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Co-operative Research Centre for Food Industry Innovation, CSIRO (Australia) Division of Human Nutrition, Adelaide 5000, Australia
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Twelve young male pigs consumed a purified diet containing wheat bran as fiber source. Starch provided 50% of total daily energy either as a low amylose cornstarch or as a high amylose (amylomaize) starch. The pigs were given a supplement of a freeze-dried probiotic organism (Bifidobacterium longum CSCC 1941). A block crossover design was used so that at any one time two groups of three pigs consumed either the high or low amylose cornstarch without probiotic and a further two groups of three pigs consumed either high or low amylose cornstarch with probiotic. Neither food intake nor body weight gain was affected by diet. Fecal output was higher when pigs were fed the high amylose cornstarch, but moisture content was unaffected. Fecal concentrations and excretion of total volatile fatty acids were higher when pigs were fed the high amylose cornstarch. Concentrations of acetate were unaffected by dietary starch, but those of propionate and butyrate were higher when the high amylose cornstarch was consumed. Fecal excretion of all three acids was higher during high amylose cornstarch feeding. Bifidobacteria were detected in the feces only when pigs were fed Bifidobacterium longum. Fecal bifidobacteria counts (expressed per gram of wet feces) and their daily fecal excretion were higher when pigs were fed high amylose cornstarch. Feeding the probiotic did not alter fecal starch or volatile fatty acids. None of the variables studied was affected by the order of feeding of starch or probiotic. The data show that a high amylose starch acts as a prebiotic in promoting the fecal excretion of probiotic organisms.
KEY WORDS: pigs · prebiotics · probiotics · starch · volatile fatty acidsINTRODUCTION
Probiotic microorganisms (Lactobacillus sp., Bifidobacterium sp. and others) have been defined as live microbial food supplements that affect the host animal beneficially by improving its intestinal microbial balance (Fuller 1989
). It has been suggested that probiotics may be of therapeutic or preventative benefit for a number of pathological states, including gastroenteritis, diarrhea, constipation and hypercholesterolemia (Goldin and Gorbach 1992). Consumption of yogurt containing Bifidobacterium longum lowers the frequency of antibiotic-induced gastrointestinal disorders (Colombel et al. 1987). Experimental studies in mice fed bifidobacteria have shown lower numbers of chemically induced tumors in the large bowel (Koo and Rao 1991), which is consistent with a possible reduction in the risk of cancer in that viscus. These and other observations indicate that probiotics have the potential to improve human colonic health (Playne 1995).
While these data are suggestive of benefit from the standpoint of disease reduction and health promotion, a number of criteria need to be met before increased consumption of probiotics can be recommended to the general population. One of these is survival of the live organisms in the gastrointestinal tract. The large bowel is the major site of bacterial colonization and metabolism in the human gut and contains a wide range of species. These include bacteria that metabolize dietary carbohydrates to volatile fatty acids (VFA)7 that are thought to mediate some of the health benefits normally ascribed to the carbohydrates themselves. (Annison and Topping 1994, Cummings and Macfarlane 1991) These actions include stimulation of electrolyte and fluid transport, enhancement of colonic muscular activity and promotion of a normal cell phenotype in colonocytes. Potentially pathogenic species such as clostridia and coliforms also reside in the large bowel and may proliferate and produce adverse reactions (Tancrede 1992). The hind gut is the region where probiotics could be expected to colonize and where some of their beneficial actions could originate, e.g., through altering of VFA profiles. Studies of the effects of probiotics on VFA excretion have been inconclusive and Bartram et al. (1995) noted that the fecal flora and VFA excretion of normal humans were extremely stable with respect to dietary perturbation through ingestion of live B. longum. These authors attempted to enhance the survival of the probiotic culture by adding lactulose as a metabolic substrate for the bifidobacteria. This illustrates the concept of prebiotics, which are nondigestible food ingredients that affect the host beneficially by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improving host health (Gibson and Roberfroid 1995). Certain oligosaccharides are used selectively by probiotic microorganisms, and the fructo-oligosaccharides seem to act as prebiotics in humans (Gibson et al. 1995). Other, nutritionally important, complex carbohydrates including starches and nonstarch polysaccharides (NSP) have been viewed as having a low potential as prebiotics (Gibson and Roberfroid 1995). In the case of starches, this is understandable because they may be digested completely by human digestive enzymes, in contrast to fructo- and galacto-oligosaccharides and NSP, which are resistant. However, it seems that this is an oversimplification because a large proportion of starch escapes small intestinal digestion and enters the large bowel, where it is fermented by the bacteria, yielding VFA (Cummings and Macfarlane 1991). This so-called resistant starch (RS) arises for a variety of reasons, including cooking, the presence of NSP and the degree of mastication (Annison and Topping 1994). Of particular interest is the fact that a high amylose content in corn lowers the small intestinal enzymic hydrolysis of the starch. Feeding trials with high amylose starch in humans have shown loss of starch from the ileum (Muir et al. 1994) and studies in pigs have shown increased starch concentrations in the proximal colon (Topping et al. 1997). Fecal VFA excretion is higher in humans consuming high amylose starch (Noakes et al., 1996) or another RS manufactured by a commercial process (van Munster et al. 1994). These changes are consistent with enhanced large bowel bacterial fermentation and we considered it possible that RS might act as a prebiotic. This hypothesis runs contrary to current opinion and was tested by examining the fecal excretion of bifidobacteria in pigs fed freeze-dried Bifidobacterium longum with a high amylose starch. In view of the fact that important health benefits of RS (and NSP) are mediated through VFA, we examined the effect of feeding this probiotic on fecal VFA as an index of their production by the colonic microflora.
METHODS
). All of the procedures described were approved formally by the Animal Care and Ethics Committee of the Division of Human Nutrition and conformed to published guidelines (National Health and Medical Research Council, CSIRO and Australian Agricultural Council 1985).
Diets and feeding procedures.
Twelve pigs were fed a diet composed of commercially available human foods that met all their macronutrient needs at a daily intake of 400 kJ/kg body wt. In brief, the diet was formulated from cornstarch or a high amylose cornstarch and casein, sucrose, wheat bran (Laucke Mills, Strathalbyn, SA, Australia) and safflower oil. A commercial vitamin and mineral supplement was also added to the diet (Topping et al. 1993). The diet contained 8.8% of fiber as wheat bran. It was necessary to provide dietary fiber so as to prevent constipation and discomfort and wheat bran was chosen because it is consumed widely by humans. However, the quantity that was fed was considerably less than the normal intake of pigs fed commercial pig diets. The cornstarch or high amylose starch, safflower oil, sucrose, casein, wheat bran, vitamins and minerals were mixed and then weighed into individual meals. The pigs received two meals per day (morning and afternoon) and consumed water ad libitum. They received 40 g of freeze-dried Bifidobacterium longum CSCC 1941 culture in two equal doses of 20 g with their morning and afternoon meals. There were four experimental groups, each of three animals. One group was fed the diet with the low amylose (i.e., low resistant starch) cornstarch. In the second group, the low amylose starch was fed in addition to the bifidobacteria. The third group of pigs was fed the diet with the high amylose starch (HimaizeTM, Starch Australasia Ltd, Botany, NSW, Australia). In the fourth group, the high amylose starch was fed in addition to the bifidobacteria. A block crossover design was used so that eventually all groups were fed the two starches with and without bifidobacteria. The pigs were fed each experimental diet for 7 d, followed by a period of 1 wk during which they were fed pig pellets (Laucke, Mills) containing olaquindox, a broad-spectrum antibiotic for both Gram-positive and Gram-negative bacteria.
Bacterial supplementation.
All bacterial counts are expressed as the log10 colony-forming units (cfu). The freeze-dried culture of Bifidobacterium longum (1941) was provided kindly by Gist Brocades Australia Pty Ltd (Moorebank, NSW, Australia) in sealed bags. The culture (20 g) was mixed evenly into the experimental diet immediately before feeding and the dry mixture tipped into the feeding bowl with 2 L of water. The presence of live bifidobacteria in the freeze-dried culture was confirmed by standard microbiological techniques. The concentration of B. longum in the culture was 11.0 colony-forming units/g (log10 cfu/g), and each pig was given 12.6 cfu/d.
Sampling procedures.
At the start of the experiment and during each period when the commercial ration was fed, fecal samples were taken for microbiological assessment to establish the baseline level of naturally occurring and residual bifidobacteria. Fresh feces were collected each morning after feeding and analyzed for VFA concentrations. Additional samples were taken aseptically after 6 d for microbiological enumeration of bifidobacteria. To account for any variation in bifidobacteria in the feces excreted during the day, four pigs were selected randomly and fresh feces sampled as they dropped between 0900 and 1300 h. These samples showed no significant variation in counts during the time examined. On d 6 of the feeding period, a 1- to 2-g sample of feces was taken for determination of moisture content. Total fecal output for the last 3 d for each pig was pooled and air-dried. A representative sample was freeze-dried and analyzed for total starch. During the experiment, one pig became lame and required analgesics. This pig was fed and sampled with the others but the samples were not analyzed so the data are reported for 11 animals.
Analytical procedures.
A fresh fecal sample (1.5-2.0 g) was weighed accurately into a 10-mL plastic centrifuge tube and diluted threefold with water containing 3.52 mmol/L oenanthic acid as internal standard. The sample was then centrifuged for 15 min at 3000 × g and 5°C. An aliquot was taken, acidified with phosphoric acid and distilled in vacuo and the distillate was analyzed for VFA by gas-liquid chromatography (Topping et al. 1993). A sample of fresh feces was freeze-dried for total starch determination using the Megazyme total starch assay procedure (Amyloglucosidase/
-amylase method; Megazyme Ltd, Sydney, NSW, Australia). The bacteriological examination of feces was performed within 2 h of collection. A 10-g sample of feces was suspended homogenously in 1% buffered peptone and was further diluted in 10-fold dilutions. Of the appropriate dilutions, 0.1 mL was plated in duplicate onto the surface of Bifidus Blood Agar (Pachenari et al. 1997, Reuter 1963) and spread evenly over the plate. The plates were incubated under anaerobic conditions using an Anaerocult A mini-procedure (Merck Pty Ltd, Kilsyth, VIC, Australia) and Anaerojar (Oxoid Australia Pty Ltd, West Heidelberg, VIC, Australia). Plates were incubated at 37°C for a period of 5-6 d and counted. The numbers of bifidobacteria per gram of wet feces were calculated. Bifidobacteria were distinguished from other bacteria present on the plates by their distinctive raised, copper-pigmented colonies (Pachenari et al. 1997). These isolates were then Gram-stained and examined for Gram-positive slightly bifurcated club-shaped rods, indicative of bifidobacteria species (Wood and Holzapfel 1995).
Statistical methods.
The data were analyzed by ANOVA using the residual maximum likelihood procedure of Genstat 5 (release 3.1; Genstat 5 committee 1993) on a Sun Ultra workstation. This procedure accounted for random variation due to time, experimental group and animal. The significance of the fixed effects of starch type and the feeding of Bifidobacterium longum in the model then was assessed by testing the deviance change with and without the treatment effects in the model against a chi-square distribution. Data are shown as the means of 11 observations for each of the main treatments (cornstarch, amylomaize starch, no Bifidobacterium longum or feeding of Bifidobacterium) with the standard error of the difference. A value of P < 0.05 was taken as the criterion of significance.
RESULTS
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Table 1. Fecal concentrations and daily excretion of bifidobacteria of pigs fed either a low amylose or high amylose (amylomaize) cornstarch with live Bifidobacterium longum1 |
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Table 2. Fecal concentrations of volatile fatty acids (VFA) of pigs fed either a low amylose or high amylose (amylomaize) cornstarch with or without live Bifidobacterium longum1 |
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Table 3. Fecal pools of volatile fatty acids (VFA) of pigs fed either a low amylose or high amylose (amylomaize) cornstarch with or without live Bifidobacterium longum1 |
DISCUSSION
) showing that ingestion of a resistant starch increased total fecal bulk. In the present experiment, fecal wet weight was approximately 45% higher in pigs fed high amylose cornstarch and was unaffected by the presence of probiotic bacteria. As in previous studies with RS (Noakes et al. 1996, van Munster et al. 1994), there was no difference in fecal water content, which means that the greater fecal excretion was as dry matter. Some of this dry matter was starch, but the quantities excreted were small even when the pigs ate the high amylose cornstarch. However, the amount of fecal starch was insufficient to account for the difference in wet stool weight between pigs fed the low and high amylose cornstarches.
were the first to show in humans that the increase in fecal bulk due to dietary fiber consumption was due to greater bacterial numbers as well as excretion of NSP and lignin. In the present experiment, fecal bifidobacteria numbers expressed per unit fecal weight were higher when the probiotic was fed with RS. We could not distinguish between the B. longum that was fed and other bifidobacteria species. However, it is likely that the increase reflected greater growth of the bacteria administered orally, because no bifidobacteria were detected in pigs when they were not fed the supplement. Possibly, this could reflect the presence of antibiotic in the standard ration and also the lability of bifidobacteria colonization of the gut. The latter possibility has been confirmed in a subsequent experiment in pigs in which we used the same design as in the present study. In this subsequent experiment, the standard ration was manufactured from the same ingredients but without the antibiotic. In that experiment, the fecal bifidobacteria numbers and total excretion were comparable to those found in pigs fed the low and high amylose cornstarches in this study (Bird, A. R., Warhurst, M., Crittenden, R., Hayakawa, T., Playne, M. J., Illman, R. J., Brown, I. L. and Topping, D. L., unpublished observations). Other variables (including stool mass and fecal VFA) were similar, indicating that the inclusion of olaquindox did not influence the outcome. In that experiment, fecal bifidobacteria declined within a few days of stopping the feeding of live organisms when the diet contained low amylose starch, but the decline was much slower when high amylose starch was fed.
, the high amylose starch acted as a prebiotic even though these authors concluded that starches were unlikely to perform this function. However, the increase in fecal mass noted in this experiment was of such a magnitude as to suggest that the feeding of high amylose cornstarch increased the numbers of other bacterial species as well as the probiotic organisms.
, Muir et al. 1994, Topping et al. 1997). This is supported by the observation that alginate, which is not degraded by human small intestinal enzymes, raises fecal bifidobacteria in humans (Terada et al. 1996). However, the fact that other NSP, which also affect the dynamics of the small intestine, do not seem to act as prebiotics tends to make this possibility less likely. Secondly, the bacteria may have been protected by adhesion to undigested starch or through entry into the pits formed in the starch granules during small intestinal amylolysis (Topping et al. 1997). A final possible mechanism is that the high amylose starch was simply a substrate for the bifidobacteria. Generally, it is thought that bifidobacteria do not metabolize starches efficiently (Sgorbati et al. 1995). This is supported by the lack of difference in fecal starch excretion between pigs fed RS alone and those fed RS with probiotic.
) and pigs (Topping et al. 1997), the fecal concentrations of VFA were higher in pigs fed high amylose cornstarch. Also as in other previous studies, VFA excretion was higher during the feeding of a high amylose starch (Mazur et al. 1990, Noakes et al. 1996) and another RS (van Munster et al. 1994). These differences are consistent with enhanced large bowel bacterial fermentation through provision of extra substrate. As we have noted recently in pigs (Topping et al., 1997), fecal acetate, propionate and butyrate were raised during consumption of high amylose starch. This differs from findings in humans and rats in which feeding of RS raises fecal butyrate more than the other acids (Mazur et al. 1990, Noakes et al. 1996, van Munster et al. 1994), apparently through the preferential production of this acid during bacterial starch fermentation (Weaver et al. 1992). Butyrate is thought to exert protective effects on the colonic mucosa, including a diminished risk of neoplasms (Kruh et al. 1994). Epidemiologic data suggest a protective role for greater starch consumption in colorectal carcinogenesis (Cassidy et al. 1994) and one attractive possibility is through fermentation of starch to butyrate. However, there seems to be a species difference between pigs on the one hand and humans and rats on the other. The reason for the difference may lie in the bacterial population present in the gut or in the physiology of the gut itself. The cecum and colon are a larger proportion of the total gastrointestinal tract in pigs compared with humans and other model species such as rats and dogs (van Soest 1995). Given that butyrate is used preferentially by colonocytes and that its supply may be limiting to utilization (Illman and Topping 1986), it is possible that any increase in its production could be offset by increased utilization. Alternatively, it may be an effect of the type of starch, because the feeding of brown rice to pigs increases the large bowel pool of butyrate, apparently through the fermentation of starch (Marsono et al. 1993).
, there was no effect of probiotic ingestion on either total or individual VFA excretion when fed with either starch even though bifidobacteria numbers were higher when pigs consumed RS. However, it must be noted that fecal excretion is a net process and represents a balance between production and utilization. Studies in pigs have shown that the distribution of digesta and VFA along the large bowel cannot be predicted from values in the distal colon (Topping et al. 1993). More recently, Bartram and co-workers (1994) made a similar suggestion and proposed that there may be changes in VFA in the proximal bowel that might not be detected in the distal colon or feces. However, most degenerative bowel disease is expressed in the distal colon, suggesting that, if probiotic organisms were to be protective against them, VFA are not the likely mediators.
). In addition to this health benefit it has been noted that RS may offer a technological advantage in that their presence in human foods raises their content of "fiber" without necessarily altering their organoleptic properties (Annison and Topping 1994). In the case of high amylose starches, their capacity to act as prebiotics seems to extend the scope for their use in food so as to enhance delivery of probiotic microorganisms to the distal bowel.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 29 January 1997. Initial reviews completed 24 February 1997. Revision accepted 20 May 1997.
LITERATURE CITED
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