Anthony R Bird1, Ian L Brown2 and David L Topping1
1CSIRO Health Sciences and Nutrition, GPO Box 10041, Kintore Avenue, Adelaide BC 5000, Australia
2Starch Australasia Ltd, 170, Epping Road, Lane Cove 2066, Australia
Starches are important as energy sources for humans and also for their interactions with the gut microflora throughout the digestive tact. Largely, those interactions promote human health. In the mouth, less gelatinised starches may lower risk of cariogensis. In the large bowel, starches which have escaped small intestinal digestion (resistant starch), together with proteins, other undigested carbohydrates and endogenous secretions are fermented by the resident microflora. The resulting short chain fatty acids contribute substantially to the normal physiological functions of the viscera. Specific types of resistant starch (e.g. the chemically modified starches used in the food industry) may be used to manipulate the gut bacteria and their products (including short chain fatty acids) so as to optimise health. In the upper gut, these starches may assist in the transport of probiotic organisms thus promoting the immune response and suppressing potential pathogens. However, it appears unlikely that current probiotic organisms can be used to modulate large bowel short chain fatty acids in adults although resistant starch and other prebiotics can do so. Suggestions that starch may exacerbate certain conditions (such as ulcerative colitis) through stimulating the growth of certain pathogenic organisms appear to be unfounded. Short chain fatty acids may modulate tissue levels and effects of growth factors in the gut and so modify gut development and risk of serious disease, including colo-rectal cancer. However, information on the relationship between starches and the microflora is relatively sparse and substantial opportunities exist both for basic research and food product development.
The early work of Metchnikoff (1907), linking the consumption of live organisms in fermented foods by certain ethnic groups with their greater longevity, carried the implicit assumption that the normal colonisation of the gut was potentially deleterious. The current view is rather different in that the linkage between the microflora is with chronic non-infective diseases and not acute outbreaks of food or water borne infections. Colonic microorganisms have been designated as putrefactive under certain circumstances while other gut bacteria may contribute to pathologies such as gastritis (McColl 1999) and inflammatory bowel disease (IBD) (Ardizzone et al., 1999). More importantly, the view is emerging that most of the activities and products of the microflora are, in fact, beneficial to the host and they should be promoted. Any deleterious effect is subtle and contributes to ill health of the host through those chronic diseases which these beneficial bacteria help to diminish. The activities of these beneficial organisms can be modified by the substrates which they receive, predominantly through the diet. Some of the most important of these are the starches which contribute substantially to energy intake both as processed and unprocessed cereals, pulses, fruits and vegetables. It is the aim of this review to examine the interactions between the gut microflora and human health and the ways in which starches can influence these relationships.
Gastrointestinal Bacteria and Human Health
The microbial population of the digestive tract is largest in the caecum and proximal colon with fewer numbers and species in the mouth, stomach and small intestine. However, these smaller populations can be important for health outcomes. Oral colonisation (especially by S. mutans) varies by individual and is thought to contribute to cariogenesis through leaching of calcium from enamel by organic acids (lactate, acetate) produced by metabolism of dietary carbohydrate (Bibby, 1982). This is a condition of apparently low mortality. Even so, a median estimated cost of dental caries in Australia in 1992 was $478,000,000 (Crowley et al., 1992), which gives some indication of the costs which can be associated with diet-related disease. Fluoridation of the water supply is an effective mechanism for control and limiting damage (Morgan et al., 1998) but does not attack the primary problem. An effective preventive strategy appears to be oral hygiene plus limiting the supply of substrate to minimise production of organic acids from fermentable carbohydrates at or near the tooth surface.
The relatively sparse flora of the healthy stomach is usually less than 103 organisms per gram of contents, comprising chiefly facultative anaerobes such as lactobacilli, staphylococci and streptococci. A specific organism (Helicobacter pylori) appears to infect about 60% of the adult population in more affluent countries. Rates of carriage are much higher in some populations which may reflect the possibility that the organism is a zoonosis derived from domestic animals (Dore et al., 1999). H. pylori is found adhering to the gastric epithelium and infections with particularly virulent strains are associated with reflux, gastritis, surface ulceration and cancer (Ernst, 1999). The bacterium secretes an enzyme, urease, which liberates free NH3 from urea. NH3 is a known cytotoxic agent and carcinogen and it is thought that it (or more accurately, NH4+) contributes to the local tissue necrosis, ulceration and, ultimately, cancer (Tsuji et al., 1997). In global terms, gastric carcinoma is a major cause of morbidity and mortality (World Cancer Research Fund and American Institute for Cancer Research, 1997). Eradication of the bacterium through antibiotic therapy is an obvious option for primary prevention but may not be entirely satisfactory for two reasons. Firstly, a direct connection between infection and gastritis which proceeds through ulceration to gastric carcinoma has yet to be shown. Secondly, it is possible that H. pylori may be involved to some degree in the maintenance of normal gastrointestinal motility. Affected individuals in whom the organism has been suppressed or eradicated can develop reflux oesophagitis (Richter et al., 1998). This may be associated with a rise in the incidence of Barrett's oesophagus - believed to predispose to increased risk of oesophageal carcinoma (Heatley and Guillou, 1997). It appears that control, rather than eradication, of H. pylori may be a preferred option. In addition, there is evidence implicating H. pylori (and other organisms) in the pathogenesis of human cardiovascular disease. Treatment of patients with unstable angina with an antibiotic against Chlamydia pneumoniae for 30 days lowers the clinical endpoints for up to 6 months (Gurfinkel et al., 1999). H. pylori been shown to be present in atheromatous plaques (Ellis, 1997). However, a direct causal role for it and other candidate infectious agents in atherogenesis has yet to be proven (Danesh et al.,1998) . The gut microflora, however, may influence coronary heart disease (CHD) risk by modulating serum lipid concentrations. Circulating levels of LDL and HDL-cholesterol, which are established risk factors for CHD, appear to be influenced by the population profile and metabolic activity of the colonic microflora (Jenkins et al., 1999).
Rapid peristalsis, and the bactericidal actions of gastric secretions, limit colonisation of the upper small intestine to usually less than 105 cells/ml of fluid. Bacterial numbers, especially of the strict anaerobes, increase along the small intestine, attaining a density of between 106 - 108/ml in the distal ileum. Small intestinal infections, principally due to food and drinking water contaminated with various microorganisms, are common in developing countries (Farthing 1999). Hypochlorhydria and intestinal motor dysfunction often result in substantial bacterial overgrowth of the small bowel (Brasitus and Sitrin 1990) and illustrate the dynamic nature of the balance between the host and the microflora.
The prime focus for any health-related interaction between gut bacteria and starches is the large bowel. This is the major site of bacterial colonisation (Gibson and Roberfroid, 1995) and fermentation (van Soest, 1995) in the gastrointestinal (GI) system of omnivorous animal species including humans. In adult humans the bacterial population is very large, consisting of about 1014 viable cells, which account for about 100 grams or 50% of the wet mass of colonic contents (Cummings and Macfarlane, 1997) and of which about 15 grams of biomass is shed daily in faeces (Cummings and MacFarlane 1991; Hill, 1995). The colonic microflora is taxonomically diverse, consisting of over 50 genera and 400 species (Drasar, 1988; Salminen et al., 1995; Savage, 1986; Mitsuoka 1996) in which only a relatively few genera predominate. Bacteria are implicated in the aetiology of several important human large bowel pathologies including colorectal cancer (Rowland et al., 1985; Roberton 1993), irritable bowel syndrome (IBS) (King et al., 1998), and inflammatory bowel disease (IBD) (Lisby et al., 1994). The nutritional influences on these chronic bowel conditions have been reviewed recently (Topping and Bird, 1999). In westernised countries, large bowel cancer is a serious malignancy in which lifestyle factors, especially diet, are believed to play a causative role in the majority of cases (Szilagyi 1998, Topping and Bird, 1999). Putrefactive species may play a role in the initiation and promotion of carcinogenesis through their ability to metabolise luminal substances to potentially harmful toxins, including mutagens and carcinogens. Various nitrogenous compounds, principally ammonia, amines, indoles and phenols, are products of protein and amino acid degradation by putrefactive bacteria (Roberton 1993) and are considered putative risk factors for neoplastic transformation in the large bowel (Bingham 1999). A specific bacterium, Streptococcus bovis, has been implicated in cancer of the large bowel (Macaluso et al., 1998). This organism has been shown to colonise large bowel carcinomas, adenomas and polyps but recent studies indicate that the carriage rate of S. bovis in patients with confirmed colorectal neoplasms is low (Norfleet and Mitchell 1993) and not significantly different from controls (Potter et al., 1998). Overall, there is little evidence supporting a direct role for this or other bacterial agents in initiating or promoting colorectal cancer. The same appears to be true for the aetiology of constipation and diverticular disease which are manifest as inadequate defaecation and, in the case of the latter, by herniation of the colon. These conditions relate principally to a sub-optimal fibre intake and can be prevented and managed by increasing fibre consumption (Topping and Bird, 1999). IBS is characterised by disordered gut motility and pain with bloating, diarrhoea and/or constipation and while it can affect the whole gut, its primary focus appears to be the large bowel. The condition appears to be to be unrelated to fibre intake. IBD occurs in two main forms - ulcerative colitis (UC) and Crohn's disease. The former occurs mostly in the distal colon and is characterised by surface ulceration and bleeding. Crohn's disease may be found throughout the GI tract but has a primary focus at the ileo-caecal junction. The microflora have been implicated in both conditions but it appears that disordered cytokine production (possibly due to inadequate n-3 fatty acid intake) is involved in Crohn's disease (Topping and Bird, 1999).
The composition and state of the intracolonic environment is largely a product of the metabolic actions of the microflora and their interactions with their nutrient supply. Generally, these are viewed as positive and occur through the production of organic acids, especially short chain fatty acids (SCFA). These acids are important regulators of colonic physiological processes and appear essential for maintaining normal bowel function (Cummings and Macfarlane 1991; Velazquez et al., 1996).
Large Bowel Microflora - Substrates and Carbohydrate Fermentation
Supply of Fermentable Substrates
Normally the bacteria which ferment carbohydrates escaping small intestinal digestion and also any undigested proteins of exogenous or endogenous origin predominate in the large bowel ecosystem. This bacterial profile reflects the supply of fermentable substrates which reach the large bowel (Table 1). Of dietary plant polysaccharides and oligosaccharides, humans possess intrinsic hydrolytic enzymes to digest only starch and its breakdown products. All other non-starch polysaccharides (NSP, major components of dietary fibre) and oligosaccharides (OS) such as fructo-oligosaccharides (FOS), are not digested in the small intestine and enter the large bowel. Depending on age and genetic factors, a variable fraction of lactose may be digested in the small intestine. Monosaccharides (e.g. fructose and sorbitol) which are absorbed incompletely in the small intestine also may be fermented. A variable, but substantial, fraction of dietary starch (resistant starch, RS) also enters the human large bowel where it is fermented (Annison and Topping, 1994; Asp et al., 1996). RS may be an important substrate for the human colonic microflora and its fermentation may convey substantial benefits to the host (Cummings et al. 1996).
The passage of NSP into the colon helps to explain the enhanced faecal bulking and promotion of laxation which is one of the best-documented effects of fibre-rich foods such wheat bran (Topping and Bird, 1999). Nevertheless, faecal excretion of NSP is generally incomplete as a fraction is fermented by the microflora (Stephen and Cummings, 1981). This enables bacteria to proliferate with the nitrogen requirement being met by protein and peptide fermentation and, to a lesser extent, by urea. Thus, the voided stool is a composite of undigested dietary components, endogenous secretions as well as bacteria. The degree of susceptibility of carbohydrates to bacterial digestion is termed "fermentability" and varies between foods. Wheat bran is of relatively low fermentability (<50%) while fruit and vegetable fibres appear to be more susceptible to fermentation (Stephen and Cummings, 1981). There appears to be an important gender difference in that transit is slower and fermentability greater in women than men (Stephen et al., 1986; Lampe et al., 1993). Whether this difference extends to the bacterial population is uncertain. If it does, then it raises important issues including the age at which any difference becomes established.
It was assumed that for both sexes, NSP were the principal substrates for fermentation and that ileal starch digestibility was complete as little appears in human faeces under normal circumstances. This was despite indirect evidence (e.g. from breath H2 evolution) that there was escape of starch into the colon (Anderson et al., 1981). However, when it became clear that NSP alone could not meet the substrate requirements of the microflora - the "carbohydrate gap" - RS became a candidate to meet the deficit (Stephen, 1991). It had been shown by direct intubation of the human small intestine that RS could contribute to large bowel fermentation (Stephen et al., 1983). Other carbohydrates, such as oligosaccharides derived from wheat, onions, endive and similar sources (Moshfegh et al., 1999) not measured commonly in food analysis may make a smaller contribution to filling this gap. However, RS is thought to be the largest contributor to the missing carbohydrate and hence to SCFA production.
As in the rumen or large bowel of obligate herbivores, human large bowel bacteria hydrolyse complex carbohydrates to their constituent monosaccharides. These are then metabolised to the major end products providing energy for bacterial growth. In addition to heat of fermentation and gases (CO2, H2 and CH4), the main metabolic products are SCFA which, in normal adults, are principally acetate, propionate and butyrate (Cummings and Macfarlane, 1991). These acids are the principal anions in normal large bowel digesta and faeces. Generally, they are found in gut contents at concentrations in excess of 80 mmol/L and are thought to play a substantial role in maintaining colonic integrity.
Factors Influencing Starch Digestion
Potentially, starches may be hydrolysed completely to glucose by human small intestinal a-amylase and associated enzymes. The degree of hydrolysis is raised considerably by cooking with water (gelatinisation). Heating with restricted moisture may effect a limited internal rearrangement of the starch polymers in the granule which may increase their resistance to amylolysis. Various factors, including physical inaccessibility of the starch in the food, and its granular structure and degree of retrogradation can limit ileal starch digestibility to less than 100% (Annison and Topping, 1994; Asp et al., 1996). Chemical modifications (etherisation, esterification and cross-bonding) used by the food industry to produce starches with desirable food technological properties also lower ileal starch digestibility (Brown et al., 1995). The classification of RS into four main types reflects these influences (Table 2) and the RS content of a food can vary with each classification according to the nature and type of components in the food, food manufacturing processes and conditions, storage and how the food is consumed. Obviously, for RS1 the degree of milling can be a prime determinant of digestibility. Starch is found in two main forms, amylose and amylopectin (Annison and Topping, 1994; Baghurst et al., 1996). The former is a relatively small (100-10,000 monomer units), essentially linear, polymer in which glucose units are linked by a (1Æ4) linkages (Shannon and Garwood, 1984). In contrast, amylopectin is an extremely large (normally 10,000-100,000 monomer units) glucose polymer containing many a (1Æ6) branch points. The positions of the branch points relative to the linear regions of amylopectin dictate its cluster and structural properties in the crystalline region of the starch granule. The majority of the amylose exists in the amorphous regions of the starch granules. Most starches available commercially contain at least 70-80% of the total as amylopectin which means that they gelatinise relatively easily on heating with water. Starches from specific maize cultivars can contain much more amylose. Those available currently contain approximately 70% of the total as amylose but newer maize cultivars having >80% of starch as amylose are appearing. The amylose:amylopectin ratio is significant for RS2 and RS3. All high amylose maize starches have a structure and conformation which elevates gelatinisation temperatures compared with regular maize starch. However, once gelatinised, the high proportion of amylose leads to rapid retrogradation of the starch paste (Brown et al.,1998). Retrogradation is a phenomenon which occurs on cooling (e.g. potato salad) and involves reassociation of the linear regions of the polymers to form insoluble crystallites which resist enzymatic hydrolysis (Sievert and Pomerantz, 1989). These consist of small amylose domains between amylopectin regions in the cooled cooked starch (Gidley et al., 1995). The RS4 content of a food can be affected by the degree of substitution (Wurzburg, 1986) which means that processed foods containing modified starches can vary in RS content on both this variable and the absolute degree of incorporation. Normally these starches are limited to degrees of substitution which are quite low (<2.5% for acetylated starch). In addition to these reasonably well defined factors, there are physiological influences on RS which can be equally strong but also hard to quantify. For example, large particles (e.g. those which have been chewed relatively little) move through the gut more quickly than small ones (Lewis and Heaton 1999; McIntyre et al., 1997). Thus, starch in a small food particle may be hydrolysed quite rapidly compared with that in a large particle. If the transit time from mouth to ileocaecal valve were shorter for the former, then its effective RS content could be quite high compared with the more rapidly digested starch. Obviously, chewing and transit vary by individual and are hard to incorporate into a purely chemical determination of RS which rely principally on the in vitro incubation of starches with amylase and/or amyloglucosidase (Englyst et al., 1996). These personal influences have led to the description of RS as "physiological" and "chemical" RS (Annison and Topping, 1994). The former term embraces RS as it occurs in the body and the latter the values obtained by chemical analysis. The differences between physiological and chemical RS may be considerable for specific foods. For example, baked beans or brown rice contain low amounts of RS when analysed by standard procedures (Cheng and Yu 1997; Parchure and Kulkarni 1997). However, in animals the contribution of starch escaping into the large bowel is enough to double the effective "fibre" content of these foods (measured as digesta mass or SCFA) when compared with high fibre/low starch foods such as wheat or rice bran at the same level of dietary fibre (Topping et al., 1993; Marsono et al., 1993). Muir and colleagues (1993) and Åkerberg et al., (1998) have attempted to replicate physiological influences, specifically chewing, in RS analysis and their data suggest that it is an important factor. This means that it is quite difficult to give an exact figure for a particular person's RS intake. Generally, it is assumed that about 10% of ingested starch may enter the colon (Baghurst et al., 1996).
SCFA and the Large Bowel
Effects of SCFA may be subdivided into the general and specific. The former relate substantially to the fact that the major SCFA are organic acids with pKa values of approximately 5 so that their production leads to acidification of the intracolonic environment. It is a common finding that promotion of SCFA production through provision of additional fermentable carbohydrate gives lower pH values in the large bowel contents of animals (e.g. Topping et al., 1993) and in faecal samples of humans (e.g. Noakes et al., 1996) compared with diets low in fermentable carbohydrate. Greater acidity inhibits the growth of potentially pathogenic pH-sensitive organisms (Prohaszka et al., 1990). Lowering of pH leads also to the dissociation of alkaline compounds with toxic or carcinogenic potential and so inhibits their absorption. Other effects which appear to be common to the major SCFA include the stimulation of colonic blood flow (through the dilation of resistance blood vessels) and enhancement of colonic muscular contraction (for more extensive reviews see Cummings et al., 1995). These changes raise muscular tone and large bowel oxygenation and nutrient transport (Kvietys and Granger, 1981). Uptake of SCFA by the large bowel is accompanied by that of fluid and electrolytes. It was thought that this was limited to K+ and Na+ but now it seems likely that substantial uptake of Ca2+ occurs also and that this may be enhanced by greater provision of fermentative substrate such as OS (Coudray et al., 1997). SCFA promote colonocyte proliferation and thereby assist in reversing the colonic atrophy which is a feature of low-fibre diets. SCFA also maintain a normal phenotype in this population of frequently replaced cells. In vitro data indicate that SCFA are important metabolic fuels for colonocytes with butyrate being their preferred substrate (Roediger 1982) and that their capacity to oxidise butyrate is modulated by the microflora (Cherbuy et al., 1995). Cell culture studies show that the presence of butyrate at physiological concentrations enhances growth of normal cells and inhibits that of malignant ones. These actions are effected by a variety of mechanisms including promotion of DNA repair and differentiation of tumour cells (Smith et al., 1998). One important action of butyrate is to induce apoptosis in malignant cells (Smith et al., 1998). In normal cells, the absence of butyrate produces the same effect. Although an unequivocal in vivo demonstration of a direct role for butyrate in the maintenance of normality in colo-rectal mucosal cells has yet to be made, there are supporting data. Thus, raising of large bowel butyrate with a pelletised preparation has been shown to increase apoptosis in aberrant crypt foci in a rat cancer model (Caderni et al., 1998). One form of inflammatory bowel disease, distal ulcerative colitis, may also relate to SCFA metabolism and/or availability. Colonocytes isolated from patients with this condition have an impaired capacity to oxidise butyrate in vitro (Burke et al., 1997) and faecal butyrate levels are higher in adult and infant colitis patients than in controls (Holtug et al., 1988 ; Treem et al., 1994). This suggests that there is a defect in butyrate metabolism which could be overcome through provision of more substrate because SCFA enemas (including butyrate) have been reported to induce remission in affected individuals but final confirmation is yet to be obtained (Scheppach et al., 1997). Cancer of the large bowel accounts for a large proportion of cancer-related deaths in westernised countries and the incidence is increasing in developing regions, especially in Asia. With increasing affluence, there is a shift in location with large bowel cancers no longer being confined to the most distal region of the colon (Szilagyi 1997), the region where SCFA availability is lowest (Topping and Bird, 1999). Now cancers are being found in greater numbers towards the right (proximal) side of the colon.
The role of SCFA in large bowel physiology raises an important issue which is that both the fermentative substrates and the bacterial population which metabolise them may be secondary in importance to the effective delivery of SCFA, especially to those sites at greatest risk of disease. This is important for RS as it affects the large bowel principally through fermentation and those individuals at greater risk for bowel cancer (designated by the presence of large bowel polyps) were found to be more efficient at digesting and absorbing dietary starch than low risk patients (Thornton et al., 1987).
RS and SCFA Production
Several colonic bacterial groups have the metabolic capacity to ferment starch in vitro (Table 3). However, this does not mean that they do so in vivo as all of the influences (gelatinisation, amylose content, retrogradation) which contribute to RS in the small intestine apply to large bowel amylolysis. That RS modified large bowel bacterial activity was shown by early studies in rats where high amylose starches (Morand et al., 1992) or legumes (Key and Mathers, 1995) raised total caecal digesta mass and SCFA concentrations and pools. In vitro studies showed that starch fermentation by faecal inocula appeared to favour butyrate production (Weaver et al., 1992). Human feeding trials accord with these findings. Increasing the effective intake of RS either through inhibition of small intestinal starch digestion with acarbose (Scheppach et al., 1988) or feeding foods high in RS (Noakes et al., 1996; van Munster et al.,1994) raises faecal butyrate.
These data have attracted much interest in view of the potential role of butyrate in promoting a normal colonocyte phenotype. They may help to account for some of the epidemiological data linking diet to lowered risk of large bowel cancer. Much of the early epidemiological work in Africa linked their consumption of unrefined plant foods to diminished risk of this malignancy and other diseases (Burkitt, 1995). Accordingly, considerable research effort was directed at the protective effects of fibre itself and there have been a large number of human population studies to determine its role. A meta-analysis of these studies showed, rather surprisingly, that the reduction in risk was relatively modest (Trock et al., 1990). A subsequent analysis of published studies showed that substantial protection was conferred by increased starch consumption (Cassidy et al., 1994). A recent study has shown that a low-risk population of Africans had lower fibre but higher starch intakes than a high-risk population of Europeans (O'Keefe et al., 1999). The Africans had higher rates of large bowel fermentation as measured by breath H2 evolution which implies greater SCFA production. However, it appears simplistic to suppose that increased RS consumption will automatically confer benefit. There are not only a large number of determinants of starch digestibility but also individual variations in the microflora. In vitro data show that faecal inocula from some subjects are less capable of fermenting starch than others (Weaver et al., 1989). This has been confirmed in a more recent study (Cummings et al., 1996) which suggested that the variability was a property of both the individual's microflora and the type of RS. It remains to be established which properties of the starch and the mix of bacterial species in the large bowel ecosystem allow amylolysis to proceed. Other animal studies have shown that the absolute levels of SCFA, the relative proportions of the major acids and their distribution along the colon varied between different sources of RS (Annison and Topping, 1994). Overall, these observations have led to the hypothesis that specific types of starch may be especially effective in raising butyrate (Topping and Bird, 1999). One of these appears to be maize as the in vitro studies, in which preferential production of this acid was shown, used starch from this cereal.
Starches, RS and the Microflora
Starches can influence the gut microflora simply through the provision of extra substrate. This may occur in the mouth as fermentable carbohydrates (particularly sucrose and other disaccharides and monosaccharides) have been implicated in caries formation (Bibby, 1982). The highly gelatinised starches found in processed foods may be even more important. Highly gelatinised starches stick to teeth on chewing (Åkerberg et al., 1998) and oral carboxylic acid production is greater with foods which favour this adhesion (Kashket et al., 1996). It seems that factors affecting starch adhesion and/or fermentation could lower caries risk so that processed and unprocessed foods containing RS would affect risk of cariogenesis.
Adverse reactions to RS have been reported under some circumstances. One area of considerable economic significance is the weaning of piglets where significant mortality accrues from swine dysentery due to a specific organism, Serpulina hyodysenteriae. Feeding of RS and fermentable NSP (guar gum) leads to raised large bowel SCFA, colonisation with this bacterium and appearance of pathological clinical symptoms (Pluske et at., 1998). These diets increase the number of total culturable bacteria and colonisation by synergistic Fusobacterium (Durmic et al., 1998). It should be noted that commercial weaning of pigs is effected very abruptly through introduction of the new diet. It may be this rapid transition, and the concomitant destabilisation of the enteric microflora, which contributes to the syndrome. Possibly, the phenomenon may be relevant to human gut disease as starches (and NSP) have been implicated also in human IBD. Total carbohydrate (starch and sugar) consumption seems to be higher in IBD patients than in controls (Tragnone et al., 1995). An interaction between the gut microflora and the mucosa is a possible contributor to this condition which could be enhanced by fermentable carbohydrate. Immunoreactive antibodies to specific organisms, specifically Klebsiella, have been found in patients with ankylosing spondylitis, a population affected also by IBD (Tiwana et al., 1997). A diet low in starch has been shown to lower circulating IgA in these patients leading to the suggestion that the presence of potentially pathogenic organisms in the faecal microflora may be a trigger for these conditions (Ebringer and Wilson, 1996). If this were to occur, it could be a serious contraindication for greater RS consumption. However, further study has not supported a primary role for K. pneumoniae in spondylitis (Smith et al., 1997) or this organism, Escherichia coli or Listeria monocytogenes in IBD (Walmsley et al., 1998). Indeed, it appears that these responses may be a consequence of active disease rather than causative. Removal of RS from the diet induces temporary remission through bowel rest as does a low fibre diet (Klurfeld, 1999). Sulphur-reducing bacteria have been implicated in UC through the production of H2S and hence blocking of SCFA metabolism by colonocytes (Roediger et al., 1997). This remains to be established as does any role for RS. Disordered fermentation and, possibly, a disordered large bowel microflora have been implicated in IBS. It has been reported that both faecal SCFA concentrations and production rates in vitro are lower in patients with diarrhoea-predominant IBS (Treem et al., 1996). More recently, a study in vivo showed that H2 evolution was more rapid in IBS patients although total gas production was the same as in affected individuals (King et al., 1998). Both of these interesting studies were performed with very limited numbers of volunteers and require replication with larger populations.
Modulation of the Gut Microflora and the Role of Starches
The majority of colonic bacteria are saccharolytic, and several bacterial groups, including Eubacterium, Bacteroides, Bifidodobacterium and Escherichia can ferment starch (Brown et al., 1998; Wang and Gibson 1993). Clostridium butyricum and species of Bifidobacterium in particular are effective starch utilisers in vitro (Brown et al., 1998). There are data from studies with "human microflora-associated rats" showing that a retrograded high amylose starch raised large bowel butyrate and the numbers of lactobacilli and bifidobacteria (Silvi et al., 1999). These data demonstrate the potential for RS to modify the microflora specifically (i.e. to act as prebiotics).
Probiotics and the Role of Starches
Probiotic use centres on a specific colonisation of the human (or animal) gastrointestinal tract by one or several exogenous bacterial species which act to improve health (Naidu et al., 1999). Probiotic organisms commonly used in commercial foods are strains of the lactic acid bacteria Lactobacillus and Bifidobacterium. Streptococcus thermophilus and L. delbrueckii subsp. bulgaricus are used in yoghurt manufacture. Demonstrated physiological effects resulting from regular ingestion of probiotic bacteria include immune system stimulation (De Simone 1993), modulation of the enteric microflora (Fooks et al., 1999) and decreased faecal bacterial enzyme activities (Rafter, 1995). Probiotics have been claimed to be effective for preventing or treating a raft of intestinal and systemic disorders including infantile and travellers' diarrhoea, lactose maldigestion, hypercholesterolemia, constipation, and colon and other cancers (Goldin 1998; Sanders 1993). However it must be recognised that much of the investigation has been in animals and there have been relatively few well-controlled trials in humans. In the absence of such studies, many of the effects of probiotics remain unsubstantiated. When one effect, reduction of plasma cholesterol, was examined in humans no effect of probiotics was seen despite positive results in rodents (de Roos et al., 1999). Possible therapeutic use of probiotic lactic acid bacteria (LAB) to control H. pylori appears feasible. Various LAB strains have been shown to inhibit the growth of H. pylori in vitro (Aiba et al., 1998; Michetti 1999) and Lactobacillus johnsonii, administered to humans for 14 days in a randomized, double-blind clinical trial, suppressed but did not eradicate H. pylori infection (Michetti et al., 1999). The latter is an important finding given the potentially adverse outcomes of complete eradication, as referred to earlier. The inhibitory effect of probiotics on H. pylori may be due to the bactericidal actions of organic acids and other substances released by the probiotic microorganism (Brassart and Schiffrin 1997) or from direct competition of the probiotic with H. pylori for adhesion sites on gastric mucosal cells (Kabir et al., 1997). Effective use of probiotics in humans is limited by the apparent refractoriness of the human gut microflora to exogenous bacteria resulting in only relatively transient colonisation. In part this could reflect substantial loss of viability of the organisms on passage through the relatively hostile environment of the stomach and small intestine. This drawback has led to the development of the concept of prebiotics.
Starches as Prebiotics
Prebiotics are defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and (or) activity of one or a limited number of bacterial species already resident in the colon, and, thus, attempt to improve host health" (Gibson and Roberfroid 1995). Perhaps one of the best known of these agents is fructo-oligosaccharide (FOS) but certain other carbohydrates appear to have prebiotic potential. Starch and NSP have been regarded as unlikely prebiotics, largely because many of the colonic bacterial species are capable of metabolising these carbohydrates. However, several in vivo studies in experimental animals and humans suggest otherwise. Alginates, which are used widely in the food industry, appear to be prebiotics (Terada et al., 1996) and RS may also be useful. During studies with a high amylose RS it was noted that the granules developed a particular etching pattern on passage through the upper gut of either pigs or humans (Topping et al., 1997a). This resulted in surface pitting (Figure 1a, b) and it was thought that this could enhance the viability of probiotics by providing a surface for physical adhesion. It was confirmed that specific bifidobacteria adhered to this starch (Figure 1c) (Brown et al., 1998). Feeding trials in mice (Brown et al., 1998) and pigs (Brown et al., 1997) showed that faecal excretion of bifidobacteria was higher when the animals were fed live Bifidobacterium longum with this high amylose starch. In pigs, faecal bifidobacterial numbers rose from 10.76 CFU/day in pigs fed a control (waxy maize) starch to 11.73 CFU/day in animals fed the high amylose starch (Brown et al., 1997). Total faecal SCFA and butyrate excretion was significantly higher when the pigs were fed the high amylose starch. In the absence of B. longum, excretion of total SCFA and butyrate were 25.4 and 3.2 mmol/day respectively with low amylose starch, and 60.7 and 9.1 with the high amylose starch. There was no effect of the probiotic on SCFA levels. A further experiment showed that the effects of FOS and the RS on faecal bifidobacteria numbers were additive (Topping et al., 1997b) - suggesting that they acted by different mechanisms (Table 4). Possibly the former acted as a substrate and the latter as a physical protector. This concept is supported by the longer survival and maintenance of high numbers of viable organisms in yoghurts containing high amylose starch where the effective shelf life was improved by several weeks (Brown et al., 1998). Prebiotic action does not seem to be limited to high amylose starches per se but also to chemically modified variants (i.e. RS4) which are also effective. Studies in vitro have shown that bifidobacteria can adhere to starches modified through acylation, octenylsuccinylation, carboxymethy-lation and succinylation (Brown et al., 1998). This adhesion varied by strain and did not extend to Lactobacillus casei. These data show that RS2 and RS4 have the potential to act as prebiotics - an attribute which could be especially useful in delivering organisms to regions where they are needed (e.g. to the distal colon). Several other studies support the prebiotic action of RS and that the effect varies with type. Kleessen et al., (1997b) have shown that retrograded potato starch (RS3), but not native potato starch (RS2), promotes faecal and caecal Lactobacillus counts in rats. A preliminary report showed that consumption of foods containing high amylose starch (RS2) by volunteers resulted in lower faecal counts of Bifidobacterium, and higher butyrate levels, compared to a cornflake control diet (Rao et al., 1997). In young pigs fed rice-based diets formulated to increase the quantity of starch (RS1 and 3) flowing into the caecum, counts of bifidobacteria and lactobacilli in the proximal colon were similar to those in pigs fed a digestible starch diet (Bird et al., 1997). Numbers of coliforms, especially E. coli, were several log units lower in pigs offered the RS-rich diet. These limited data suggest that certain starches alter the species composition and metabolic activity of the colonic microbiota. Not only is the total amount of starch reaching the colon important, but the chemical and physical properties of the resistant starch seem to be important in determining the prebiotic properties of RS.
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Figure 1. High amylose starch granule after passage though (a: left panel) human and (b: centre panel) porcine small intestine and (c: right panel) showing adherent bifidobacteria. |
Infant SCFA and Weaning to Starches
Probiotics do not seem to modify faecal SCFA (e.g. Brown et al., 1997) and, in any event, on current evidence they would seem unlikely to do so. Bifidobacteria and, to a lesser extent, lactobacilli are found in the human gastrointestinal system in their relatively greatest numbers in milk-fed infants (Mitsuoka, 1996). Studies using NMR and 13C-glucose as a substrate have confirmed that in infants a bifidobacterial fermentation appears to predominate (Wolin et al., 1998a). Examination of faecal samples from these infants show also that the SCFA profile is unlike that in adults (Edwards et al., 1994). Although acetate is the predominant SCFA in adults and pre-weaned bottle or breast-fed infants, propionate is present at much lower concentrations in the latter while butyrate is virtually absent. Other products such as ethanol, formate, succinate and lactate appear in infant faeces but not in appreciable quantities in adults (Wolin et al., 1998b). These metabolites may play an important role in controlling infection while the absence of butyrate could be significant for neonatal gut development. Faecal butyrate is rather higher in bottle-fed babies than in those receiving breast milk and the former may be at greater risk of developing IBD in later life than infants who were breast-fed (Szilagy, 1998). However, this predisposition may stem from a heightened sensitivity to milk formula proteins in infancy rather than any effect of butyrate. In adults, SCFA are trophic and appear to play a role in promoting a normal colonocyte phenotype. Diets high in a high amylose starch promote colon length in adult pigs (Topping et al., 1997a) and the increase in length is proportional to the amount of resistant starch entering the colon (Bird AR, Trimble RP, Brown, IL, Topping DL and Illman RJ - unpublished observations). It appears reasonable to assume that this reflects greater SCFA availability.
In infants, the role SCFA play in adults in promoting intestinal growth appears to be accomplished instead by growth factors in milk and those expressed within the colon. Insulin-like growth factors (IGF-I and IGF-II) are important regulators of somatic cell growth and differentiation and gut tissues seem to be particularly responsive to their trophic actions. IGF-I promotes tissue accretion in the small and large bowel of normal suckling (Steeb et al., 1997) and young adult rats (Conlon et al., 1995) and IGF-II appears to have similar although less potent effects than IGF-I (Conlon et al., 1995). However, not all studies have confirmed an increase in gut size in response to IGF-I administration (Bird et al., 1994a; Young et al., 1990). Short-term subcutaneous administration of long R3 IGF-I, a potent IGF-I analog, decreased overall length of the small intestine in mice and tended to reduce the thickness of the bowel wall (Bird et al., 1994a). In mice, short-term systemic administration of another growth factor, epidermal growth factor (EGF) had no effect on small bowel mass and length, or on jejunal architecture, yet this peptide elicited a substantial increase in active jejunal glucose absorption (Bird et al., 1994b). In humans, foetal plasma IGF concentrations increase with gestational age, especially during the final trimester although levels are markedly less than those found in adults (Lee and Han 1990). Concentrations continue to rise during infancy and childhood. Plasma IGF-I levels reach a maximum at puberty, and then decline progressively with age (Cohen et al., 1992, Sara and Hall 1990, Yamamoto et al., 1991). Plasma IGF-II concentrations are relatively low before birth and increase during the first year of life (Lee and Han 1990, Sara and Hall 1990). In adults, IGF-II is present in the bloodstream at levels fourfold greater than those of IGF-I (Sara and Hall 1990). It appears reasonable to assume that the weaning process leads to a shift in the large bowel towards SCFA both as metabolic substrates and as modulators of development and physiological activity. Maintenance of a pre-weaning colonic population of bacteria (and their fermentation products) in adults may not be desirable for long term bowel health. Similarly, maintenance of elevated plasma IGF-I may predispose to adenocarcinoma of the colon (Holly et al., 1999). Lower plasma levels of IGF binding protein (IGFBP), especially IGFBP-3, can increase the tissue availability of IGF-I (Ma et al., 1999). However, SCFA seem to interact with them to lower the risk of disease. Thus, in well differentiated cells of the colonic cancer cell line CaCo-2, butyrate stimulates the secretion of IGFBP-2 without altering that of IGFBP-3 (Nishimura et al., 1998). Butyrate also appears to lower the in vitro responsiveness of colon cancer cells to EGF (Archer et al., 1998). This could contribute to diminished tissue availability of IGF and so may modulate the carcinogenic process. Studies in animals have shown that IGF I and II are highest in neonates and decline with age. When adult rats were fed dietary fibre as wheat bran, caecal epidermal growth factor (EGF) levels were lower than in animals fed a low-fibre diet (Schaudies et al., 1991). This change was accompanied by an increase in the mass of cecal tissue. Under these circumstances wheat bran raises digesta concentrations of SCFA, including butyrate (Topping et al., 1993) which would be expected to increase substrate supply and also promote colonocyte growth and turnover. It could be hypothesised that fermentation provides SCFA which obviates the need for growth factors and leads to down regulation of their synthesis. If this is correct, it has a number of important corollaries. The first is to add support for breastfeeding as faecal butyrate concentrations in infants fed by this route are lower than in bottle-fed infants. Secondly, the weaning process itself could be optimised by the consumption of foods which promote a favourable bacterial profile while enhancing the production of SCFA, especially butyrate. RS (and other prebiotics) could assist in risk reduction by enhancing probiotic delivery and the generation of a favourable SCFA profile in the colon.
Recently, there has been concern about RS4 in infant foods (Lanciers et al., 1997) even though they may be prebiotics. As acylated SCFA, they may deliver SCFA to the large bowel (Annison et al., 1998). Thus, their presence in infant foods may be of assistance in the maturation of the developing large intestine. The final corollary is that efforts to modify the large bowel microflora so as to raise the populations of lactobacilli and bifidobacteria would be counterproductive if they were to lower the production of desirable products such as SCFA.
Conclusions and Future Directions
Starches and RS are emerging as important factors in health, especially through their interactions with gut bacteria. These interactions seem to occur both physically and through the metabolism of starches by the micro-organisms. It appears likely that starch fermentation in both the mouth and the large bowel can be modulated so as to lower disease risk. In the mouth, slower starch breakdown can reduce caries risk. In the colon, fermentation can promote SCFA production so as to optimise physiological function and manage and prevent important pathologies. It appears likely that the latter will involve tailoring the supply of RS to meet individual variations in the microflora which may limit starch fermentation. Newer varieties of starch high in RS are one of the means whereby this can be achieved. In this regard the parallels with NSP are striking. Dietary fibre used to be regarded as a single entity with multiple actions. Now, different NSP are known to affect gut physiology in disparate ways. The same appears to be true for RS1-4. Thus, identifying the factors which mean that the mix of bacteria in the microbial population which metabolise the various forms of RS appears to be an important research objective. Equally, identifying the ways in which the various types of RS can assist in delivering probiotics to the upper gut (to deal with infections) and to the large bowel (to optimise the microflora and to deliver SCFA) also is important. The relationships between the microflora in preweaned infants and those in adults, especially as influenced by starches seems to be important in promotion of gut health throughout life. The gaps in knowledge in these significant aspects of gut physiology are large and warrant exploration, especially as they relate to the gut microflora. Much of the technology (animal models, human faecal and in vitro measures, chemical and biological assays) is available to study microbial starch metabolism but the same cannot be said about the impact of starch on the microflora. While conventional culture and microscopic methods have provided the basic knowledge of the large bowel microlfora, they are limited both in scope and their demands for labour (Tannock, 1999). It is to be hoped that the application of new molecular methodologies will give a fuller understanding of the ways in which starches and other carbohydrates influence bacterial populations in the human gut.
Aiba, Y., Suzuki, Kabir, A.M., Takagi, A., and Koga, Y. 1998. Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model. Am. J. Gastroenterol. 93: 2097-2101.
Åkerberg, A.K.A., Liljeberg, H.G.E., Granfeldt, Y.E., Drews, A.W., and Björck, I.M.E. 1998. An in vitro method, based on chewing, to predict resistant starch content in foods allows parallel determination of potentially available starch and dietary fiber. J. Nutr. 128: 651-660.
Anderson, A.H., Levine, A.S., and Levitt, M.D. 1981. Incomplete absorption of the carbohydrate in all-purpose wheat flour. New Engl. J. Med. 304: 891-892.
Annison, G., and Topping, D.L. 1994. Resistant starch: chemical structure vs physiological function. Ann. Rev. Nutr. 14: 297-320.
Annison, G., Topping, D.L., Illman, R.J., Trimble, R.P., McGrath, L. 1998. Novel food ingredients for delivering specific short chain fatty acids to the large bowel. Proc. Nutr. Soc. Aust. 22: 91.
Archer, S., Meg, S.F., Woo, J., Johnson, J., Tang, R., and Hooding, R. 1998. Butyrate inhibits colon carcinoma cell growth through two distinct pathways. Surgery. 124: 248-253.
Ardizonne, S., Bollani, S., Manzionna, G., and Porro, G.B. 1999. Inflammatory bowel disease approaching the 3rd millenium: pathogenesis and therapeutic applications? Eur. J. Gastroenterol. Hepatol. 11: 27-32.
Asp, N.G., van Amelsvoort, J.M.M., and Hautvast, J.G.A.J. 1996. Nutritional implications of resistant starch. Nutr. Res. Rev. 9: 1-31.
Baghurst, P.A., Baghurst, K.I., and Record, S.J. 1996. Dietary fibre, non-starch polysaccharides and resistant starch - a review. Food Aust. 48: S3-S35.
Bibby, B.G. 1982. Changing perspectives on dental caries. In: Diet and Dental Caries Changing Perspectives. E. Story, ed. University of Melbourne, Melbourne. p. 1-17.
Bingham, S.A. 1999. High-meat diets and cancer risk. Proc. Nutr. Soc. 58: 243-248.
Bird, A.R., Croom, W.J., Brown, B.R., and Daniel, L.R. 1994a. The effect of long R3 insulin-like growth factor-I on jejunal glucose absorption in mice. Nutr. Res. 14: 1101-1112.
Bird, A.R., Croom, W.J., Fan, Y.K., Daniel, L.R., Black, B.L., McBride, B.W., Eisen, E.J., Bull, L.S., and Taylor, I.L. 1994b. Jejunal glucose absorption is enhanced by epidermal growth factor in mice. J. Nutr. 124: 231-240.
Bird, A.R., Illman, R.J., Hayakawa, T., and Topping, D.L. 1997. Brown rice increases faecal and large bowel short-chain fatty acid levels in pigs. Proc. Nut. Soc. (Aust). 21: 135.
Brassart, D., and Schiffrin, E.J. 1997. The use of probiotics to reinforce mucosal defence mechanisms. Trends Food Sci. Technol. 8: 321-326.
Brasitus, T.A., and Sitrin, M.D. 1990. Intestinal malabsorption syndromes. Annu. Rev. Med. 41: 339-347.
Brown, I.L., McNaught, K.J., and Moloney, E. 1995. Hi-maizeTM: new directions in starch technology and nutrition. Food Aust.47: 272-275.
Brown, I.L., Wang, X., Topping, D.L., Playne, M.J., and Conway, P.L. 1998. High amylose maize starch as a versatile prebiotic for use with probiotic bacteria. Food Aust. 50: 602-609.
Brown, I.,Warhurst, M., Arcot, J., Playne, M., Illman, R.J., and Topping, D.L. 1997. Fecal numbers of bifidobacteria are higher in pigs fed Bifidobacterium longum with a high amylose cornstarch than with a low amylose cornstarch. J. Nutr. 127: 1822-1827.
Burke, A., Lichtenstein, G.R., and Rombeau, J.L. 1997. Nutrition and ulcerative colitis. Baillieres Clin. Gastroenterol. 11: 153-174.
Burkitt, D.P. 1995. Historical aspects. In: Kritchevsky D, Bonfield C. Dietary fiber in health and disease. St Paul, Eagan Press, p. 3-10.
Caderni, G., Luceri, C., Lancioni, L., Tessitore, L., and Dolara, P. 1998. Slow-release pellets of sodium butyrate increase apoptosis in the colon of rats treated with azoxymethane, without affecting aberrant crypt foci and colonic proliferation. Nutr. Cancer 30: 175-181.
Cassidy, A., Bingham, S.A., and Cummings, J.H. 1994. Starch intake and colorectal cancer risk: an international comparison. Br. J. Cancer. 69: 937-942.
Cheng, H.H., and Yu, W.W. 1997. Lipid metabolism is altered by nebacitin in rats fed cooked-stored polished rice as the only dietary carbohydrate with or without exogenous cholesterol. J. Nutr. 1997 127: 153-157.
Cherbuy, C., Darcy-Vrillion, B., Morel, M.T., Pegorier, J.P., and Duee, P.H. 1995. Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-butyrate, glucose, and glutamine. Gastroenterology. 109: 1890-1899.
Cohen, P., Ocrant, I., Fielder, P.J., Neely, E.K., Gargosky, S.E., Deal, C.I., Ceda, G.P., Youngman, O., Pham, H., Lamson G., Guidice L.C., and Rosenfeld R.G. 1992 Insulin-like growth factors (IGFs): implications for aging. Psychoneuroendocrinology 17: 335-342.
Conlon, M.A., Francis, G.L., Tomas, F.M., Wallace, J.C., Howarth G.S., and Ballard F.J. 1995. Continuous 14 day infusion of IGF-II increases the growth of normal female rats, but exhibits a lower potency than IGF-I. J. Endocrinol. 144: 91-98.
Crowley, S., Antioch, K., Carter, R., Waters, A.-M., Conway, L., and Mathers, C. 1992. The cost of diet-related disease in Australia. Canberra, Australian Institute of Health and Welfare.
Coudray, C., Bellanger, J., Castiglia-Delavaud, C., Remesy, C., Vermorel, M., and Rayssiguier, Y. 1997. Effects of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. Eur. J. Clin. Nutr. 51: 375-380.
Cummings, J.H., Beatty, E.R., Kingman, S.M., Bingham, S.A., and Englyst, H.N. 1996. Digestion and physiological properties of resistant starch in the human large bowel. Br. J. Nutr. 75: 733-747.
Cummings, J.H., and Macfarlane, G.T. 1991. The control and consequences of bacterial fermentation in the human colon. J. Appl. Bacteriol. 70: 443-459.
Cummings, J.H., and Macfarlane, G.T. 1997. Role of intestinal bacteria in nutrient metabolism. J. Parenter. Enteral. Nutr. 21: 357-365.
Cummings, J.H., Rombeau, J.L., and Sakata, T., eds. 1995. Physiological and clinical aspects of short-chain fatty acids. Cambridge: Cambridge University Press.
Danesh, J., and Peto, R. 1998. Risk factors for coronary heart disease and infection with Helicobacter pylori - meta-analysis of 18 studies. Br. Med. J. 316: 1130-1132.
De Roos, N.M., Schouten, G., and Katan, M.B. 1999. Yoghurt enriched with Lactobacillus acidophilus does not lower blood lipids in healthy men and women with normal to borderline high serum cholesterol levels. Eur. J. Clin. Nutr. 53: 277-280
De Simone, C., Vesely, R., Bianchi, Salvadori, B., and Jirillo, E. 1993. The role of probiotics in modulation of the immune system in man and in animals. Int J. Immunotherapy. 9: 23-28.
Dore, M.P., Bilotta, M., Vaira, D., Manca, A., Massarelli, G., Leandro, G., Atzei, A., Pisanu, C., Graham, D.Y., and Realdi, G. 1999. High prevalence of Helicobacter pylori infection in shepherds. Dig. Dis. Sci. 44: 1161-1164.
Drasar, B.S. 1988. The bacterial flora and the intestine. In: Rowland I.R., editor. Role of the Gut Flora in Toxicity and Cancer. Academic Press, London.
Durmic, Z., Pethick, D.W., Pluske, J.R., and Hampson, D.J. 1998. Changes in bacterial populations in the colon of pigs fed different sources of dietary fibre, and the development of swine dysentery after experimental infection. J. Appl. Microbiol. 85: 574-582.
Ebringer, A., and Wilson, C. 1996. The use of a low starch diet in the treatment of patients suffering from ankylosing spondylitis. Clin. Rheumatol. 15: 62-6.
Edwards, C.A., Parrett, A.M., Balmer, S.E., and Wharton, B.A. 1994. Faecal short chain fatty acids in breast-fed and formula-fed infants. Acta Paedatr. 83: 459-462.
Ellis, R.W. 1997. Infection and coronary heart disease. J. Med. Microbiol. 46: 535-539.
Englyst, H.N., Kingman, S.M., Hudson G.J., and Cummings J.H. 1996. Measurement of resistant starch in vitro and in vivo. Br. J. Nutr. 75: 749-755.
Ernst, P. 1999. The role of inflammation in the pathogenesis of gastric cancer. Aliment. Pharmacol. Therapeutics. 13: 13-18.
Farthing, M.J.G. 1999. Gut infections. Eur. J. Gastroenterol. Hepatol. 11: 17-20.
Fooks, L.J., Fuller, R., and Gibson, G.R. 1999. Prebiotics, probiotics and human gut microbiology. Int. Dairy J. 9: 53-61.
Gibson, G.R., and Roberfroid, M.B. 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125: 1401-1412.
Gidley, M.J., Cooke, D., Darke, A.H., Hoffmann, R.A., Russell, A.L., and Greenwell, P. 1995. Molecular order and structure in enzyme-resistant retrograded starch. Carb. Polymers. 28: 23-31.
Goldin, BR. 1998. Health benefits of probiotics. Br. J. Nutr. 80: S203-S207.
Gurfinkel, l.E., Bozovich, G., Beck, E., Testa, E., Livellara, B., and Mautner, B. 1999. Treatment with the antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. The final report of the ROXIS Study. Eur. Heart J. 20: 121-127.
Heatley, R.V., and Guillou, P.G. 1997. Barrett's oesophagus - a ray of hope. Eur. J. Gastroenterol. Hepatol. 9: 873-875.
Hill, MJ. 1995. Bacterial fermentation of complex carbohydrate in the human colon. Eur. J. Cancer Prevention. 4: 353-358.
Holly, J.M.P., Gunnell, D.J., and Davey-Smith, G. 1999. Growth hormone, IGF-I and cancer. Less intervention to avoid cancer? More intervention to prevent cancer. J. Endocrinol. 162: 321-330.
Holtug, K., Rasmussen, H.S., and Mortensen, P.B. 1988. Short chain fatty acids in inflammatory bowel disease. The effect of bacterial fermentation of blood. Scand J. Clin. Lab. Invest. 48: 667-671
Jenkins, D.J., Vuksan, V., Rao A.V., Vidgen, E., Kendall, C.W., Tariq, N., Wursch, P., Koellreutter, B., Shiwnarain, N., and Jeffcoat, R. 1999. Colonic bacterial activity and serum lipid risk factors for cardiovascular disease. Metabolism. 48: 264-268.
Kabir, A.M.A., Aiba, Y., Takagi, A., Kamiya, S., Miwa, T., and Koga, Y. 1997. Prevention of Helicobacter pylori infection by lactobacilli in a gnotobiotic murine model. Gut. 41: 49-55.
Kashket, S., Zhang, J., and Van Houte J. 1996. Accumulation of fermentable sugars and metabolic acids in food particles that become entrapped on the dentition. J. Dent. Res. 75: 1885-1891.
Key, F.B., and Mathers, J.C. 1995. Digestive adaptations of rats given white bread and cooked haricot beans (Phaseolus vulgaris): large-bowel fermentation and digestion of complex carbohydrates. Br. J. Nutr. 74: 393-406
King, T.S., Elia, M., and Hunter, J.O. 1998. Abnormal colonic fermentation in irritable bowel syndrome. Lancet. 352: 1187-1189.
Kleessen, B., Sykura, B., Zunft, H.-J., and Blaut, M. 1997. Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. Am. J. Clin. Nutr. 65: 1397-1402.
Klurfeld, D.M. 1999. Colonic diseases and disorders: nutritional management. In: Encyclopaedia of Human Nutrition. Sadler MJ, Strain JJ., and Caballero B, ed. Academic Press, San Diego. p. 427-433.
Kvietys, P.R., and Granger, D.N. 1981. Effect of volatile fatty acids on blood flow and oxygen uptake by the dog colon. Gastroenterology. 80: 962-969.
Lampe, J.W., Fredstrom, S.B., Slavin, J.L., and Potter, J.D. 1993. Sex differences in colonic function: a randomised trial. Gut. 34: 531-536.
Lanciers, S., Mehta, D.I., Blecker, U., and Lebenthal, E. 1997. The role of modified food starches in baby food. J. La State Med. Soc.149: 211-214.
Lee, D.C., and Han, K.M. 1990. Expression of growth factors and their receptors in development. In: Peptide Growth Factors and Their Receptors II (Sporn M.B., and Roberts A.B., eds.) p. 611-654. Springer-Verlag, Berlin, Germany.
Lewis, S.J., and Heaton, K.W. 1999. Roughage revisited: the effect on intestinal function of inert plastic particles of different sizes and shape. Dig. Dis. Sci. 4: 744-788.
Lisby, G., Andersen, J., Engbaek, K., and Binder, V. 1994. Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction. Scand. J. Gastroenterol. 29: 923-929.
Ma, J., Pollak, M.N., Giovannucci, E., Chan, J.M., Tao, Y., Hennekens, C.H., and Stampfe,r M.J. 1999. Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J. Natl. Cancer Inst. 91: 620-625.
Macaluso, A., Simmang, C., and Anthony, T. 1998. Streptococcus sanguis bacteremia and colorectal cancer. South Med. J. 9: 206-7.
Marsono, Y., Illman, R.J., Clarke, J.M., Trimble, R.P., and Topping, D.L. 1993. Plasma lipids and large bowel volatile fatty acids in pigs fed white rice, brown rice and rice bran. Br. J. Nutr. 70: 503-513.
McColl, K.E.L. 1999. Helicobacter pylori 1988-1998. Eur. J. Gastroenterol. Hepatol 11: 13-16.
McIntyre, A., Vincent, R.M., Perkins, A.C., and Spiller, R.C. 1997. Effect of bran, ispaghula, and inert plastic particles on gastric emptying and small bowel transit in humans: the role of physical factors. Gut. 140: 223-227.
Metchnikoff, E. 1907. The prolongation of life: optimistic studies. P. Chalmers Mitchell (ed). Heinemann, London.
Michetti, P., Dorta, G., Wiesel, P.H., Brassart, D., Verdu, E., Herranz, M., Felley, C., Porta, N., Rouvet, M., Blum, A.L., and Corthesy-Theulaz, I. 1999. Effect of whey-based culture supernatant of Lactobacillus acidophilus (johnsonii) La1 on Helicobacter pylori infection in humans. Digestion. 60: 203-209.
Mitsuoka, T. 1996. Intestinal flora and human health. Asia Pacific J. Clin. Nutr. 5: 2-9.
Morand, C., Remesy, C., Levrat, M.A., and Demigne, C. 1992. Replacement of digestible wheat starch by resistant cornstarch alters splanchnic metabolism in rats. J. Nutr. 122: 345-354.
Morgan, M.V., Campain, A.C., Adams, G.G., Crowley, S.J., and Wright F.A. 1998. The efficacy and effectiveness of a primary preventive dental programme in non-fluoridated areas of Victoria, Australia. Community Dent. Health. 15: 263-271.
Moshfegh, A.J., Friday, J.E., Goldman, J.P., and Ahuja, J.K. 1999. Presence of inulin and oligofructose in the diets of Americans. J. Nutr. 129 (Suppl): 1407S-1411S.
Muir, J.G., and O'Dea, K. 1993. Validation of an in vitro assay for predicting the amount of that escapes digestion in the small intestine of humans. Am. J. Clin. Nutr: 57: 540-546.
Naidu, A.S., Bidlack, W.R., and Clemens, R.A. 1999. Probiotic spectra of lactic acid bacteria (LAB). Crit. Rev. Food Sci. Nutr. 39: 13-126.
Nishimura, A., Fujimoto, M., Oguchi, S., Fusunyan, R.D., MacDermott R.P., and Sanderson I.R. 1998. Short-chain fatty acids regulate IGF-binding protein secretion by epithelial cells. Am. J. Physiol. 275 (Endocrinol Metab): E55-E63.
Noakes, M., Clifton, P., Nestel, P.J., Le Leu, R., and McIntosh, G.H. 1996. Effect of high amylose starch and oat bran on metabolic variables and bowel function in subjects with hypertriglyceridemia. Am. J. Clin. Nutr. 64: 944-951.
Norfleet, R.G., and Mitchell, P.D. 1993. Streptococcus bovis does not selectively colonize colorectal cancer and polyps. J. Clin. Gastroenterol. 17: 25-8.
O'Keefe, S.J., Kidd, M., Espitalier-Noel, G., and Owira, P. 1999. Rarity of colon cancer in Africans is associated with low animal product consumption, not fiber. Am. J. Gastroenterol. 94: 1373-1380.
Parchure, A.A., and Kulkarni, P.R. 1997. Effect of food processing treatments on generation of resistant starch. Int J. Food Sci. Nutr. 48: 257-260
Pluske, J.R., Durmic, Z., Pethick, D.W., Mullan, B.P., and Hampson, D.J. 1998. Confirmation of the role of rapidly fermentable carbohydrates in the expression of swine dysentery in pigs after experimental infection. J. Nutr. 128: 1737-1744.
Potter, M.A., Cunliffe, N.A., Smith, M., Miles, R.S., Flapan, A.D., and Dunlop, M.G. 1998. A prospective controlled study of the association of Streptococcus bovis with colorectal carcinoma. J. Clin. Path. 51: 473-474.
Prohaszka, L., Jayarao, B.M., Fabian, A., and Kovacs, S. 1990. The role of intestinal volatile fatty acids in the Salmonella shedding of pigs. Zentralbl. Veterinar. Med. [B] 37: 570-574.
Rafter, J.J. 1995. The role of lactic acid bacteria in colon cancer prevention. Scand. J. Gastroenterol. 30: 497-502.
Rao, A.V., Shiwnarain, N., and Jenkins, D.J.A. 1997. Effect of high amylose resistant starch on the composition of intestinal microflora. FASEB J. 11: 3527.
Richter, J.E., Falk, G.W., and Vaezi, M.F. 1998. Helicobacter pylori and gastroesophageal reflux: the bug may not be all bad. Am. J. Gastroenterol. 93: 1800-1802.
Roberton, A.M. 1993. Roles of endogenous substances and bacteria in colorectal cancer. Mut. Res. 290: 71-78.
Roediger W.E.W. 1982. Utilisation of nutrients by isolated epithelial cells of the rat colon. Gastroenterology. 83: 424-429.
Roediger, W.E., Moore, J., and Babidge, W. 1997. Colonic sulfide in pathogenesis and treatment of ulcerative colitis. Dig. Dis. Sci. 42: 1571-1579.
Rowland, I.R., Mallett, A.K., and Wise, A. 1985. The effect of diet on the mammalian gut flora and its metabolic activities. CRC Crit. Rev. Toxicol. 16: 31-103.
Salminen, S., Isolauri, E., and Onnela, T. 1995. Gut flora in normal and disordered states. Chemotherapy. 41: 5-15.
Sanders, M.E. 1993. Effect of consumption of lactic cultures on human health. Adv. Food Nutr. Res. 37: 67-130.
Sara, V.R., and Hall, K. 1990. Insulin-like growth factors and their binding proteins. Physiol. Rev. 70: 591-614.
Savage, D.C. 1986. Gastrointestinal microflora in mammalian nutrition. Ann. Rev. Nutr. 6: 155-178.
Schaudies, R.P, Satchithanandam, S., and Calvert, RJ. 1991. Alteration in levels of immunoreactive epidermal growth factor in the gastrointestinal mucosa of Fischer rats fed a diet containing 10% wheat bran. J. Nutr. 121: 800-805.
Scheppach, W. 1994. Effects of short chain fatty acids on gut morphology and function. Gut. 35: S35-S38.
Scheppach, W., Christl, S.U., Bartram, H.P., Richter, F., and Kasper, H. 1997. Effects of short-chain fatty acids on the inflamed. colonic mucosa. Scand. J. Gastroenterol. 222: 53-57.
Scheppach, W., Fabian, C., Sachs, M., and Kasper, H. 1988. The effect of starch malabsorption on fecal short chain fatty acid excretion in man. Scand J. Gastroenterol. 23: 755-759.
Shannon, J.C., and Garwood, D.L. 1984. Genetics and physiology of starch development. In: Starch: chemistry and technology. Whistler RL, BeMiller J.N., and Paschall E.F., eds. Academic Press, London p. 25-86.
Sievert, D., and Pomerantz, Y. 1989. Enzyme-resistant starch I. Characterisation and evaluation by enzymatic, thermoanalytical, and microscopic methods. Cereal Chem. 66: 342-347.
Silvi, S., Rumney, C.J., Cresci, A., and Rowland, I.R. 1999. Resistant starch modifies gut microflora and microbial metabolism in human flora-associated rats inoculated with faeces from Italian and UK donors. J. Appl Microbiol 86: 521-530.
Smith, G.W., Blackwell, C.C., and Nuki, G. 1997. Faecal flora in spondyloarthropathy. Br. J. Rheumatol 36: 850-854.
Smith, J.G., Yokoyama, W.H., and German, J.B. 1998. Butyric acid from the diet: Actions at the level of gene expression. Crit. Rev. Food Sci. 38: 259-29.
Steeb, C.-B., Shoubridge, C.A., Tivey, D.R., and Read, L.C. 1997. Systemic infusion of IGF-I or LR3IGF-I stimulates visceral organ growth and proliferation of gut tisses in suckling rats. Am. J. Physiol. 272: G522-G533.
Stephen, A.M. 1991. Starch and dietary fibre: their physiological and epidemiological relationships. Can. J. Physiol. Pharmacol. 69: 116-120.
Stephen, A.M., and Cummings, J.H. 1981. Mechanism of action of dietary fibre in the human colon. Nature 284: 283-284.
Stephen, A.M., Haddad, A.C., and Phillips, S.F. 1983. Passage of carbohydrate into the colon: direct measurement in humans. Gastroenterol. 85: 589-595.
Stephen, A.M., Wiggins, H.S., Englyst, H.N., Cole, T.J., Wayman, B.J., and Cummings, J.H. 1986. The effect of age, sex and level of intake of dietary fibre from wheat on large-bowel function in thirty healthy subjects. Br. J. Nutr. 56: 349-361.
Szilagyi, A. 1998. Altered colonic environment, a possible predisposition to colorectal cancer and colonic inflammatory bowel disease: Rationale of dietary manipulation with emphasis on disaccharides. Can. J. Gastroenterol. 12: 133-146.
Tannock, G.W. A fresh look at the intestinal microflora. 1999. In: Probiotics: A critical review. G.W. Tannock , ed. Horizon Scientific Press, Wymondham. p 5-14.
Terada, A., Hara, H., and Mitsuoka, T. 1996. Effect of dietary alginate on the faecal microbiota and faecal metabolic activity in humans. Microbial Ecol. Health Dis. 8: 259-266.
Thornton, J.R., Dryden, A., Kelleher, J., and Losowsky, M.S. 1987. Super-efficient starch absorption. A risk factor for colonic neoplasia? Dig. Dis. Sci. 32: 1088-1091.
Tiwana, H., Wilson, C., Walmsley, R.S., Wakefield, A.J., Smith, M.S., Cox, N.L., Hudson, M.J., and Ebringer, A. 1997. Antibody responses to gut bacteria in ankylosing spondylitis, rheumatoid arthritis, Crohn's disease and ulcerative colitis. Rheumatol. Int. 17: 11-16.
Topping, D.L., and Bird, A.R. 1999. Foods, nutrients and digestive health. Aust. J. Nutr. Dietet. 56: S22-S42.
Topping, D.L., Illman, R.J., Clarke, J.M., Trimble, R.P., Jackson, K.A., and Marsono, Y. 1993. Dietary fat and fiber alter large bowel and portal venous volatile fatty acids and plasma cholesterol but not biliary steroids in pigs. J. Nutr. 123: 133-143.
Topping, D.L., Gooden, J.M., Brown, I.L., Biebrick, D.A., McGrath, L., Trimble, R.P., Choct, M., and Illman, R.J. 1997a. A high amylose (amylomaize) starch raises proximal large bowel starch and increases colon length in pigs. J. Nutr. 127: 615-622.
Topping, D.L., Warhurst, M., Illman, R.J., Brown, I.L., Playne, M.J., and Bird, A.R. 1997b. A high amylose (amylomaize) starch and fructooligosaccharide increase faecal excretion of bifidobacteria in pigs fed live Bifidobacterium longum. Proc. Nutr. Soc. Aust. 21: 134.
Tragnone, A., Valpiani, D., Miglio, F., Elmi, G., Bazzochi, G., Pipitone, E., and Lanfranchi, G.A. 1995. Dietary habits as risk factors for inflammatory bowel disease. Eur. J. Gastroenterol. Hepatol. 7: 47-51.
Treem, W.R., Ahsan, N., Shoup, M., and Hyams, J.S. 1994. Fecal short-chain fatty acids in children with inflammatory bowel disease. J. Pediatr. Gastroenterol. Nutr. 18: 159-164.
Trock, B., Lanza, E., Greenwald, P. 1990. Dietary fiber, vegetables, and colon cancer: critical review and meta-analyses of the epidemiologic evidence. J. Natl. Cancer Inst. 82: 650-661.
Tsuji, S., Tsuji, M., Sun, W.H., Gunawan, E.S., Murata, H., Kawano, S., and Hori M. 1997. Helicobacter pylori and gastric carcinogenesis. J. Clin. Gastroenterol. 25: S186-197.
Van Munster, I.P., Tangerman, A., and Nagengast, F.M. 1994. Effect of resistant starch on colonic fermentation, bile acid metabolism, and mucosal proliferation. Dig. Dis. Sci. 39: 834-842.
Van Soest, P.J. 1995. Comparative aspects of animal models. In: Dietary fibre in health and disease. D Kritchevsky and C Bonfield, eds. Eagan Press, St. Paul. p. 321-329.
Velazquez, O.C., Lederer, H.M., and Rombeau, J.L. 1996. Butyrate and the colonocyte. Implications for neoplasia. Dig. Dis. Sci. 41: 727-739 .
Walsmley, R.S., Anthony, A., Sim, R., Pounder, R.E., and Wakefield, A.J. 1998. Absence of Escherichia coli, Listeria monocytogenes, and Klebsiella pneumoniae antigens within inflammatory bowel disease tissues. J. Clin. Pathol. 51: 657-661.
Wang, X., and Gibson, G.R. 1993. Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J. Appl. Bacteriol. 75: 373-380.
Weaver, G.A., Krause, J.A., Miller, T.L., and Wolin, M.J. 1989. Constancy of glucose and starch fermentations by two different human faecal microbial communities. Gut. 30: 19-25.
Weaver, G.A., Krause,J.A., Miller,T.L., and Wolin, M.J. 1992. Cornstarch fermentation by the colonic microbial community yields more butyrate than does cabbage fiber fermentation; cornstarch fermentation rates correlate negatively with methanogenesis. Am.J. Clin. Nutr. 55: 70-77.
Wolin, M.J., Zhang, Y., Bank, S., Yerry, S., and Miller, T.L. 1998a. NMR Detection of 13CH313COOH from 3-13C-glucose: a signature for Bifidobacterium fermentation in the intestinal tract. J. Nutr. 128: 91-96.
Wolin, M.J., Yerry, S., Miller, T.L., Zhang, Y., and Bank, S. 1998b. Changes in production of ethanol, acids and H2 from glucose by the fecal flora of a 16- to 158-d-old breast-fed infant. J. Nutr. 128: 85-90.
World Cancer Research Fund and American Institute for Cancer Research. 1997. Food, nutrition and the prevention of cancer: a global perspective. American Institute for Cancer Research. Washington DC, ISBN: 1 899533 05 2.
Wurzburg, O.B. 1986. Nutritional aspects and safety of modified food starches. Nutr. Rev. 44: 74-79.
Yamamoto, H., Sohmiya, M., Oka, N., and Kato, Y. 1991. Effects of aging and sex on plasma insulin-like growth factor I (IGF-I) levels in normal adults. Acta Endocrinol. 124: 497-500.
Young, G.P., Taranto, T.M., Jonas, H.A., Cox, A.J., Hogg, A., and Werther, G.A. 1990. Insulin-like growth factors and the developing and mature rat small intestine: receptors and biological actions. Digestion. 46: 240-252.