The Journal of Nutrition Vol. 128 No. 4 April 1998, pp. 688-693

Fermentation Products of Sugar-Beet Fiber by Cecal Bacteria Lower Plasma Cholesterol Concentration in Rats¹

Hiroshi Hara², Satoko Haga, Takanori Kasai, and Shuhachi Kiriyama³

Laboratory of Nutritional Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Plasma cholesterol concentration is reduced by feeding some dietary fibers but the mechanism is not fully understood. We examined whether cecal fermentation products are involved in lowering plasma cholesterol by feeding rats a highly fermentable sugar-beet fiber (SBF) in four separate experiments. These were designed to investigate the effects on plasma cholesterol of oral ingestion of fermentation products on plasma cholesterol, the effects of the products in comparison with that of a short-chain fatty acid (SCFA) mixture, effects of individual SCFA and effects of alteration of energy and nitrogen ratio in the diet by the addition of the SCFA mixture. Cecal contents of rats were cultured with SBF by using a jar fermenter under anaerobic conditions, and the supernatant from the culture medium, containing fermentation products of SBF, was collected and freeze-dried before feeding to rats. Yield of fermentation products as dry weight from the fiber was 80-90%. In rats fed a diet containing fermentation products (80 g/kg diet), plasma cholesterol concentrations were lower than in rats of the fiber-free group 3, 7 and 14 d after feeding the test diet. Major SCFA in the fermentation products were sodium salts of acetic, propionic and butyric acids. Plasma cholesterol concentration in rats fed the diet containing a mixture of equal amounts of the three SCFA salts (66 g/kg diet) as the fermentation products diet was also lower than that in the fiber-free group and was not different from those in rats fed SBF (100 g/kg diet) and the fermentation products. In rats fed an acetate-containing diet but not in rats fed diets without acetate, plasma cholesterol was significantly lower than in the fiber-free group. In conclusion, absorption of SCFA from cecal fermentation products lowers plasma cholesterol. Acetate, and not propionate, may be responsible for lowering plasma cholesterol concentration.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Feeding some dietary fibers lowers plasma cholesterol concentration (Chen and Anderson 1979, Kiriyama et al. 1969). The mechanism is not fully understood. It is proposed that cecal and colonic fermentation of dietary fiber is associated with cholesterol-lowering effects of fibers (Anderson 1985, Moundras et al. 1995). Feeding of sugar-beet fiber (SBF), a highly fermentable dietary fiber, lowered plasma cholesterol concentration in rats (Aritsuka et al. 1989, Overton et al. 1994); Nishimura et al. (1993) reported that the lowering effect of the fiber disappeared when the cecum was resected. This finding shows that the large intestine is involved in the reduction of plasma cholesterol in rats fed SBF.

Two possible mechanisms are proposed for the ceco-colonic-dependent decrease in plasma cholesterol. As the first mechanism, fiber or its fermentation products stimulate the large intestine, and humoral factors secreted from the large intestine or enteral nervous system modify cholesterol metabolism. Goodlad et al. (1989) showed that colonic fermentation of dietary fiber elevates plasma enteroglucagon, and it is known that the large intestine is important for production of this hormone (Kennedy et al. 1982). In addition, the enteric nervous system in the large intestine is possibly stimulated by fiber or its fermentation products. Some neuropeptides are known to influence cholesterol and bile acid metabolism (Cho et al. 1997, Farouk et al. 1992, Gebhard et al. 1981). In these cases, the large intestine is essential for the beneficial effects of dietary fiber. Second, propionate, a fermentation product of SBF, absorbed from the intestine can modify cholesterol synthesis directly (Lin et al. 1995, Nishina and Freedland 1990). Bridges et al. (1992) suggested that acetate is involved in the serum cholesterol-lowering effect of oat bran in humans.

The purpose of this study was to determine whether the cecal fermentation products of SBF decrease plasma cholesterol concentration. We prepared fermentation products of this fiber source with the cecal contents (as a source of cecal bacteria) of rats using a jar fermenter.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Fermentation products.  Fermentation products of SBF (Nippon Sugar Beet Manufacturing, Obihiro, Japan) were prepared by incubating the SBF with the cecal contents of rats fed a SBF diet (100 g/kg diet) for 10 d. That is, the cecal contents of two rats were cultured with SBF (70 g/3 L) in M 10 media (Itoh and Mitsuoka 1985) without agar, cellobiose and SCFA in a jar fermenter (Tokyo Rikakikai, Tokyo, Japan) at 37°C for 72 h under anaerobic conditions with CO₂ gas. Concentrations of acetic, propionic and butyric acids were increased in the culture medium, and pH of the medium was decreased over 72 h of culture as shown in Figure 1. After an incubation, the supernatant was separated by centrifugation and freeze-dried (fermentation products). The pellet was washed, dried and milled (fermented residue). To obtain sufficient amounts of the preparations, the procedure was repeated several times. Yields of fermentation products and residue used in Experiment 1 were 80.0 and 14.6% for the initial weight of SBF, and that of fermentation products used in Experiment 2 was 91.9%. Total fiber concentrations of fermentation products used in Experiments 1 and 2 were 14 and 17 g/kg product, respectively, and of residue and SBF were 830 and 816 g/kg dry matter, respectively. Acetic, propionic and butyric acid content (as sodium salt) in dried fermentation products used in Experiment 1 were 346 (4.22 mol), 148 (1.54 mol) and 101 (0.91 mol) g/kg product, and those used in Experiment 2 were 386 (4.71 mol), 245 (2.55 mol) and 100 (0.90 mol) g/kg product, respectively.

View larger version (20K): [in this window] [in a new window]   Fig 1. Representative changes in short-chain fatty acid concentrations and pH in a culture medium. The cecal contents of rats were cultured with sugar-beet fiber. Details are described in Materials and Methods.

Animals and diets.  Male Wistar-ST rats (Japan SLC, Hamamatsu, Japan), weighing ~100 g, were fed a semipurified sucrose-based diet containing casein, 250 g/kg diet (basal diet, Table 1) for 7 d. Four separate experiments were conducted to examine effects of the fermentation products on plasma cholesterol concentration. Rats were divided into diet groups on the basis of plasma cholesterol concentration and body weight, and fed test diets. Fiber, fermentation products and artificial short-chain fatty acid (SCFA) mixture were added to the fiber-free diet at the expense of whole diet (Experiments 1, 2 and 3). Throughout all experiments, rats were housed in individual cages in a temperature-controlled room at 22°C. The study was approved by the Hokkaido University Animal Committee, and animals were maintained in accordance with the guidelines for the care and use of laboratory animals of Hokkaido University.

Experimental protocol.  In Experiment 1, rats of four groups were fed fiber-free diet (basal diet), and three test diets containing SBF (100 g/kg diet), soluble fermentation products (80.0 g/kg diet) and fermented residue (14.6 g/kg diet) for 14 d. Amounts of fermentation products and fermented residue in the diet corresponded to yields of these preparations from SBF. Tail blood was sampled before and 3, 7 and 14 d after feeding the test diets, and plasma was separated to measure total cholesterol concentration. Feces were collected for the last 3 d. On the last day, rats were killed under anesthesia (nembutal/sodium pentobarbital, 50 mg/kg body weight, Abbott Laboratories, North Chicago, IL). The cecum with its contents was removed. The cecal contents were collected, weighed and stored at 40°C. The cecal walls were washed with saline and weighed.

In Experiment 2, rats of four groups were fed fiber-free diet (basal diet), and three test diets containing SBF (100 g/kg diet), fermentation products (91.9 g/kg diet); a mixture of short-chain fatty acids [SCFA diet, sodium salts of acetic, propionic and butyric acids (average of purity of three reagents was 96%, Wako Pure Chemical, Osaka, Japan) was added up to 35 g (0.43 mol), 22 g (0.23 mol) and 9.0 g (0.082 mol)/kg diet, respectively] for 8 d. Other details were as in Experiment 1 except for blood sampling. In this case, aortic blood was collected on the last day.

Experiment 3 was designed to examine effects of SCFA, acetic, propionic and butyric acids on concentrations of plasma cholesterol. In two separate experiments, rats were fed fiber-free diet, diet containing the three SCFA and diets containing one or two of the three SCFA for 14 d. The amount of each SCFA added to each test diet was the same as that in the diet used in Experiment 2. Other details were the same as Experiment 2.

In Experiments 2 and 3, additions of the SCFA mixture to a basal diet altered to some extent the proportion of nitrogen to energy in the diet. In Experiment 4, the ratio of nitrogen (casein) to energy in the fiber-free diet was matched to that in the SCFA mixture diet used in Experiments 2 and 3. Rats in one of two groups were fed the same SCFA diet used in Experiment 2 [69 g of the SCFA mixture (net amount of SCFA sodium salt is 66 g as shown above) and 931 g of basal diet]; in one group, rats were fed a diet that contained sucrose (54 g of sucrose and 931 g of basal diet) in place of SCFA at an equivalent energy level. The casein content of the sucrose-added, fiber-free diet was 230 g/kg test diet. These diets were fed isocalorically (weight ratio of given diets, SCFA diet/sucrose diet = 0.985:1). Tail blood was sampled before and 7 and 15 d after feeding the test diets.

Analyses.  Total fiber content of SBF and preparations was measured by the enzyme-gravimetric method of Prosky et al. (1985).

Plasma total and HDL cholesterol concentrations were measured using the enzymatic procedure (T-CHO, Kainos Laboratories, Tokyo, Japan) and HDL cholesterol-test (Wako Pure Chemical).

The cecal contents were homogenized with 9 volumes of distilled water. SCFA concentrations in the homogenate and cultured medium were evaluated by the previously described method (Hara et al. 1994). Individual SCFA were measured by gas-liquid chromatography (Shimadzu GC-14A) with a prepacked glass column [1600 mm × 3 mm, SP-1220 + H₃PO₄ (15% + 1%) on 80-100 mesh chromosorb W-AW DMCS, Shimadzu, Kyoto, Japan] after adding phosphoric acid (final concentration 0.67 mol/L).

The feces were freeze-dried, weighed and milled. Total bile acid was measured by an enzymatic method (Sheltawy and Losowsky 1975) using 3-hydroxysteroid dehydrogenase after chloroform/methanol (2:1, v/v) and alkaline-methanol extraction (Eneroth et al. 1968) from powdered feces.

Statistics.  Data were analyzed by one-way or two-way (Diet and Day, Figs. 2 and 4) ANOVA, and significant differences between diet groups were determined by Duncan's multiple range test (Duncan 1955, P < 0.05, SAS version 6.07, SAS Institute, Cary, NC). If the variance was unequal, log transformations of the data were performed before ANOVA. Values in the text are untransformed means ± SEM.

View larger version (23K): [in this window] [in a new window]   Fig 2. Plasma total cholesterol concentrations in rats fed fiber-free, sugar-beet fiber or fermented products diet 3, 7 and 14 d after feeding. Plasma cholesterol concentrations were decreased by feeding of diets containing sugar-beet fiber (100 g/kg diet) and its fermentation products (80.0 g/kg diet), but not after fermented residue (14.6 g/kg diet) in Experiment 1. These fermentation materials were dried supernatant and precipitate of a cultured medium of the cecal contents with sugar-beet fiber. Each value represents a mean ± SEM, n = 6. P-values estimated by two-way ANOVA were <0.001 for diet and day. Mean values not sharing a common letter are significantly different between diet groups on the same day (P < 0.05).

View larger version (49K): [in this window] [in a new window]   Fig 4. Effects of acetate (Ac), propionate (Prop) and butyrate (But) or their combination on plasma cholesterol concentration with two separate experiments (Experiment 3). Each value represents a mean, n = 6. Pooled SEM for total cholesterol and HDL cholesterol were 0.142 and 0.098 mmol/L in the left panel, and 0.087 and 0.085 mmol/L in the right panel, respectively. P-values estimated by one-way ANOVA were 0.006 for total cholesterol and 0.003 for HDL cholesterol in the left panel experiment, and 0.001 for total cholesterol and 0.035 for HDL cholesterol in the right panel experiment. Mean values not sharing a common letter are significantly different between diet groups (lowercase letters in HDL cholesterol and uppercase letters in total cholesterol, P < 0.05).

	RESULTS

Abstract Introduction Methods Results Discussion References

In all experiments, body weight gain and food intake were not different among groups as follows: 101 ± 1.7 g/14 d and 17.8 ± 0.26 g/d in Experiment 1, 51.2 ± 1.1 g/8 d and 15.9 ± 0.19 g/d in Experiment 2 and 105 ± 1.3 g/14 d and 16.8 ± 0.18 g/d in Experiment 3. Experiment 4 was designed so that energy and nitrogen intakes were the same for rats fed fiber-free and SCFA mixture diets. Body weight gain was 83.3 ± 2.05 g/15 d, and energy intake in the fiber-free and the SCFA groups was 245 ± 0.42 kJ/d and 244 ± 0.46 kJ/d, respectively, in the experimental period (15 d).

Plasma total cholesterol concentrations were significantly lower in rats fed fermentation products of SBF than in the fiber-free group 3, 7 and 14 d after feeding the test diet (Fig. 2). Feeding the diet containing fermented residue had no effect on plasma cholesterol. As shown in Figure 3, HDL cholesterol was decreased mainly by feeding of fermentation products. Total and HDL cholesterol concentrations were lower in rats fed the diet containing a mixture of major three SCFA than in rats fed a fiber-free diet and were not different from those in rats fed fermentation products. In Experiment 3 (Fig. 4), plasma cholesterol was again lower in the group fed the three SCFA than in the fiber-free group and not different than that in the SBF group. In rats fed diets containing propionate or butyrate alone and propionate and butyrate in combination, plasma cholesterol levels were not lower compared with rats fed the fiber-free diet. The plasma cholesterol concentrations were significantly lower in rats fed acetate-containing diets (acetate, acetate + propionate and acetate + butyrate) than in the fiber-free group. In Experiment 4, in which nitrogen and energy intake were matched in the diet groups, plasma total cholesterol concentration was significantly lower in rats fed the diet with added SCFA than in rats fed a basal diet (Fig. 5).

View larger version (32K): [in this window] [in a new window]   Fig 3. Plasma total and HDL cholesterol concentrations in rats fed fiber-free, sugar-beet fiber, fermented products or short-chain fatty acid (SCFA) mixture diet. Plasma total and HDL cholesterol concentrations were lower in rats fed sugar-beet fiber (100 g/kg diet), fermentation products (91.2 g/kg diet) and SCFA mixture diets than in rats fed a basal diet (Experiment 2). The SCFA mixture diet contained sodium salts of acetic (35 g/kg diet), propionic (22 g/kg diet) and butyric (9.0 g/kg diet) acids. Each value represents a mean, n = 6. Pooled SEM for total cholesterol and HDL cholesterol were 0.109 and 0.078 mmol/L, respectively. P-values estimated by ANOVA were 0.015 for total cholesterol and 0.001 for HDL cholesterol concentration. Mean values not sharing a common letter are significantly different between diet groups (lowercase letters in HDL and uppercase letters in total cholesterol, P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig 5. Plasma cholesterol concentrations in rats fed fiber-free or short-chain fatty acid (SCFA) mixture diet 0, 7 and 15 d after feeding. Plasma cholesterol concentrations were decreased after consumption of the three SCFA mixture diets in comparison with the sucrose-added, fiber-free diet (230 g casein/kg diet), in which the ratio of nitrogen to energy was the same for both diets (Experiment 4). Each value represents a mean ± SEM, n = 6. P-values estimated by two-way ANOVA were 0.002 for diet and <0.001 for day. Mean values not sharing a common letter are significantly different between diet groups on the same day (P < 0.05).

Wet weight of the cecal contents and wall, shown in Table 2, were higher in rats fed the SBF diet than in rats fed other test diets in both Experiments 1 and 2. The total and individual SCFA pool in the cecal contents of the SBF group was greater than those of the fiber-free and fermentation products groups in Experiment 1 (Table 3).

Fecal excretion of total bile acid was significantly higher in the SBF group than in the fiber-free and SCFA groups (Table 4). Bile acid excretion was slightly higher in rats fed the diet containing the fermentation products than in rats fed the fiber-free diet (P < 0.05). Fecal dry weight was significantly greater in the SBF group than in the other groups.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study shows that fermentation products of SBF evoke a sustained decrease in plasma cholesterol concentration. The fermentation products contained only a small amount of measurable fiber, as shown in Materials and Methods. This finding suggests that some components other than fiber in the fermentation products reduce plasma cholesterol concentration.

The fermentation products prepared from SBF contained large amounts of SCFA as shown in Materials and Methods. Plasma total and HDL cholesterol in rats fed amounts of three major SCFA corresponding to the fermentation products diet was also lower than in rats of the fiber-free group, and these cholesterol levels were similar to those in rats fed fermentation products (Fig. 3). This result shows that the major SCFA, acetic, propionic and butyric acids, produced by ceco-colonic fermentation, are responsible for the plasma cholesterol reduction by the fermentation products, and shows that the fermentation products, SCFA, are possibly involved in the cholesterol-lowering effects of SBF, at least in part. We confirmed that the decrease in the ratio of casein to energy in the test diet from the addition of SCFA was not involved in the lowering of cholesterol (Fig. 5). Furthermore, the cecal content of SCFA in rats fed the diet containing the fermentation products or the SCFA mixture was low compared with SBF-fed rats, but comparable to that in the fiber-free group (Table 3). This reveals that orally ingested SCFA were absorbed in the upper gastrointestinal tract and did not reach the large intestine. Illman et al. (1988) showed that dietary propionate is absorbed in the stomach. Disappearance of the cholesterol-lowering effect of SBF by cecal resection as shown in a report described above (Nishimura et al. 1993) may be due to decreases in absorption of SCFA produced by cecal fermentation and not associated with the resection of the large intestine itself.

Fermentation products of SBF were added to the diet according to yields of a culture using a jar fermenter, and amounts of SCFA sodium salts in a SCFA diet used in Experiments 2 and 3 were the same as those in the fermentation products diet. We previously estimated that the in vivo fermentable energy of SBF in rats fed an SBF diet (100 g/kg diet) was about 60% of gross energy in the fiber source measured by a previously described method (Hara et al. 1994), with SCFA produced in the large intestine contributing the major part of the in vivo fermentable energy in the fiber. Thus the energy value of SCFA added to the test diet is comparable to that produced by large intestinal fermentation in rats fed the SBF diet (100 g/kg diet). This confirms that the amount of SCFA added to the test diet (66 g/kg diet as sodium salts) is at a physiologic level. In a human study, MacNeil (1984) reported that the energy of SCFA produced by colonic fermentation contributes 5-10% of the daily requirement of energy intake.

As shown in Table 4, fecal dry weight and bile acid excretion were slightly, but significantly, higher in rats fed fermentation products than in the fiber-free group. These increases may be due to a small amount of fiber and/or oligosaccharides (not detectable by a fiber estimation) in the fermentation products. It is reported that increases in bile acid excretion cause reduction in plasma cholesterol (Kay and Truswell 1977, Reddy et al. 1980). However, plasma cholesterol was decreased similarly in the SCFA mixture groups as in the fermentation products group, and bile acid excretion in the SCFA mixture group was not increased. The small increase in fecal bile acid in the fermentation products group may not contribute to the lowering of plasma cholesterol.

The effects of individual and combined SCFA on plasma cholesterol are shown in Figure 4. The results are of two separate experiments; thus we estimated relative changes in plasma cholesterol concentration on the basis of differences between means of the fiber-free and the three SCFA mixture groups in each experiment (Figure 6). Feeding the propionate, butyrate or propionate plus butyrate diet did not lower plasma cholesterol, but feeding diets containing acetate or acetate plus one of two other SCFA did. In cultured liver cells, propionic acid decreases cholesterol synthesis (Chen et al. 1984, Demigné et al. 1995, Lin et al. 1995). However, in studies in vivo, lowering effects of propionic acid on plasma cholesterol are controversial. That is, Levrat et al. (1994) and Beaulieu et al. (1992) reported that administration of propionic acid did not decrease plasma cholesterol and cholesterol synthesis, and Kishimoto et al. (1995) showed that a continuous infusion of a small amount of propionic acid decreased plasma cholesterol. Our results show that acetate rather than propionate is involved in the cholesterol-lowering effects of SCFA. Acetate in the cecal lumen is absorbed into the portal vein and metabolized in the liver (Morand et al. 1992). These observations show that acetate reached the liver, possibly affecting hepatic cholesterol metabolism. However, the mechanism of the lowering of plasma cholesterol by acetate feeding is unknown. At least, our results show that the effects on cholesterol metabolism differ among individual SCFA. The absorption ratio of individual SCFA ingested orally may be different from those produced in the large intestine, which may have an effect on cholesterol metabolism.

View larger version (29K): [in this window] [in a new window]   Fig 6. Changes in plasma total cholesterol concentration by feeding diets containing individual short-chain fatty acids or their combination, referenced to mean values of the fiber-free and the acetate (Ac) + propionate (Prop) + butyrate (But) group 14 d after feeding the test diet. The differences between means of the Ac + Prop + But and the fiber-free groups were presented as 100% in each experiment.

Energy derived from SCFA in the test diet is possibly associated with the lowering of plasma cholesterol. As shown in Figure 6, however, energy supplied from SCFA in the acetate + butyrate group was the same as that in the propionate + butyrate group. In the former group, plasma cholesterol concentration was lower than that of the fiber-free group; however, the latter group did not show any decrease in plasma cholesterol concentration. This finding also shows that a cholesterol-lowering effect is specific for acetic acid among major SCFA.

In conclusion, fermentation products of SBF with cecal bacteria reduced plasma cholesterol concentration by oral feeding. Absorption of acetate from the fermentation products contributes to the reduction in plasma cholesterol concentration.

	ACKNOWLEDGMENTS

We thank Elizabeth K. Lund and Anthony J. A. Wright, Institute of Food Research, Norwich Laboratory, Norwich, UK, for useful suggestions on the manuscript and for assistance in data analysis.

	FOOTNOTES

Initial reviews completed 11 July 1997. Revision accepted 19 December 1997

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