The Journal of Nutrition Vol. 128 No. 5 May 1998, pp. 848-854

Hydroxypropyl-Modified Potato Starch Increases Fecal Bile Acid Excretion in Rats¹

Kiyoshi Ebihara², Rumiko Shiraishi, and Kazuhiro Okuma^*

Department of Biological Resources, Faculty of Agriculture, Ehime University, Matsuyama 790, Japan; and ^* Research Institute, Matsutani Chemical Industry Co., Ltd., Itami 664, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The effects of hydroxypropyl potato starches (HPS) of three different degrees of substitution (DS) on concentration of plasma cholesterol, apparent digestibility of protein, fecal excretion of bile acids, fecal output and cecal pool of organic acids such as acetic, propionic, butyric, lactic and succinic acid were studied in rats in Experiment 1. In Experiment 2, the effects of hydroxypropyl distarch phosphate (HDP) of three different degrees of cross-linking (DC) on the same indexes were studied. Gelatinized potato starch that was not modified chemically (PS) was used as a control. Rats were fed a fiber-free, purified diet containing either HPS, HDP or PS (100 g/kg) for 21 d. In each experiment, fecal output was greater and fecal excretion of bile acids was higher in rats fed the HPS diets with higher DS and the HDP diets compared with control rats fed the PS diet. Apparent protein digestibility in rats fed the HPS diets with higher DS and the HDP diets with higher DC was lower than that in control rats fed the PS diet. The pool size of cecal organic acids was not affected by diet. In Experiment 1, apparent protein digestibility, fecal output and fecal bile acids excretion were significantly correlated with DS (r = 0.994, P = 0.0059; r = 0.976, P = 0.0236; and r = 0.899, P = 0.0077, respectively). The plasma cholesterol concentration was significantly lower in rats fed the HPS diets than in control rats fed the PS diet. The HPS diets resulted in higher proportions of propionic acid, lactic acid and succinic acid and a lower proportion of n-butyric acid than the PS diet. In Experiment 2, apparent protein digestibility was significantly correlated with DSP (r = 0.996, P = 0.0028), which was inversely related to DC. The HDP diets did not affect the plasma cholesterol concentration. The HDP diets resulted in higher proportions of acetic acid, lactic acid and succinic acid and a lower proportion of n-butyric acid than the PS diet. These results suggest that the physiological effects of chemically modified starches are affected by the type of modification.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Chemically modifiedstarches (CMS)³ are more resistant to retrogradation, have higher viscosity and are more stable to acid and high temperatures than native starch. Therefore, they are increasingly used by the food industry to improve the physical properties of various food items, including baby food. Their consumption is increasing as the consumption of processed foods increases. Many investigators have observed a reduced susceptibility of CMS to enzymes in vitro and in vivo compared with the corresponding unmodified starch (Björck et al. 1989, Ebihara 1992a, Östergård et al. 1988, Whistler and Belfort 1961, Wootton and Chaudhry 1981). The enzymic susceptibility of CMS was affected by the type of modification such as substitution and cross-linking. The presence of CMS in the gut digesta may influence gastric emptying, digestion of other nutrients, satiety and absorption of other compounds. CMS that reach the large bowel would influence bowel habits, cell proliferation and bile acid metabolism as a result of fermentation. Therefore CMS may exert physiological effects similar to those of dietary fiber. However, there is very little published information on the physiological effects of CMS in animals and humans, or on whether various types of CMS have different physiological effects.

We evaluated the physiological effect of two different types of CMS, potato starch modified either by substitution only or by a combination of substitution and cross-linking. Three kinds of hydroxypropyl starch (HPS) with different degrees of substitution and three kinds of hydroxypropyl distarch phosphate (HDP) with different degrees of cross-linking were used as CMS. These materials were chosen for this study because they are widely used by the food industry.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Chemically modified starches (CMS).  The following chemically modified and gelatinized potato starches were used: three kinds of HPS, namely, HPS-a, HPS-b and HPS-c and three kinds of HDP, namely, HDP-A, HDP-B and HDP-C (Table 1). The hydroxypropyl substituents were introduced by causing the starch to react with propylene oxide at alkaline pH in the presence of Na₂SO₄. Cross-linking of the etherified starch was performed with Na₃(PO₃)₃ (0.5, 1.2 or 3.0 g/600 mL) at alkaline pH. All derivatives, as well as the unmodified potato starch, were also added to diets in a pregelatinized powdery form. The degree of substitution (DS) was measured according to the method of Johnson (1969). The degree of swelling power (DSP) for HDP was as follows: HDP-A > HDP-B > HDP-C. Kainuma et al. (1975) have shown that there is a good inverse relationship (r = 0.861, P = 0.0275) between the degree of cross-linking (DC) and the DSP of starch granules. Therefore the DC for HDP is as follows: HDP-A < HDP-B < HDP-C. The DSP of HDP was measured using a slight modification of the method described by Leach et al. (1959). Briefly, 1 g of dry sample was accurately weighed into a 50-mL graduated centrifugal tube. After adding 1 mL of methanol, 25°C distilled water was added by mixing with a glass rod until the total volume was 50 mL. A graduated centrifugal tube was shaken occasionally in a water bath at 25°C for 20 min to prevent the formation of a precipitate of HDP. After centrifugation at 1200 × g for 30 min at room temperature, the volume of aqueous supernatant was measured. Then the total sugar in the aqueous supernatant was analyzed with phenol/H₂SO₄ reagent (Dubois et al. 1956). Weights of precipitates of swollen granules were measured. The DSP of HDP was calculated as follows: where A is the total sugar in a supernatant (mg), B is the precipitate (mg) and S is the solubility (%).

Measurement of amylase-resistant starch.  Amylose-resistant starch is that which is not hydrolyzed to glucose by amylotic enzymes. For the determination of amylase-resistant starch in HPS and HDP, 1 g of triplicate samples was incubated at pH 6.0 for 30 min at 100°C with heat-stable -amylase (Termamyl 120L; Novo Nordisk, Copenhagen, Denmark) and then allowed to cool. After cooling, the pH was adjusted to 4.5, and the samples were incubated with amyloglucosidase (Sigma A-9913; Sigma Chemical, St. Louis, MO) for 30 min at 60°C. After incubation, the amounts of glucose in the hydrolysates were enzymatically measured using pyranoseoxidase (Determiner GL-E; Kyowa Medix, Tokyo, Japan). The concentration of amylase-resistant starch was calculated as follows: where wt sample is the initial weight (g) and G is the the hydrolysate (g).

Animals and diets.  This study was approved by the Laboratory Animal Care Committee of Ehime University, and rats were maintained in accordance with the guidelines for the care and use of laboratory animals of Ehime University.

Male Wistar rats (Japan SLC, Hamamatsu, Japan) with an initial weight of ~80 g were used in these experiments. Rats were housed individually in cages with screen bottoms of stainless steel in a room maintained at 23 ± 1°C with a 12-h light:dark cycle (light, 0700-1900 h). Rats were acclimated by feeding a commercial solid diet (MF, Oriental Yeast, Osaka, Japan) for 7 d. After acclimation, rats were divided into four groups of six (each with similar mean body weight) in Experiments 1 and 2. The experimental diets contained the following ingredients (g/kg diet): casein, 200; corn, 50; AIN-76 mineral mixture (AIN 1977), 40; AIN-76 vitamin mixture, 10; sucrose, 600; and starch, 100. PS, HPS and HDP were used as starch. The AIN-76 vitamin mixture used in this study contained 20 g/100 g choline bitartrate. Rats were given free access to the experimental diets and water for 21 d. Body weights and food intakes were recorded daily for each rat in the morning before replacing the diet. Then, the condition of feces was observed for each rat.

Sampling and analytical procedures.  Before the rats were killed, feces were collected during the final 3 d of the experimental period from individual rats, freeze-dried and weighed. Nitrogen (N) in diets and feces was analyzed in duplicate for each collection by using the Kjeldahl method (Miller and Houghton 1945). The apparent digestibility of protein (N·6.25) was calculated by the measurement of N contents in the food and feces. Total fecal steroids were extracted with a mixture of chloroform:methanol (1:1, v/v) at 70°C for 60 h (Eneroth et al. 1968). Total fecal bile acids were determined enzymatically by the 3-hydroxysteroid dehydrogenase assay method of Sheltaway and Losowsky (1975) with taurocholic acid as the standard.

Blood was collected from the abdominal aorta of rats under sodium pentobarbital anesthesia (50 mg/kg body mass, Nembutal, Abbot Laboratories, North Chicago, IL) in a blood collection tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) that contained heparin as anticoagulant. Plasma was separated by centrifugation at 1400 × g for 15 min at 4°C and stored at 50°C until analyzed. After blood collection, the ileocecal and cecocolonic junctions were ligated and the cecum was removed. After removal, the cecum was weighed with contents (total cecal weight); the contents were drained from the cecocolonic junction into a cooled 50-mL vial and mixed well under CO₂ gas. Cecal pH was immediately measured with a compact pH meter using a sampling sheet (Model C-1, Horiba, Tokyo, Japan; calibrated at 20°C). The cecal wall was flushed with ice-cold saline, blotted on filter paper and weighed (cecal wall weight). The liver was also removed, weighed and then stored at 50°C for further analyses.

Cecal organic acids (acetic, propionic, n-butyric, succinic and lactic) were measured by HPLC (Shouwa Denko, Tokyo, Japan). Cecal contents (~500 mg) were homogenized by ultrasonication (USC-6; Iwaki Glass, Chiba, Japan) in a threefold weight of distilled water and then centrifuged at 1500 × g for 5 min. The supernatant was mixed with an equivalent amount of methanol and then centrifuged at 10,000 × g for 15 min. The supernatant was filtered through a membrane filter (cellulose acetate, pore size 0.45 µm; DISMIC-13cp; Toyo Roshi, Tokyo, Japan). These samples were applied to HPLC (Shouwa Denko, Tokyo, Japan) for an analysis of organic acids. Organic acids were separated with two ion exclusion columns (Ion-pack KC-811 and KC-810p, 8 mm i.d. × 30 cm long and 6 mm i.d. × 5 cm; Shouwa Denko), column temperature 45°C, a mobile phase of 2 mmol/L perchloric acid (flow rate, 1 mL/min, 45°C) and then reacted with a commercial detection reagent (ST3-R; 15 mmol/L disodium hydrogenphosphate, 0.2 mmol/L bromothymol blue, 2 mmol/L sodium hydroxide; Shouwa Denko). The optical density at 430 nm was recorded with the use of a spectrophotometer (UV-8000, Toyo Soda, Tokyo, Japan).

Triglyceride and cholesterol in plasma were enzymatically determined with a commercial diagnostic kit (Triglyceride E-Test Wako and Cholesterol E-Test Wako, Wako Pure Chemical, Osaka, Japan). Liver total lipids were determined gravimetrically after extraction by the method of Folch et al. (1957). Liver total cholesterol was measured colorimetrically with a commercially available kit (Cholesterol E-Test Wako, Wako Pure Chemical). Lipids were extracted from 500 mg liver with chloroform:methanol (2:1,v/v) according to the method of Folch et al. (1957). After lipid extraction, the lipid solution volume was adjusted to 20 mL with the same solution. From this extract, 1 mL was dried under a nitrogen stream; the residue obtained was mixed with 100 µL isopropyl alcohol containing 10% Triton-X 100 (Wako Pure Chemical). From this mixture, 30 µL was mixed with 3 mL of aqueous enzyme solution according to the standard procedure of the assay kit, and cholesterol concentration was determined colorimetrically. In a preliminary study, 30 µL of isopropyl alcohol containing 100 g Triton-X 100/L did not affect the enzymatic reactions (data not shown).

Statistical analysis.  Data are expressed as means ± SEM. Data were analyzed by ANOVA using Super ANOVA (Abacus Concepts, Berkeley, CA), and the differences among groups were examined using Duncan's multiple range test (Shibata 1974) when the F-value was significant. A P-value  0.05 was considered significant. Correlation coefficients were determined by linear regression (Snedecor and Cochran 1967)

	RESULTS

Abstract Introduction Methods Results Discussion References

The properties of HPS and HDP.  The amount of amylase-resistant starches in HPS and HDP was markedly greater than in PS, which was used as a control (Table 1). In HPS, the amount of amylase-resistant starch increased with increasing DS (r = 0.952, P = 0.0479). In HDP, the amount of amylase-resistant starch decreased with decreasing DSP (r = 0.929, P = 0.0326).

Experiment 1. Body mass gain, food intake and feed efficiency were unaffected by diet (Table 2). HPS caused diarrhea. The severity of diarrhea increased with increasing DS, but intestinal adaptation took place during wk 1 of feeding. Apparent protein digestibility was significantly lower in rats fed HPS-b and HPS-c, but not in rats fed HPS-a, than in the control rats fed PS. Apparent protein digestibility was inversely and significantly correlated with DS (r = 0.994, P = 0. 0059). Fecal output was significantly greater in rats fed HPS-b and -c than in the control rats fed PS and increased with increasing DS (r = 0.976, P = 0.015).

The plasma cholesterol concentration was significantly lower in rats fed the HPS diets than in rats fed the PS diet (Table 3). The plasma triacylglycerol concentration and total liver lipids were not affected by the diets. Fecal excretion of bile acids was significantly higher in rats fed HPS-b and -c than in the control rats fed PS, but not in rats fed HPS-a. Fecal excretion of bile acids increased with increasing DS (r = 0.899, P = 0.0077).

Feeding of HPS-c led to an increase in the weight of cecal contents (Table 4). Feeding of HPS-a led to an increase in the weight of the cecal wall. These cecal alterations caused by HPS were gradually intensified with increasing DS. The pH value of cecal contents in rats fed the HPS diets was significantly lower than that in rats fed the PS diet. The cecal pool of organic acids, expressed as micromoles per cecum, did not differ in rats fed the HPS diets and rats fed the PS diet. However, the proportion of n-butyric acid in the cecal pool of organic acids was significantly lower in rats fed the HPS diets compared with rats fed the PS diet. The proportions of propionic, lactic and succinic acids were higher in rats fed the HPS diets compared with rats fed the PS diet.

Experiment 2. Body mass gain, food intake and feed efficiency were unaffected by the diets (Table 2). HDP caused diarrhea. The severity of diarrhea increased with increasing DC, but intestinal adaptation took place during wk 1 of feeding. Apparent protein digestibility was significantly lower in rats fed HDP-B and HDP-C, but not in rats fed HDP-A, than in the control rats fed PS. Apparent protein digestibility was significantly correlated with DSP (r = 0.996, P = 0.028). Fecal output was significantly greater in rats fed the HDP diets than in rats fed the PS diet.

The plasma cholesterol concentration was unaffected by the diets (Table 3). The plasma triacylglycerol concentration, total liver lipids and liver cholesterol were also not affected by the diets. Fecal excretion of bile acids was significantly higher in rats fed the HDP diets than in rats fed the PS diet.

Feeding HDP-B and HDP-C increased the weight of cecal contents (Table 4). Feeding the HDP diets increased the weight of the cecal wall. The pH values of cecal contents in rats fed the HDP diets were significantly lower than values in those fed the PS diet. The cecal pool of organic acids, expressed as micromoles per cecum, did not differ in rats fed the HDP diets and those fed the PS diet. However, the percentage of n-butyric acid in the cecal pool of organic acids was significantly lower in rats fed the HDP diets compared with rats fed the PS diet. The proportions of acetic acid, lactic acid and succinic acid were higher in rats fed the HDP diets than in rats fed the PS diet.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The contents of amylase-resistant starch in HPS and HDP were greater than in PS, which was used as the control. The content of amylase-resistant starch in HPS gradually increased with increasing DS, in agreement with the results of earlier studies of the in vitro digestibility by pancreatin (Leegwater and Luten 1971, Wootton and Chaudhry 1979 and 1981). This can be explained by the greater restriction of enzyme action by increasing numbers of hydroxypropyl groups, because the hydroxypropyl glucopyranose units prevent hydrolysis of adjacent (1,4)glucosidic bonds by pancreatic enzymes (Leegwater and Luten 1972). On the other hand, the content of amylase- resistant starch in HDP gradually decreased with increasing DC. This may have resulted from a slight decrease in starch crystallinity caused by the alkaline conditions used in the introduction of cross-linking with phosphate, which allowed better access to the enzyme. The content of amylase-resistant starch in HPS-b and HDP-B was about the same. This shows that the cross-linking with phosphate had little effect on the in vitro enzymic hydrolysis. Björck et al. (1989) reported that the cross-linking with phosphate did not have a major effect on in vivo digestibilities of acetyl distarch phosphate and hydroxypropyl distarch phosphate from potato starch.

A summary of investigations concerning the in vivo digestibility of various CMS has been published by FAO/WHO (WHO 1972). In some reports, a reduced digestibility was observed after modification, often in association with diarrhea and cecal enlargement, particularly at high levels of intake. In this study, both HPS and HDP, at 100 g/kg diet, caused diarrhea. The severity of diarrhea increased with increasing DS and with increasing DC. However, body mass gains in rats fed the HPS and HDP diets did not differ from those of rats fed the PS diet. Whistler and Belfort (1961) showed that chemically modified cornstarch, hydroxyethyl starch and cornstarch phosphate do not adversely affect rat growth. The apparent protein digestibility was lower in rats fed the HPS and HDP diets than in rats fed the PS diet. Generally, the decrease of feed efficiency is caused by a decrease in the digestibility of proteins and/or energy sources, and by a decrease in the retention of nutrients resulting from various metabolic disturbances. However, significantly lower feed efficiencies were not found in rats fed the HPS and HDP diets compared with that in rats fed the PS diet. Thus, it does not appear that HPS and HDP interfered with the digestion and absorption of protein and energy sources. On the other hand, fecal N excretion and fecal output were greater in rats fed the HPS and HDP diets than in rats fed the PS diet. The greater fecal N excretion is caused by a decrease in protein digestibility, an increase in production of endogenous protein (e.g., digestive enzymes or sloughed cells) and/or more fecal biomass. Bacteria in the large bowel have a major role in fecal bulking and constitute a major proportion of fecal N (Scheppach et al. 1988). However, the amylase-resistant starch in HPS and HDP was not a good substrate for bacteria in the large bowel (Björck et al. 1988, Ebihara 1992b). Thus the greater fecal N excretion and the lower apparent protein digestibility in rats fed HPS and HDP diets might be explained by an increase of fecal output as a result of a shorter transit time in the large bowel.

Both HPS and HDP had laxative effects, as does any carbohydrate that reaches the colon. When the magnitude of the effect was calculated as an increase in fecal wet weight per gram of amylase-resistant starch consumed, the effect was as follows (g/g): 0.481 for HPS-a, 0.594 for HPS-b, 0.645for HPS-c, 0.826 for HDP-A, 1.131 for HDP-B and 1.597 for HDP-C. The effect of HPS increased with increasing DS (r = 0.958, P = 0.0346). The amylase-resistant starch is water insoluble and has no water-holding properties (data not shown). Thus it is unlikely that the laxative effects of HSP and HDP are due to the water-holding capacities of amylase-resistant starch structures.

HPS lowered the plasma cholesterol concentration, but HDP did not. Because the experimental diet was not supplemented with cholesterol, the hypocholesterolemic effect of HPS must involve changes in endogenous sterol metabolism. One possible explanation concerns the increase in fecal excretion of cholesterol and bile acids. Binding of bile acids by HPS in the small intestine might have occurred, because starch has been shown to bind bile acids in vitro (Bianchini et al. 1989). The fecal excretion of bile acids was significantly greater in rats fed the HPS and HDP diets than in rats fed the PS diet. It seems that HPS and HDP, by enhancing fecal bile acid excretion, cause an increased hepatic synthesis of bile acids and reduce serum cholesterol concentrations. But it is still unclear whether HPS and HDP can effectively bind bile acids. Another possible explanation concerns propionic acid production from fermentation of HPS and HDP in the large bowel.

When propionic acid was fed to rats, it significantly decreased serum cholesterol concentration (Illman et al. 1988). Propionic acid infused into the cecum prevented the increase of plasma cholesterol concentration in rats fed a cholesterol-free, casein diet (Ebihara et al. 1993). Further, in vitro studies using rat liver cells have shown that propionic acid may attenuate hepatic cholesterol synthesis (Wright et al. 1990). The amount of propionic acid in cecal contents was significantly higher in rats fed the HPS diet than in rats fed the PS diet, but not in rats fed the HDP diet. Thus, the hypocholesterolemic effect of HPS might be due to the greater production of propionic acid. However, other studies have shown that propionic acid has no hypocholesterolemic effects (Beaulieu and McBurney 1992, Venter et al. 1990). Therefore HPS may affect the plasma cholesterol concentration by a combination of increases in fecal bile acid excretion and production of propionic acid. Further investigation is required to clarify the mechanism for the hypocholesterolemic effect of HPS.

Cecal enlargement was observed in rats fed the HPS-c, HDP-A, HDP-B and HDP-C diets, but not in those fed the HPS-a and HPS-b diets. Oku et al. (1981) speculated that cecal enlargement depends on the amount of maldigested materials reaching the cecum. The content of amylase-resistant starch in HPS-c, HDP-A, HDP-B and HDP-C was larger than that in HPS-a and HPS-b. Therefore the cecal enlargement caused by the HPS-c, HDP-A, HDP-B and HDP-C diets may be explained by an increased influx of the unabsorbed starch fraction into the cecum. Cecal tissue weight in rats fed the HPS and HDP diets was heavier than that in rats fed the PS diet. Nonfermentable bulk predominantly increases the thickness of the muscularis externa (Dowling et al. 1967). There was a positive correlation between cecal contents and cecal tissue weight in rats fed diets containing resistant starch (Verbeek et al. 1995). Thus the increase in cecal contents may contribute to the heavier cecal tissue weight in rats fed the HPS and HDP diets.

A strong negative correlation was found between cecal pH and the cecal pool of short-chain fatty acids (Berggren et al. 1993). However, in spite of a similar cecal pool size of organic acids, the cecal pH in rats fed the HPS and HDP diets was lower than that in rats fed the PS diet. Lactic acid is poorly and slowly absorbed (Giesecke and Stangassinger 1980, Macfarlane and Cummings 1991). Hoshi et al. (1994) suggested that the higher concentration of cecal succinic acid appears to contribute predominantly to the lower cecal pH. Thus a probable explanation for lower pH in rats fed the HPS and HDP diets could be the greater proportion of lactic and succinic acids to total organic acids.

The cecal organic acid pool size was not affected by diet, but the proportion of each organic acid in the cecal pool of organic acids was affected. Thus the proportion of each organic acid in the cecal pool of organic acids is probably more reliable as an indicator of dietary change than the total pool size. A recent in vitro study examined carbohydrate fermentation in human fecal slurries and found that starch fermentation increased butyric acid production (Cummings and Macfarlane 1991, Høverstad and Bjørneklett 1984). In this study, the proportions of n-butyric acid in rats fed the HPS and HDP diets were lower than that in rats fed the PS diet. This lower proportion of n-butyric acid could be explained by the cecal enlargement, because butyric acid is so readily oxidized by cecal and colonic epithelial cells (Roediger 1982). Also, this lower proportion of n-butyric acid could be explained by lower concentrations of butyric acid-producing bacteria.

The amylase-resistant starch in HPS and HDP may be not a good substrate for butyric acid-producing bacteria. The proportions of lactic acid in rats fed HPS and HDP diets were higher than that in rats fed the PS diet. This result would indicate that HPS and HDP are significantly fermented by bifidobacteria. However, the pancreatin-indigestible fractions in HPS and HDP were not good substrates for lactic acid-producing bacteria in the in vitro bacterial fermentation (Ebihara 1992a). The proportions of succinic acid in rats fed HPS and HDP diets were higher than that in rats fed the PS diet. The proportion of succinic acid increased with increasing DS and DC. This increase could depend on a higher concentration of amylase-resistant starch. On the other hand, the proportion of propionic acid was significantly higher in rats fed the HPS diet than in rats fed the PS diet, but not in rats fed HDP diet. It has been reported that the apparent digestibility of CMS and the structure of the maldigested fraction in feces after administration of CMS depend on the type of modification (Björck et al. 1989).

These results show that the amylase-resistant starch in HPS is different in quality and quantity from that in HDP and suggest that the physiological effects of CMS are affected by the type of modification.

	ACKNOWLEDGMENT

The authors thank Taro Kishida for the analysis of cecal organic acids.

	FOOTNOTES

Manuscript received 20 May 1997. Initial reviews completed 14 July 1997. Revision accepted 20 January 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977; 107:1340-1348
• Beaulieu K. E., McBurney M. I. Changes in nutrient digestibility and sterol excretion during cecal infusion of propionate. J. Nutr. 1992; 122:241-245
• Berggren A. M., Björck I. M. E., Nyman E.M.G.L., Eggum B. O. Short-chain fatty acid content and pH in caecum of rats given various sources of carbohydrates. J. Sci. Food Agric. 1993; 63:397-406
• Bianchini F., Caderni G., Dolara P., Fantetti L., Kriebel D. Effect of dietary fat, starch and cellulose on fecal bile acids in mice. J. Nutr. 1989; 119:S121-S122
• Björck, I., Gunnarsson, A. & Östergård, K. (1989) A study of native and chemically modified potato starch. Part II: Digestibility in the rat intestinal tract. Starch/Stärke 41: 128-134.
• Cummings J. H., Macfarlane G. T. The control and consequences of bacterial fermentation in the human colon. J. Appl. Bacteriol. 1991; 70:443-459 [Medline]
• Dowling R. H., Riecken E. O., Laws J. W., Booth C. C. The intestinal response to high bulk feeding in the rat. Clin. Sci. (Lond.) 1967; 32:1-9 [Medline]
• Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. Calorimetric method for determination of sugars and related substances. Anal. Chem. 1956; 28:350-356
• Ebihara K. In vitro -amylase hydrolysis of modified starch and postprandial plasma glucose response. J. Jpn. Soc. Nutr. Food Sci. 1992a; 45:551-553
• Ebihara K. Utilization of pancreatin-indigestible parts of modified starch by various intestinal bacteria. J. Jpn. Soc. Nutr. Food Sci. 1992b; 45:554-559
• Ebihara K., Miyada T., Nakajima A. Hypocholesterolemic effect of cecally infused propionic acid in rats fed a cholesterol-free, casein diet. Nutr. Res. 1993; 13:209-217
• Eneroth P., Hellstrom K., Sjovall J. A method for quantitative determination of bile acids in human feces. Acta Chem. Scand. 1968; 22:1729-1744 [Medline]
• Folch J., Less M., Sloane-Stanley G. H. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 1957; 226:497-509 ^{[Free Full Text]}
• Giesecke, D. & Stangassinger, M. (1980) Lactic acid metabolism. In: Digestive Physiology and Metabolism in Ruminant (Ruckebush, Y. & Thivend, P., eds.), pp. 523-539, M.T.P. Press, Lancaster, UK.
• Hoshi S., Sakata T., Mikuni K., Hashimoto H., Kimura S. Galactosylsucrose and xylosylfructoside alter digestive tract size and concentrations of cecal organic acids in rats fed diets containing cholesterol and cholic acid. J. Nutr. 1994; 124:52-60
• Høverstad T., Bjørneklett A. Short-chain fatty acids and bowel functions in man. Scand. J. Gastroenterol. 1984; 19:1059-1065 [Medline]
• Illman R. J., Topping D. L., McIntosh G. H., Trimble R. P., Storer G. B., Taylor M. N., Cheng B.-Q. Hypocholesterolemic effects of dietary propionate: studies in whole animals and perfused rat liver. Ann. Nutr. Metab. 1988; 32:97-107
• Johnson D. P. Spectrophotometric determination of the hydroxy-propyl group in starch esters. Anal. Chem. 1969; 41:859-860
• Kainuma K., Miyamoto S., Suzuki S. Studies on structure and physico-chemical properties of starch. Part 2. Reaction of Epichlorohydrin with corn starch. Denpun Kagaku 1975; 22:66-71
• Leach H. W., McCowen L. D., Schoch T. J. Structure of the starch granule I. Swelling and solubility patterns of various starches. Cereal Chem. 1959; 36:534-544
• Leegwater, D. C. & Luten, J. B. (1971) A study on the in vitro digestibility of hydroxypropyl starches by pancreatin. Starch/Stärke 23: 430-432.
• Leegwater, D. C. & Luten, J. B. (1972) A model for the structure of hydroxy-propyl starch. Starch/Stärke 24: 11-15.
• Macfarlane, G. T. & Cummings, J. H. (1991) The colonic flora, fermentation, and large bowel digestive function. In: The Large Intestine: Physiology, Pathophysiology, and Disease (Phillips, S. F., Pemberton, J. H. & Shorter, R. G. eds.), pp. 51-91. Raven Press, New York, NY.
• Milller L., Houghton J. A. The micro-Kjeldahl determination of the nitrogen content of amino acids and proteins. J. Biol. Chem. 1945; 159:373-380 ^{[Free Full Text]}
• (in Japanese) Oku T., Konishi F., Hosoya N. Effect of various unavailable carbohydrate and administration periods on several physiological functions of rats. J. Jpn. Soc. Nutr. Food Sci. 1981; 34:437-443
• Östergård, K., Björck, I. & Gunnarsson, A. (1988) A study of native and chemically modified potato starch. Part I: Analysis and enzymic availability in vitro. Starch/Stärke 40: 58-66.
• Roediger W.E.W. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982; 83:424-429 ^[Medline]
• Scheppach W., Fabian C., Ahrens F., Spengler M., Kasper H. Effect of starch malabsorption on colonic function and metabolism in humans. Gastroenterology 1988; 95:1549-1555 [Medline]
• Sheltaway M. J., Losowsky M. S. Determination of fecal bile acids by an enzymic method. Clin. Chem. Acta 1975; 64:127-132 ^[Medline]
• Shibata, K. (1974) Basic Statistics for Biologist (in Japanese), p. 64, Shoubun Publishing Co., Tokyo, Japan.
• Snedecor, G. W. & Cochran, W. G. (1967) Statistical Methods, 6th ed., pp. 166-190. Iowa State University Press, Ames, IA.
• Venter C. S., Vorster H. H., Cummings J. H. Effects of dietary propionate on carbohydrate and lipid metabolism in healthy volunteers. Am. J. Gastroenterol. 1990; 85:549-553 [Medline]
• Verbeek M.J.F., De Deckere E.A.M., Tijburg L.B.M., Van Amelsvoort J.M.M., Beynen A. C. Influence of dietary retrograded starch on the metabolism of neutral steroids and bile acids in rats. Br. J. Nutr. 1995; 74:807-820 ^[Medline]
• Whistler, R. L. & Belfort, A. M. (1961) Nutritional value of chemically modified corn starch. Science (Washington, DC) 133: 1599-1600.
• Wootton, M. & Chaudhry, M. A. (1979) Enzymic digestibility of modified starches. Starch/Stärke 31: 224-228.
• Wootton, M. & Chaudhry, M. A. (1981) In vitro digestion of hydroxypropyl derivatives of wheat starch. I. Digestibility and action pattern using porcine pancreatic alpha-amylase. Starch/Stärke 33: 135- 137.
• World Health Organization (1972) Food Additive Series No 1. Toxicological Evaluation of Some Enzymes, Modified Starches and Other Substances. WHO, Geneva, Switzerland.
• Wright R. S., Anderson J. W., Bridges S. R. Propionate inhibits hepatocyte lipids synthesis. Proc. Soc. Exp. Biol. Med. 1990; 195:26-29 [Medline]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences