾ 0.1b
228 ⫾ 5 63 ⫾ 2b 3.0 ⫾ 0.2b 7.2 ⫾ 0.0b
199 ⫾ 4a 50 ⫾ 2a 5.2 ⫾ 0.4c 5.9 ⫾ 0.1a
84.4 ⫾ 4.7ab 36.1 ⫾ 2.1a 10.7 ⫾ 1.1a 3.6 ⫾ 1.6a 1.1 ⫾ 0.7
74.3 ⫾ 5.1a 31.4 ⫾ 2.5a 11.9 ⫾ 1.1a 5.1 ⫾ 1.1a 2.1 ⫾ 2.0
112.2 ⫾ 7.9bc 56.7 ⫾ 6.6b 45.8 ⫾ 4.3b 19.0 ⫾ 4.3b 4.9 ⫾ 3.3
141.2 ⫾ 11.5c 68.5 ⫾ 4.9b 33.7 ⫾ 2.8b 36.6 ⫾ 5.0b ND
83.4 ⫾ 17.0abc 100.6 ⫾ 8.3c 23.1 ⫾ 9.1ab 19.3 ⫾ 3.0b 347.9 ⫾ 78.9
b
b
b
b
CEB, cellobiose; FOS, fructo-oligosaccharide; ND, not detected. * Data are expressed as mean ⫾ SE (n ⫽ 8); values in a row not sharing a common superscript are significantly different (P ⬍ 0.05) when analyzed using one-way analysis of variance, followed by the Tukey-Kramer test.
CEB diet were almost the same as in those fed the control diet. Because the administration of 25 g CEB to Japanese subjects induced a remarkable excretion of breath hydrogen without any glycemic response, Nakamura et al. [3] concluded that little CEB was hydrolyzed, and therefore CEB was readily fermented by intestinal microbes. However, two distinct lactase phenotypes exist in humans. One is persistent high lactase activity throughout life, dominating in Caucasians, particularly those with ancestry in Northern and Western Europe [5], and the other is adult-onset lactase decline, which is prevalent in many human populations, such as Asians and Blacks [4,20]. Accordingly, the lactaserelated hydrolysis of CEB in rats, if extrapolated to humans, may have important implications for the bioavailability of CEB in European populations. Whether the findings obtained from Japanese subjects [3] can be extrapolated to Caucasians with persistent high lactase activity requires further investigation. Conclusion Our results indicate that dietary CEB was extensively digested in the small intestine by -galactosidase (lactase) in rats, leading to complete digestion of CEB when dietary supplementation was limited. References [1] Watanabe T. Development of physiological functions of cellooligosaccharides. Cellulose Commun 1998;5:91–7. [2] Satouchi M, Watanabe T, Wakabayashi S, Ohokuna K, Koshijima T, Kuwahara M. Digestibility, absorptivity, and physiological effects of cellooligosaccharides in human and rat (in Japanese). J Jpn Soc Nutr Food Sci 1996;49:143– 8. [3] Nakamura S, Oku T, Ichinose M. Bioavailability of cellobiose by tolerance test and breath hydrogen excretion in humans. Nutrition 2004;20:979 – 83. [4] Bayless TM, Rosensweig NS. A racial difference in incidence of lactase deficiency: a survey of milk intolerance and lactase deficiency in healthy adult males. J Am Med Assoc 1966;197:138 – 42.
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