1. Introduction

In the 1970s Burkitt and Trowell published their revolutionary epidemiological study on non-contagious diseases in traditional African population groups whose diet was characterized by a high percentage of plant fiber. From the low incidence of these diseases in the African population groups studied, Trowell formulated the hypothesis that the low-fiber diet in the industrial developed countries is the cause of numerous civilization-related diseases (Biesalski, 1995).

The intense research carried out after this publication did not confirm the simple cause-and-effect relationship postulated by Trowell between dietary fiber and civilization-related diseases. It did have the result, however, that a vast amount of diversified information was gathered on this group of nutrients; as a result, a complex picture emerged of the many types of dietary fiber, the "digestionö of dietary fiber in the human large intestine, and the possible mechanism of action involved. This picture yielded many new insights on the bacterial colonization of the human large intestine and its role in the mechanism of action of dietary fiber.

In the early 1990s the term "prebioticsö was established to designate types of dietary fiber which are a preferred source of nutrition for intestinal bacteria with especially beneficial effects.

From the interactions between prebiotics and dietary fiber on the one hand and the intestinal flora on the other, scientists continue to gain new insights on the preventive and therapeutic possibilities resulting from a fiber-rich, i.e. prebiotic, diet.

The microbial flora of the human intestines represents a highly complex system whose equilibrium can be disturbed by various endogenous and exogenous factors (e.g. age, physiological status, medication, illness, diet and stress). The decline in the bifidobacteria populations and the increased colonization of the human large intestine by potentially pathogenic microorganisms is an indicator of an "unhealthy stateö (Mitsuoka, 1992). The resulting disorders range from mild diarrhea to serious illnesses such as pseudomembranous enterocolitis.

The inclusion of prebiotic foods in the diet can make a contribution to stabilizing – or exerting a beneficial effect on – the intestinal flora; it can also help to prevent or stop diarrhea.

Persons who are ill or weak, in particular, may benefit from the beneficial effects of prebiotic foods. From the point of view of clinical nutrition, the points of central interest are: the anti-diarrhea effect, the improvement of the glycemic index in diabetes, and the possible long-term immunomodulatory effects.

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2. Definitions

The intestinal microflora represents a complex ecosystem affected by various environmental factors. It can be altered, for example, by several constituents of the human diet as well as by bacteria contained in our food. The following definitions will provide a survey of the nutrient groups involved.

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2. Definitions

The intestinal microflora represents a complex ecosystem affected by various environmental factors. It can be altered, for example, by several constituents of the human diet as well as by bacteria contained in our food. The following definitions will provide a survey of the nutrient groups involved.

2.1 Non-digestible food ingredients

The largest part of the food taken up by human beings is already digested and absorbed in the upper gastrointestinal tract. There are only a few food ingredients, referred to as "colonic foodö (Gibson & Roberfroid, 1995), which are not broken down by the gastric acid and human digestive enzymes in the small intestine and thus arrive in the large intestine in non-digested form. The "colonic foodö comprises non-digested fats, proteins and carbohydrates as well as other food ingredients (Cf. Table 1).

This non-digested food ingredients can be broken down by the digestive enzymes produced by the bacteria in the large intestine. Furthermore, they serve as a nutritional substrate for these bacteria. The metabolic products of these bacteria are, in turn, available to the intestinal cells and gut-associated lymphatic tissue. The co-existence of human beings and bacteria is thus a form of symbiosis.

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2.2 Dietary fiber

The term dietary fiber encompasses a large number of substances with very different chemical structures. There are botanical, physiological and chemical definitions (Biesalski, 1995).

According to a very broad definition, the term "dietary fiberö refers to all those "substances which are either not directly absorbed or directly absorbed to only an insignificant extent or which can not be converted into absorbable forms by human enzymes in the intestinal tractö (Biesalski, 1995). However, most authors refer to the following narrower definition:

"Dietary fiber consists of carbohydrates and lignin which are not digested by the human enzymes in the intestinal tractö (Biesalski, 1995).

This brochure deals exclusively with dietary fiber belonging to the group of carbohydrates.

The following table provides a complete overview of the different types of dietary fiber. The information in the top half of the table (above the double line) refers to the narrower definition (i.e. dietary fiber = carbohydrates). All other nutrients which are capable of passing through the stomach and small intestine without being digested are listed below the double line.

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2.3 Prebiotics

Prebiotics are non-digestible food ingredients which are often selectively added to foods intended for human consumption with the objective of exerting a beneficial effect on gut health.

Prebiotics are non-digestible food ingredients which can have a beneficial effect on the host organism (i.e. the human being) by stimulating the growth and/or activity of one or several species of colonic bacteria and thus promote the health of the host organism (Gibson & Collins, 1995).

Prebiotics can be distinguished from dietary fiber, according to the definition, by their selectivity or specificity for a single bacterial species or for a limited group of bacterial species. Two examples of prebiotics are oligofructose and inulin; both of these substances act in the large intestine to promote the growth and activity of bifidobacteria, a genus of bacteria whose intestinal-protective properties have been described in the literature.

Other groups of dietary fiber, in contrast, possess no specific affinity for health-enhancing bacteria. Consequently, they can also promote the growth and metabolic activity of microorganisms which are potentially detrimental to health.

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2.4 Probiotics

The term "probioticsö is derived from Greek and means "for lifeö. Since this term was used for the first time back in 1965, its definition has changed repeatedly.

Probiotics are nutritional supplements consisting of a sufficient number of viable defined microorganisms which cause the intestinal microflora of the host organism to change (as a result of implantation or colonization) and thus have a beneficial effect on the host´s health (Havenaar & Huis In´t Veld, 1992; Schrezenmeir & de Vrese, 2001).

Probiotics are thus bacterial strains which are expected to bring about a change for the better in the existing intestinal flora. This change causes a shift in the microecological equilibrium toward the potentially beneficial intestinal bacteria. And prebiotics are the preferred substrates of these probiotic strains of bacteria.

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2.5 Synbiotics

Probiotic and prebiotic foods are frequently marketed in the form of yogurt and other milk products enriched with probiotic bacteria. A combination of these two nutritional supplements is referred to as a synbiotic.

Synbiotics consist of a mixture of prebiotics and probiotics which have a beneficial effect on the host organism by implanting, and promoting the survival of, viable microorganisms in the host´s gastrointestinal tract (Gibson et al., 1995).

Since the name "synbioticsö is a play on the word "synergisticö, this name should be reserved for products simultaneously containing simultaneously probiotics and the prebiotic components they prefer as food (Schrezenmeir & de Vrese, 2001).

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3. Large Intestine (Colon)

For many years it was believed that the main function of the large intestine was to solidify the semi-solid contents of the small intestine and, in the process, reabsorb fluid and salt for the body. This picture of the function of the large intestine has been subjected to constant revision as new findings emerge concerning the intestinal bacteria living in the colon and their effects on the physiological processes underway in this part of the gastrointestinal tract.

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3.1 Anatomical structure of the large intestine

At the beginning of the large intestine, we find the ileocecal valve – also referred to as "Bauhin´s valveö – separating the small intestine from the large intestine. The ileocecal valve opens into the ascending colon. Below this valve, the initial part of the large intestine, or caecum, bulges out. The ascending colon is followed by the traverse colon, descending colon, sigmoid colon and rectum. The sigmoid colon and rectum serve as a reservoir for feces (stool).

Prebiotics and dietary fiber are digested via the processes described below; these processes take place mainly in the colon. The intensity of metabolic activity in the different sections of the colon depends on the density of the bacterial population; it declines toward the end of the large intestine because of the low nutrient supply at that location.

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3.2 Functional difference between small intestine and large intestine

The bulk of the nutrients are digested and absorbed in the small intestine. This process is facilitated by the digestive enzymes present in the intestinal wall which are specific for carbohydrates as well as by the digestive enzymes secreted by the pancreas and the gall bladder. Since no digestive enzymes are synthesized and secreted in the colon, no digestion due to human enzymes takes place here.

In comparison with the small intestine, the large intestine is heavily colonized by bacteria. Various factors determine the degree of colonization in the different intestinal sections.

First, the speed with which the semi-digested food passes through the small intestine exerts a decisive impact on the bacterial density of the intestines. The rapid transit time of food in the stomach and the small intestine (approx. 4-6 hours) does not permit the development of a significant bacterial population in this part of the gastrointestinal tract. The semi-digested food remains a substantially longer period of time in the colon than in the upper gastrointestinal tract. This is a factor predisposing to a high bacterial density and the accompanying physiological processes and fermentation processes in the colon.

In addition, the pH in the various sections of the intestines is responsible for the differences in bacterial colonization. For example, the gastric acid secreted by the stomach does its part to ensure that the microorganisms ingested along with our food are rapidly killed and that, as a result, the number of pathogenic microorganisms is dramatically reduced. The effect of gastric acid continues in the proximal parts of the small intestine. The pH is between 3 and 5 in the duodenum and rises to approx. 6.5 in the ileum. Lactic acid bacteria (lactobacilli) exhibit a growth maximum in an acidic milieu; for this reason, they account for the largest part of the bacterial population in the small intestine. It is only at the end of the small intestine that the number of other intestinal bacteria increases without, however, reaching the microbial concentrations found in the large intestine.

The specific structure of the intestinal mucosa is another striking difference between the small intestine and the large intestine.

The mucosal surface in the small intestine is enlarged about 600 times by Kerckring´s folds, villi and the brush border of the enterocytes. This results in an extremely large surface for the absorption of nutrients and for reactions between nutrients and intestinal enzymes.

In comparison with the surface of the mucosa in the small intestine, the surface of the large intestine displays significantly less structural enlargement. Kerckring´s folds and villi are totally absent. The surface is enlarged only via deep furrows referred to as " Lieberkühn´s cryptsö. These consist of simple mucosal epithelia with predominantly mucigenous goblet cells and epithelial cells with a brush border. In addition, numerous lymph follicles are found in the mucosa. This anatomical structure reflects the function of the large intestine, which is designed for water absorption and not for digestion.

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3.3 Intestinal surface and surface protection

The side of the intestinal mucosa facing the lumen possesses a protective layer of mucous (consisting of a glycoprotein network) with viscoelastic properties. The complex structure of the protective layer is of vital importance for the protection of the mucosal epithelia and for the colonization of bacteria. The mucous contains several important components, such as mucin and glycolipids, which serve as a basic framework for protective and probiotic bacteria (Bengmark, 1995).

In the lumen of the large intestine the intestinal bacteria find several microhabitats which they can permanently colonize. These include the surface of the epithelial cells (which certain bacteria can adhere directly to), the epithelial mucous layer, and the mucous layer in the crypts. The indigenous bacteria (i.e. the bacteria already living in the intestinal lumen) defend the ecological niches they occupy against microbial invaders; as a result, the latter have a hard time colonizing the intestinal mucosa (Freter, 1992).

The equilibrium of the intestinal flora, and thus gut health, depends largely on the supply of nutrients available for the intestinal bacteria. The absence of a fiber-rich enteral diet over longer periods of time deprives the intestinal bacteria of an important source of nutrients. This can cause the host-specific bacterial population to decline, a situation which lessens the body´s protection against pathogenic microorganisms. Important metabolic products (Cf. Chapter 3.6) supplied by the intestinal flora are in ever shorter supply. Over the long term this leads to an inadequate supply of nutrients to the mucosa. The result is atrophy of the normal mucosa and atrophy of the mucous-producing goblet cells in combination with a deficient protective mucous layer (Bengmark, 1995).

Antibiotics have similar damaging effects on intestinal health and, after longer periods of use, can largely destroy the intestinal flora. This can pave the way for the colonization of the intestines with pathogenic microorganism, for infectious diseases, and for the destruction of normal protective functions (Finegold et al, 1983).

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3.4 Intestinal flora

The terms "intestinal floraö or "microfloraö are used to describe the entire bacterial population of the intestines regardless of type and number. The most important areas of the human gastrointestinal tract which are permanently inhabited by bacteria are the distal ileum and the entire colon (Cf. Fig. 4).

The intestinal flora (Fig. 5) contains pathogenic microbes with potentially negative effects on the intestines and the entire organism as well as microbes with both positive and negative properties and microbes with predominantly beneficial effects on the intestines and the entire organism (Gibson & Roberfroid, 1995). The total intestinal flora consists of more than 1014 microbes representing approx. 400 – 500 different microbial species. The human body, in comparison, consists of only approx. 1013 cells. Taking into consideration that every single bacteria in the intestinal flora has its own metabolism, and as a result has an impact on its environment, we can assess the total impact of the intestinal bacteria on their human host from these figures (From Kullak, 1997).

The gastrointestinal tract of the human fetus is still sterile. It is not until after the baby has been born, and had its first food, that it is gradually colonized by microorganisms. Whereas the composition of the intestinal flora changes constantly during the first two years of life, it then attains a relatively stable status called the "climax floraö (Mackie, 1999).

After reaching the "climax floraö stage, the gastrointestinal tract represents an extremely complex and by no means uniform ecosystem. A number of endogenous factors (including pH, redox potential and various intestinal secretions, some of which have an effect on microbes) exert a decisive effect on the composition and size of the microbial population (Holzapfel & Haberer, 2000).

In the distal small intestine there are still traces of oxygen and oxygen radicals. Apart from enterococci and a few gram-negative species, lactobacilli account for the largest population at this location. Lactobacilli are oxygen tolerant. In contrast, the development of strictly anaerobic bacteria, e.g. bifidobactria and clostridia, is suppressed in the small intestine.

In the large intestine the transit time of the semi-digested food is significantly slower, i.e. 60 hours on the average in a person with a Western diet. This circumstance, as well as the special pH and oxygen conditions, supports the development of a large bacterial population containing approx. 1011 – 1012 bacteria per gram of intestinal contents (Macfarlane & Cummings, 1991).

Exogenous factors such as nutrition also play a role in determining the composition of the intestinal flora. The differences noted in the colonization of the intestines in Asians, North Americans and Europeans (Mitsuoka, 1992) may be attributable at least in part to the typical diet in these regions (Holzapfel et al., 2000). It has also been shown that infants with different types of diet (e.g. breast-fed or formula-fed) display clear-cut differences in the composition of their intestinal flora.

A comparison of the bacterial flora of normal healthy individuals and persons suffering from disease, moreover, reveals differences between these two groups (Van de Waaij, 1999). In general it is desirable for the bacterial colonization of the intestines to attain an equilibrium favoring bacteria with health promoting properties, such as lactobacilli and bifidobacteria. The probiotic and prebiotic concepts (Cf. Chapters 4 and 5) support the establishment of this kind of bacterial flora.

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3.5 Functions of the human intestinal flora

(1) Metabolization of non-digestible food ingredients

One of the most important functions of the intestinal flora, a function that has been comprehensively demonstrated, is the metabolization or breakdown of food ingredients which reach the large intestine without being digested. These include a wide range of dietary fibers, including prebiotics (Cf. Chapter 5), and other non-digested food ingredients (Cf. Table 1). The metabolic products of each nutrient, e.g. short chain fatty acids and members of the group of B vitamins, perform various functions in the host organism; these are described in greater detail in Chapter 3.7.

(2) Creation of a microbial barrier

An intact intestinal flora constitutes a relatively stable ecosystem. Host-specific intestinal bacteria successfully defend the ecological niches they have occupied in the intestinal wall or mucous layer of the mucosa against pathogenic microbes attempting to colonize the intestines. The success with which they accomplish this is related, among other factors, to their superior utilization of the available nutrients and their superior adhesion capability. The defense mechanisms against non-indigenous microbes are supported by products of bacterial metabolism with a bacteriotoxic effect (Tannock, 1995).

(3) Support of the intestines-related immune system

It has been proven, moreover, that the intestinal bacteria have a large direct effect on the host´s immune system. During the maturation of the immune system in infancy, in particular, there is a close interaction between the immune system and the intestinal flora. Bifidobacteria apparently provide a strong stimulus to the immune response. However, the precise mechanisms and consequences of this stimulation remain to be explored (McCracken and Gaskins, 1999).

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3.6 Bacterial fermentation

Bacterial metabolism in the colon is characterized by anaerobiosis, i.e. the absence of molecular oxygen combined with a strong redox potential. The anaerobic degradation of nutrients by bacteria is called fermentation.

After the stomach contents have been digested, and most of the nutrients absorbed in the small intestine, they reach the colon, where they undergo fermentation. The substrates of fermentation are carbohydrates, including dietary fiber, proteins and other non-digestible food ingredients.

As a consequence of the anaerobic conditions, the degradation of organic substance does not lead, as it would under aerobic conditions, to the end products CO2 and water but to less oxidized products. These include intermediate products such as lactate and succinate, the gases CO2, hydrogen and methane and the short chain fatty acids acetate (acetic acid), propionate and butyrate (Christl, 1997).

The various fermentation products are partially secreted and partially reutilized in the host organism. Here they are transported to various target organs, where they perform a number of functions.

The nutrient concentration – and thus the fermentation rate – is highest at the beginning of the colon, i.e. in the caecum and ascending colon. The patterns of fermentation in the different sections of the colon are depicted in Fig. 7.

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3.7 Effects of short chain fatty acids

Due to the large number of viable microbes it contains, the flora of the large intestine has a metabolic capacity comparable to that of the liver.

The short chain fatty acids produced by bacterial fermentation have different effects on colon morphology and function (Fig. 8).

(1) Supply of energy to the intestinal mucosa

The flora of the large intestine obtain more than 80% of their nutrients from the intestinal lumen (Bengmark, 1995), with the largest contribution made by the group of non-digested carbohydrates. Approximately 95-99% of the short chain fatty acids produced during bacterial fermentation are absorbed rapidly from the intestinal lumen. Butyrate is the preferred energy source for the epithelial cells in the large intestine and supplies up to 70 % of their total energy requirements (Scheppach, 1994). In the absence of a fiber-rich diet, e.g. in patients on a total parenteral diet or, on some occasions, a fiber-free diet, the mucosal cells rapidly atrophy for lack of fiber (Gibson & Roberfroid, 1995).

(2) Lowering of the pH: improvement of the intestinal milieu

Short chain fatty acids cause a lowering of the pH in the large intestine. An acidic pH optimizes the growth conditions for acidophilic bacteria. These include the large group of lactic acid bacteria, e.g. lactobacilli and bifidobacteria. In contrast, an acidic milieu provides only suboptimal conditions for pathogenic microorganisms. Lowering the pH thus reduces colonization by pathogenic microbes.

(3) Trophic effects due to improved mucosal circulation

Scientific studies indicate that short chain fatty acids, in particular acetate, promote the overall mucosal circulation in addition to exerting a local trophic effect (Scheppach, 1994).

(4) Stimulation of sodium and water absorption

The absorption of short chain fatty acids is linked to the absorption of sodium. The absorption of sodium and water is enhanced by the intake of short chain fatty acids (Royall et al, 1990).

Studies conducted with human subjects have shown that water and sodium absorption is stimulated by butyrate (Roediger & Rae, 1982) and by propionate (Ruppin et al, 1980). This mechanism is responsible for the uptake of water from the colon and for an anti-diarrhea effect (Macfarlane & Cummings, 1991).

(5) Effect on the production of mucous by the mucosa

The gastrointestinal mucosa consist of a simple epithelial layer. On the side facing the lumen, the mucosa are lined with a complex protective layer; the main constituent of this layer is mucous. Short chain fatty acids cause a decrease in the mucous storage cells (probably as a consequence of a local effect). The mucous released as a result improves the properties of the mucous layer. It contains, among other substances, mucin and glycolipids, and acts as a matrix for the protective probiotic bacteria (Bengmark & Jeppson, 1995; Scheppach, 1994).

(6) Effect on cell differentiation and cell proliferation

Cell differentiation and cell proliferation are reciprocally interdependent. The new immature colonocytes are found in the lower 60% of the colonic crypts. After cell division, the cells migrate toward the lumen and simultaneously mature. Short chain fatty acids stimulate physiological cell proliferation at the base of the crypt. Butyrate has different effects on normal and on neoplastic cells. It is a strongly differentiating (and anti-proliferative) agent for various types of cells found in human colon cancer. At concentrations between 1 and 5 mmol/L, it causes the cancer cells to assume a phenotype resembling that of normal colon cells. Butyrate promotes apoptosis in tumor cells and inhibits this process in normal non-transformed cells. The result is that damaged cells are eliminated while the development of healthy cells is promoted. Butyrate also promotes the repair of damaged epithelial cells. The induction of the detoxification enzyme glutathione-S-transferase and other mechanisms can contribute to the detoxification of harmful substances and thus also play a role in the protective effect exerted by the short chain fatty acids (Scheppach, 1994; Scheppach et al, 1997; Rechkemmer & Wollinski, 2000).

(7) Improved healing following resection

Based on the hypothesis that "undernourishmentö of the intestinal mucosa, i.e. an inadequate supply of short chain fatty acids, results in atrophy of the mucosa, studies were performed on rats who had undergone intestinal resection. The surgical area where the anastomosis was placed after resection was supplied with short chain fatty acids via a catheter. The anastomoses exhibited a significantly improved ability to remain intact under pressure (Rolandelli et al, 1986).

In rats with a short bowel syndrome, the addition of pectin, a soluble fiber fermented into short chain fatty acids in the colon, to an elemental diet resulted in an improvement of the mucosal cell parameters (mucosal mass, DNA, RNA, protein contents) in the remaining intestines (Koruda et al. 1986).

Following intestinal resection and the placement of a temporary ostomy, inflammation may occur in the dormant intestinal sections. In human beings, irrigation with short chain fatty acids resulted in elimination of both macroscopic and histological evidence of colitis. Similar observations have been made in patients with ulcerative colitis following treatment of their inflammations with enemas containing short chain fatty acids (Harig et al, 1989).

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4. Probiotic Concept

One possible approach to making use of the health promoting effects of the human intestinal flora is to selectively modify their composition via the probiotic concept. The aim of the probiotic concept is the uptake of potentially beneficial microorganisms which will hopefully colonize the intestines and improve both the composition and function of the intestinal flora.

By definition probiotics are "nutritional supplements consisting of viable defined microorganisms present in numbers sufficient to modify the intestinal microflora of the host organism (as a result of implantation or colonization) and thus exert a beneficial effect on the host´s healthö (Havenaar & Huis In´t Veld, 1992; Schrezenmeir & de Vrese, 2001).

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4.1 Requirements for probiotics

Bacterial strains which are to be used as probiotics must first satisfy a number of requirements (Fuller, 1992). They must:

(1) be members of a clearly defined viable bacterial species
(2) remain viable and stable during utilization and storage
(3) survive passage through the upper gastrointestinal tract (i.e. not be destroyed by gastric or bile acids)
(4) have been demonstrated to be non-toxic and non-pathogenic
(5) be able to colonize the intestinal lumen of the host organism and multiply there in order to bring about a sustained change in the microflora of a particular intestinal segment
(6) have health promoting effects on the host organism.

4.2 Probiotic bacteria

Typical representatives of the bacterial strains currently employed to achieve a "probiotic effectö are listed in the following table:

Various lactobacilli and bifidobacteria are the bacterial species most frequently employed in probiotic products at present. Whereas scientific evidence substantiating the positive effects of probiotic cultures was lacking for many years, a large number of recent studies have convincingly demonstrated these effects (Tannock, 1999).

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4.2.1 Lactobacilli

Today the proximal ileum is considered the primary "target regionö for colonization by lactobacilli; this colonization can be achieved both by the ingestion of "probioticö products and by a diet favoring the growth of this group of microorganism (Holzapfel & Haberer, 2000).

At least four functions performed by lactobacilli in the colon are known:
(1) They help to keep potentially pathogenic microorganism under control.
(2) They produce nutrients, mainly short chain fatty acids.
(3) They remove potentially toxic substances from the intestines.
(4) They stimulate the intestinal and somatic immune system.

The genus Lactobacillus contains numerous species employed as probiotics; the effect of these bacterial species has been investigated in many different studies. One of the most promising applications is the use of lactobacilli in the treatment of various forms of diarrhea, e.g. rotavirus diarrhea in infants. In a placebo-controlled study performed with 71 young children, Isolauri et al (1991) demonstrated that the average duration of rotavirus diarrhea can be significantly shortened by the administration of Lactobacillus casei GG. These results were conformed by a study carried out by Shornikowa et al (1997).

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4.2.2 Bifidobacteria

The beneficial biological properties of bifidobacteria have aroused intense interest during the past several years. Bifidobacteria are one of the main groups of viable bacteria in the colon and account for up to 25% of the total bacterial population in adults and up to 95% in newborns. The potential beneficial effects of bifidobacteria on human health are listed in Fig. 9.

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5. Prebiotic Concept

A different approach to exploit the advantages of probiotic bacteria can be the administration of prebiotics. Prebiotics are not viable organisms but specific substrates which are selectively metabolized by the potentially beneficial bacteria which have already colonized the intestines; as a result, they enhance the growth of these specific bacterial populations.

Prebiotics are defined as "non-digestible food ingredients which can exert a positive effect on the host organism (i.e. the human being) by promoting the growth and/or activity of one or several species of bacteria in the colon and thus the health of their hostö (Gibson & Collins, 1995).

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5.1 Requirements for prebiotics

To be classified as a prebiotic, a food ingredient must possess the following characteristics (Gibson & Roberfroid, 1995):
(1) It may not be hydrolyzed or absorbed in the upper gastrointestinal tract.
(2) It must specifically promote the growth and/or metabolic activity of a limited number of beneficial bacteria which are usually present in the intestinal tract.
(3) It should alter the colonic microflora in favor of a composition more favorable to the individual´s health.
(4) It must induce luminal or systemic effects which effect a beneficial change in the health of the host organism.

Although many different nutrients reach the colon (Cf. 3.6), only a few meet the above criteria. So far only a few non-digestible oligosaccharides have been identified which can be classified as prebiotics (Gibson and Roberfroid, 1995). Table 5 provides a summary of various carbohydrates with and without a prebiotic effect. The term "colonic foodö is used to describe food ingredients which are metabolized by the intestinal bacteria but do not meet all the requirements for classification as prebiotics.

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5.2 Dietary fiber which is not specifically prebiotic

5.2.1 Resistant starch

It was long believed that dietary starch is completely broken down and absorbed in the small intestine. More recent studies have shown, however, that a substantial portion of the starch escapes digestion in the small intestine and manages to reach the large intestine. The starch fraction which is not digested in the small intestine is referred to as the "resistant starchö (RS).

It was not until the development of efficient methods of chemical analysis involving enzymatic techniques, however, that researchers were able to demonstrate that the bulk of the carbohydrates which are usually not absorbed are in fact derived from dietary starch. The resistant starch does not constitute a uniform substance class; instead, it is classified according to:
(1) whether the resistance is attributable to the physical nature of the nutrient (RS1)
(2) whether the starch is present in crystalline form (RS2)
(3) whether certain forms of food preparation have led to a retrogradation of the starch (RS3).

Physical resistance occurs, for example, when the starch is ingested in the form of intact cereal grains, e.g. in whole grain products. Crystalline starch is found in many raw products; it is classified into types A – C on the basis of its diffraction behavior when exposed to x-rays. The A-type crystalline starch (found in cereal products) is usually digestible even in its raw form. Types B and C tend to resist degradation by pancreatic amylase (Macfarlane & Cummings, 1992); the degree of resistance depends on the source of the plant.

Retrograded starch is created when wet starch is heated and subsequently cooled. The percentage of retrograded starch can be increased by repeating this process (Christl, 1997). Amylose with a linear structure exhibits a much stronger tendency to retrogradation than the branched amylopectin molecule. The starch pattern of a food and the type of food processing thus determine the percentage of retrograde starch. The amylase resistance of the retrograded starches is largely irreversible.

The different types of resistant starch also display different behavior during fermentation in the intestines. On the whole the group of resistant starches provide an important nutrient substrate (accounting for a fraction of 10-40 g) for the colon bacteria. During bacterial fermentation short chain fatty acids are produced which contribute as a non-specific substrate to the reproduction of all the microorganisms in the colon and on the whole increase the metabolic activity of the intestinal flora. There is evidence, but as yet no concrete proof, that resistant starch constitutes an especially good substrate for butyrate production in the human colon (Holzapfel & Haberer, 2000). Further studies are necessary to fully elucidate the diversified mechanisms of action and prebiotic specificity of resistant starch (Asp, 1997).

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5.2.2 Non-starch polysaccharides

The large – and extremely non-homogeneous – group of non-starch polysaccharides (NSP) is worth mentioning at this point. The NSP are the main constituents of plant cell walls. They can be subdivided on the basis of their solubility. The soluble NSP include, for example, the pectins, ß-glucane, plant gum (e.g. gum Arabic) and mucilaginous substances. The group of non-soluble NSPs include cellulose and hemicellulose. A property common to all the NSP is that they are not digested in the small intestine.

Taken together, the non-starch polysaccharides make the second largest contribution (accounting for an estimated 8-18 g) to the substrates for bacterial fermentation in the colon. The largest portion of the NSP is fermented in the colon; however, there are clear-cut differences between the various representatives of this group with respect to the completeness and rate of fermentation (Macfarlane & Cummings, 1992).

The fermentation products correspond to the products of bacterial metabolism listed in Chapter 3.6. In general they promote the growth of the bacterial population and enhance the beneficial effects associated with this population without, however, displaying any specificity for certain species in the intestinal flora.

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5.3 Prebiotic carbohydrates

In view of the relatively young research in the area of prebiotics and the deluge of new study results, current definitions and classifications of prebiotics are subject to constant revision. None of the analytical methods used to identify the individual nutrient fractions are specific for only one exact fraction. It is hoped that the further development of the analytical methods will help to narrow the definitions and make them more precise.

In conformance with the nomenclature of the International Union of Pure and Applied Chemistry – International Union of Biochemistry (IUPAC-IUB), oligosaccharides are defined by being composed of two to ten monomers linked together.
However, several of the non-digestible oligosaccharide molecules consist of a larger number of monomers (Van Loo et al, 1999). The number of single sugar molecules (monomers) in the different non-digestible oligosaccharides varies; this is described as the grade of polymerization (GP).

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5.3.1 Fructo-oligosaccharides: oligofructose and inulin

Oligofructose and inulin belong to the group of non-digestible oligosaccharides. Oligofructose and inulin consist of chains of fructose molecules and are thus given names such as fructo-oligosaccharide (FOS), fructane or – owing to their ß-2-1 links – also as ß-fructane.

The figures given in the scientific literature for the grade of polymerization of different oligosaccharides range between 2 and 20 for oligofructose and between 2 and 65 for inulin (Van Loo et al, 1995; Gibson & Collins, 1999, Van Loo et al, 1999). Nevertheless, they are frequently included in the group of "non-digestible oligosaccharidesö in the IUPAC-IUB nomenclature.

Occurrence and intake

Inulin and oligofructose are constituents of foods derived from plants. The simplest sugar compound is sucrose; the sucrose molecule consists of one fructose molecule and one glucose molecule. When more fructose molecules are added, the result is oligofructose or inulin. The variations in the number of inserted fructose molecules result in different grades of polymerization (Fig. 10).

Inulin is present as a storage polysaccharide in more than 36,000 plant species. These include bananas (0.3 – 0.7%), Belgian endive or chicory (15-20%), barley, honey, garlic (9-16%), rye (0.5-1%), asparagus (1-30%), tomatoes, Jerusalem artichoke (16-20%), triticale, wheat (1-4%), brown sugar and onions (2-6%). The estimated amount of inulin and oligofructose eaten per person per day is between 4 and 12 g in Europe and between 2 and 4 g in the USA (de Vrese, 1997). Exact figures are hard to obtain since the applicable analytical methods are fairly new and are usually not listed in food tables. The (in some cases substantial) variations in the figures quoted are attributable to the different degree of sophistication of the various analytical methods used as well the different cultivation conditions and times of harvest.

Inulin is available commercially as a food additive; it is obtained for this purpose from the chicory root by hot water extraction and, in some cases, hydrolyzed by enzymes to produce short chain polymers. Another manufacturing method is enzymatic synthesis; this method involves the addition of a fructose molecule to a sugar monomer (glucose or fructose). The fructo-oligosaccharides supplied for food production are generally not pure products: they usually consist of mixtures of FOS with different grades of polymerization. To a certain extent, they also contain the original polysaccharide or monosaccharide and disaccharide (Crittenden & Playne, 1996).

5.3.1 Fructo-oligosaccharides: oligofructose and inulin

Oligofructose and inulin belong to the group of non-digestible oligosaccharides. Oligofructose and inulin consist of chains of fructose molecules and are thus given names such as fructo-oligosaccharide (FOS), fructane or – owing to their ß-2-1 links – also as ß-fructane.

The figures given in the scientific literature for the grade of polymerization of different oligosaccharides range between 2 and 20 for oligofructose and between 2 and 65 for inulin (Van Loo et al, 1995; Gibson & Collins, 1999, Van Loo et al, 1999). Nevertheless, they are frequently included in the group of "non-digestible oligosaccharidesö in the IUPAC-IUB nomenclature.

Occurrence and intake

Inulin and oligofructose are constituents of foods derived from plants. The simplest sugar compound is sucrose; the sucrose molecule consists of one fructose molecule and one glucose molecule. When more fructose molecules are added, the result is oligofructose or inulin. The variations in the number of inserted fructose molecules result in different grades of polymerization (Fig. 10).

Inulin is present as a storage polysaccharide in more than 36,000 plant species. These include bananas (0.3 – 0.7%), Belgian endive or chicory (15-20%), barley, honey, garlic (9-16%), rye (0.5-1%), asparagus (1-30%), tomatoes, Jerusalem artichoke (16-20%), triticale, wheat (1-4%), brown sugar and onions (2-6%). The estimated amount of inulin and oligofructose eaten per person per day is between 4 and 12 g in Europe and between 2 and 4 g in the USA (de Vrese, 1997). Exact figures are hard to obtain since the applicable analytical methods are fairly new and are usually not listed in food tables. The (in some cases substantial) variations in the figures quoted are attributable to the different degree of sophistication of the various analytical methods used as well the different cultivation conditions and times of harvest.

Inulin is available commercially as a food additive; it is obtained for this purpose from the chicory root by hot water extraction and, in some cases, hydrolyzed by enzymes to produce short chain polymers. Another manufacturing method is enzymatic synthesis; this method involves the addition of a fructose molecule to a sugar monomer (glucose or fructose). The fructo-oligosaccharides supplied for food production are generally not pure products: they usually consist of mixtures of FOS with different grades of polymerization. To a certain extent, they also contain the original polysaccharide or monosaccharide and disaccharide (Crittenden & Playne, 1996).

Evidence of non-digestibility

One of the defining properties of a prebiotic is its non-digestibility.

The fructose molecules in inulin and oligofructose are connected by ß-2,1- bonds and cannot be cleaved by human digestive enzymes. Their non-digestibility was proven during in vitro studies in which they were treated with digestive enzymes from the pancreas and small intestine. Furthermore, in vivo studies were carried out in patients who had undergone resection of the large intestine with a terminal ileostomy, i.e. patients whose digestion is limited to the small intestine. The residual semi-digested food was collected in a ileostomy bag and viewed as representative residual material which, under normal circumstances, would have been transported into the large intestine. For the purposes of this study, a carbohydrate was considered non-digestible if at least 90% of the ingested amount was recovered from the ileostomy bag (Ellegård et al, 1996)

Evidence of a prebiotic mechanism of action

Evidence of specificity for a bacteria or a limited group of bacteria whose growth and/or metabolism is/are stimulated by prebiotics, is an obligatory requirement for classification as a prebiotic.

A large number of in vitro and in vivo studies have been conducted on the prebiotic mechanism of action of the FOS (Van Loo et al, 1999). During these studies experiments were conducted in fermenters under defined conditions (e.g. temperature, pH, nutrient composition); the fermenter was inoculated with individual bacterial strains, mixtures of diverse bacterial strains or human stool sample. The experimental configuration made it possible to investigate the effect of a bacterial strain or mixed stool sample on FOS. By connecting several fermenters of this type in series, it was possible the simulate the processes underway in the various sections of the colon (McBain & Macfarlane, 1997).

The results of these studies show that inulin and oligofructose can be classified as prebiotic substances since they specifically promote the reproduction of bifidobacteria. At the same time, they exert an inhibitory effect on a limited number of other bacterial strains such as bacteroides, clostridia and enterobacteria. These results have been confirmed by human studies.

During a human study carried out by Gibson et al (1995), 8 test subjects were given 15 g of oligofructose – divided among three meals a day – in exchange for 15 g of sucrose per day for a total of 15 days. Using the same study protocol, the study team administered 15 g of inulin in exchange for sucrose to four test subjects. In both groups, the examination of the stool samples showed that the bifidobacteria had advanced to the "fore-runner positionö with respect to numbers. The researchers concluded, from the results of this experiment, that the composition of the intestinal flora can be modified in the direction of a potentially healthier configuration by a relatively small and uncomplicated dietary modification.

The coworkers of the ENDO project (ENDO = European Project on Non-Digestible Oligosaccharides) came to the following consistent conclusions: on the basis of the observations made on more than 100 test subjects of different ages, sexes and dietary habits, ß-fructane can be viewed as prebiotic or bifidogenic. Regardless of the grade of polymerization, all types of inulin and oligofructose have prebiotic properties. There are no differences in the prebiotic properties with respect to the different manufacturing methods used to produce inulin or oligofructose.

Evidence of the induction of luminal or systemic effects

(1) Suppression of unfavorable intestinal microorganisms

The specific multiplication of bifidobacteria after the ingestion of inulin or fructo-oligosaccharides appears to take place at the expense of other intestinal bacteria such as bacteroides, clostridia or Escherichia coli (Gibson et al, 1995; Wang & Gibson, 1993). The mechanism responsible for the suppression of other bacterial species by bifidobacteria is based on the lowering of the pH as a result of the production of large quantities of short chain fatty acids and lactate. Harmful microorganisms such as clostridia and bacteroides react sensitively to the acidic milieu. In contrast, the growth of bacteria with beneficial effects, such as lactobacilli and bifidobacteria, is enhanced by the acidic milieu.

In addition, there is evidence that bifidobacteria secrete bactericidal substances effective against clostridia, E. coli and many other pathogenic bacteria such as listeria, shigella, salmonella and Vibrio cholerae (Gibson & Roberfroid, 1995).

(2) Increase in bacteria number and stool weight

The fermentation of FOS leads to an increase in biomass and stool weight. Gibson & Roberfroid (1995) administered 15 gram of oligofructose to eight test subjects and observed an increase in stool weight of 1.5 – 2.0 gram per gram of ingested oligofructose. A similar increase in stool weight was observed for inulin (Van Loo et al, 1999). The increased stool weight had a positive effect on intestinal function and resulted in normalization of stool frequency.

(3) Production of short chain fatty acids

The anaerobic intestinal flora degrades the FOS almost quantitatively to short chain fatty acids. In vitro fermentation experiments performed with human stool bacteria or cecal bacteria of rates showed that inulin-like fructane is typically fermented to acetate and butyrate as well as to propionic acid, lactate and gases (Wang & Gibson, 1993).

The luminal and systemic effects of short chain fatty acids are described comprehensively in Chapter 3.7.

Additional potential effects of fructanes

Absorption of minerals

Several studies performed on rats showed a high bioavailability of minerals, in particular of calcium and magnesium, following the consumption of non-digestible oligosaccharides (Scholz-Ahrens et al, 2001). Various experimental models show that the increased absorption takes place mainly in the large intestine and results in a higher bone mineral density.

These results question the validity of well established concepts of mineral absorption according to which this process occurs mainly in the small intestine. Repeated studies with fructane confirmed these results: two out of three human studies showed significantly elevated calcium absorption (Coudray et al, 1997; van den Heuvel et al, 1997). In a study performed on ovarectomized rats, a model of the menopause, Scholz-Ahrends et al (1998) demonstrated a dose-dependent relationship between the ingestion of inulin, the effectively absorbed amount of calcium, and the increase in bone density.

Further studies will have to be performed on human beings to confirm these results and to establish new preventive and therapeutic approaches to the problem of osteoporosis (Van Loo et al, 1999).

Lipid metabolism

A number of studies have been carried out on the effects of inulin or oligofructose on the parameters of lipid metabolism. The majority of these studies were performed on rats. The intake of inulin or oligofructose apparently helps lower serum lipid levels, for example; this observation applies predominantly to the VLDL and LDL fractions (Delzenne et al, 1993; Roberfroid, 1993). On the basis of these experiments, it is suspected that the lowering of serum lipid levels is based primarily on the impact on lipid metabolism exerted by the propionic acid thus produced; in particular, it is due to the lower production of VLDL and/or an accelerated conversion of VLDL to LDL (Kok et al, 1996).

The small number of human studies carried out to date have yielded non-uniform results. Canzi et al (1995) observed a decrease in serum triglycerides and serum cholesterol in subjects who had taken up 9 g of inulin. In a controlled study performed with healthy normolipemic subjects, the administration of 15 g of oligofructose, inulin or trans-galacto-oligosaccharides (TOS) per day over a period of three weeks produced no significant effects (van Dokkum, 1995).

In its consensus report, the European ENDO project (Van Loo et al, 1999) rated the results available from the animal studies as "highly promisingö. The results available from human studies are contradictory; however, they indicate that the moderate ingestion of inulin or oligofructose has an effect on human lipid metabolism. No negative effects on lipid metabolism were found.

Carcinogenesis

Several studies performed on rats and mice showed that the addition of inulin-like fructanes (inulin, oligofructose) to the animal´s feed decreased their risk of developing cancer of the large intestine.

The consensus paper of the ENDO project stated that the results of these animal studies were substantiated. Human studies will have to be performed to obtain definitive proof (Van Loo et al, 1999).

The German Nutrition Report 2000 describes the release of toxic, genotoxic and carcinogenic substances by bacterial enzymes. The activity of these enzymes has been implicated in the development of cancer of the large intestine. Distinct differences have been noted in the nature of the enzyme activity displayed by the beneficial and detrimental bacterial groups, respectively. In comparison with other anaerobic bacteria, bifidobacteria and lactobacilli exhibit little enzyme activity of the xenobiotic-metabolizing type. (Note: Xenobiotics are substances, such as antigens and toxins, which cause defense reactions.)

In human beings the intake of probiotics and prebiotics results in a modulation of the enzymes which activate carcinogenesis and excretion of mutagenic and genotoxic substances in urine and feces (Pool-Zobel, 2000). Table 6 contains a list of the mechanisms postulated to explain the protective effect of the prebiotics and probiotics.

Lactobacilli and bifidobacteria prevent mutations in bacterial test systems. Furthermore, as yet unidentified metabolites of the lactic acid bacteria apparently inhibit carcinogenic and genotoxic effects. This inhibitory property of lactobacilli has been demonstrated not only in vitro but also in vivo in rat intestines; it may result in fewer preneoplastic lesions and tumors (Pool-Zobel, 2000).

In comparison with probiotics, prebiotics have the additional potential of promoting additional microorganisms which do not necessarily belong to the lactic-acid-producing bacteria but to the butyrate-producing microorganisms. Butyrate can trigger various mechanisms in the intestinal cells, e.g. it can foster the apoptosis of tumor cells or the inhibition of this process in normal, non-transformed cells. The result is that damaged cells are eliminated and healthy cells promoted (Pool-Zobel, 2000, Wollowski et al, 2001). Butyrate is the preferred substrate of the colonocytes and can promote the repair of damaged epithelial cells, increase the rate of mucin synthesis, and thus strengthen the surface-protection system in the intestines.

Further studies are needed to provide direct evidence of these effects and to elucidate the physiological processes involved.

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5.3.2 Potential prebiotics

Prebiotic characteristics have been ascribed to several less investigated oligosaccharides in addition to inulin and oligofructose.

A few studies have explored the bifidogenic potential of the galacto-oligosaccharides. The results indicate that these oligosaccharides may have prebiotic potential. The galacto-oligosaccharides found in breast milk are collectively referred to as "the bifidus factorö. Animal studies on soybean oligosaccharides suggest that these substances possess a prebiotic mechanism of action. However, definitive scientific evidence justifying a classification of these substances as prebiotics is still to be presented (Van Loo et al, 1999).

Lactulose is a synthetic disaccharide used, for example, in the treatment of encephalopathy. Lactulose is fermented in the large intestine to produce short chain fatty acids, in particular propionate and lactic acid; it has a laxative and ammonia-reducing effect. The reduction of ammonia is attributed to several effects: (1) The fermentation of lactulose lowers pH via the production of short chain fatty acids. The acidic milieu leads to protonation of the toxic and absorbable ammonia and converts it into non-toxic, non-absorbable ammonium ions. The uptake of ammonia from the colon is thus reduced. (2) As a result of the carbohydrate surplus and the lowering of pH, the proteolytic intestinal flora are suppressed in favor of the saccharolytic; as a result, less ammonia is formed. This points to a specific prebiotic effect.

By administering lactulose, it is possible to lower the blood ammonia concentration of patients suffering from porto-systemic encephalopathy by 25-50 % within a few hours to a few days.

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5.4 Advantages of the prebiotic concept

The use of prebiotics and probiotics represents two promising approaches to improving the composition of the human intestinal flora. However, the use of prebiotics has one decisive advantage over the intake of probiotic bacteria: prebiotic carbohydrates selectively encourage the growth of health promoting intestinal bacteria (e.g. bifidobacteria) which – and this is the decisive point – have already colonized the intestines.

Concerning the probiotic bacteria, it is still not clear whether these can actually colonize the human intestinal tract in sufficient numbers in vivo and, if so, what doses are required (Döll, 1997; Reid, 1999). Furthermore, the production and storage of probiotics is generally more difficult than the production and storage of prebiotics. In numerous studies on the quality control of probiotic recipes (e.g. for yogurt products), it has been determined that the number of living bacteria drops significantly prior to the expiration or "best beforeö date; moreover, the products were found to contain bacteria other than those stated on the label (Sanders and Veld, 1999). Pronounced quality deviations of this kind have cast doubts on the effectiveness of the products in question.

None of these uncertainties apply to the use of prebiotic carbohydrates, which have been demonstrated to reach the large intestine in non-digested form and are metabolized by host-specific bacteria. The use of these substances is considered safe and their efficacy has been confirmed by numerous studies.

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6. Summary

The health of the individual depends on a large number of factors. The intestinal flora represents one of the factors exerting a decisive impact on the health of the entire organism. The term "intestinal floraö is used to describe all of the intestinal bacteria which have colonized the intestines.

The human intestinal flora develops during the first years of life until it reaches a stable plateau. In healthy persons the large intestine is inhabited predominantly by bacterial genera with protective properties (e.g. bifidobacteria and lactobacilli) or neutral characteristics (e.g. bacteroides). Potentially pathogenic microbes such as clostridia colonize the intestines in only small numbers. In persons suffering from disease, the equilibrium of the intestinal flora is often disturbed. This imbalance paves the way for the multiplication of pathogenic microorganisms which can cause infection and diarrhea.

The intestinal flora performs a number of important functions in the host organism. It constitutes a microbial barrier to the invasion of foreign microbes, supports the intestinal-associated immune system via certain interactions, and, most importantly, is responsible for the metabolization of non-digested food ingredients. These consist primarily of fiber-containing foods (including prebiotics) which reach the colon and are fermented by the intestinal bacteria. In addition to several other products, short chain fatty acids (i.e. acetate, propionate and butyrate), in particular, are produced which exert many different beneficial effects on intestinal health (Cf. 3.7).

In the past, the probiotic and prebiotic concepts were developed for the purpose of exerting selective effects on intestinal health. Because the probiotics are living bacteria and it is difficult to control their colonization of the intestines after intake (Cf. 5.4), the prebiotic concept appears to be the more effective of the two. The term "prebioticö is used to described certain types of carbohydrates which reach the colon in non-digested form and specifically promote the growth and/or metabolic activity of protective bacteria, such as the bifidobacteria, at that location. The prebiotics thus have a beneficial effect on the intestinal flora and induce health promoting effects in human beings (Cf. 5.3).

Now that this concept has already been realized in numerous products, and its effectiveness has been adequately tested, it makes sense to employ prebiotic products in enteral nutrition / tube feeding as well. In particular, the anti-diarrhea effect of prebiotics can exert a positive effect on the tolerance of these product and the well being of patients. In addition, patients being fed entirely via the enteral route (by tube feeding) may also benefit from the immunomodulatory effects, the beneficial effect on lipid metabolism, and the anti-carcinogenic action of the prebiotics.

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8. List of Abbreviations

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