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Chapter 4 PREBIOTICS AND PROBIOTICS Among the most promising targets for functional foods are the gastrointestinal functions, including those that control transit time, bowel habits, and mucosal motility as well as those that modulate epithelial cell proliferation. Promising targets are also gastrointestinal functions that are associated with a balanced colonic microflora, that are associated with control of nutrient bioavailability, that modify gastrointestinal immune activity, or that are mediated by the endocrine activity of the gastrointestinal system. Also, some systemic functions such as lipid homeostasis that are indirectly influenced by nutrient digestion or fermentation represent promising targets. Bacteriotherapy is an alternative and promising way to combat infections by using harmless bacteria to displace pathogenic microorganism. Saliva and gastrointestinal secretions, as well as beneficial microbes (probiotics) and supplied fibers (prebiotics) are important for optimal function. It is common knowledge that the intestinal flora encompasses at least 500 different types of bacteria living in symbiosis with their host: both the host and the bacteria benefit from this symbiosis. The composition of the intestinal flora is fairly constant within an individual in spite of considerable intra-individual variation in the composition of the diet. The stable composition of the flora is indicative of a balanced ecosystem, at least in healthy individuals, which is not easily disturbed. This chapter discusses the significance of the intestinal flora to our health, how the composition of our diet can affect the intestinal flora and what impact a modified intestinal flora may have on our health. Also it reviews the basics of prebiotics and probiotics and scientific data showing that prebiotics and probiotics positively affect various physiological functions in ways that will permit them to be classified as functional foods. Bacterial counts per milliliter in intestinal contents increase in a distal direction in the gastrointestinal tract from 103–104 in the stomach

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to 106–107 in the distal ileum and 1011–1012 in the colon (Figure 4.1 and 4.2). Gram-positive bacteria are the dominant microflora in the stomach, whereas the bacteria in the distal small intestine and the colon are predominantly of the Gram-negative type. The numbers of anaerobic bacteria increase distally. The most important intestinal bacteria are bacteroides, bifidobacteria, Enterobacteraceae, lactobacilli, Grampositive cocci, Clostridium species and eubacteria. In addition, streptococci and various types of molds and yeasts are also found in the intestines. FIGURE 4.1 — Composition Of Human Gastrointestinal Microflora Bacterial flora Total bacterial count (per ml)

Stomach

Jejunum 5

Ileum 3

10 –10

Faeces 7

1010 - 1012

0–10

0–10

0–102

0–103

102–106

1010–1012

0–103 0–102 0–103 0–102

0–104 0–103 0–104 0–102

102–106 102–105 102–105 102–103

103 –1010 104 –107 106 –1010 102 –106

rare rare rare rare rare

0–102 0–103 0–103 rare rare

103–107 103–105 102–103 102–104 rare

1010–1012 108 –1012 108 –1011 106 –1011 109 –1012

Aerobic or facultative bacteria Enterobacteria Streptococci (including Peptostreptococcus) Staphylococci Lactobacilli Fungi Anaerobic bacteria Bacteroides Bifidobacteria Gram-positive cocci Clostridium spp. Eubacteria

FIGURE 4.2 — Density And Nature Of Bacteria In The Human Gastrointestinal Tract. Site Stomach and proximal ileum Terminal ileum Colon

Density 103 –104 /ml 106 –107 /ml 1011–1012/ml

Type predominantly Gram-positive predominantly Gram-negative predominantly Gram-negative

Studies with aseptically grown animals have provided insight into the physiological significance of the intestinal flora. The findings suggest that the intestinal flora affects both the immune system and the intestinal function. The intestinal flora has also been found to improve resistance to the colonization by enteropathogenic micro-organisms such

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as Salmonella bacteria. Further, the intestinal flora aids the digestion of food components, in particular of poorly digestible carbohydrates. The butyric acid formed in that process has a favorable effect on the intestinal epithelium. The intestinal flora also plays a role in fat metabolism (bile acid and cholesterol metabolism) and the synthesis of vitamin K. A variety of conditions may disturb the intestinal flora and induce colonization of the intestines by undesirable bacteria. Such conditions include treatment with antibiotics, food contamination (e.g., by Salmonella spp., Campylobacter spp. or Escherichia coli), viral infections, stress, shortage of gastric juice and diminished intestinal motility. The latter two factors play a major role in bacterial overgrowth in the small intestine leading to impaired food digestion (malabsorption of fats, carbohydrates, amino acids, and vitamin B12). A balanced intestinal flora is a precondition for a fairly stable ecosystem in which both host-related factors and antagonistic interactions among intestinal bacteria play a role (Figure 4.3). Food has limited influence on the composition of the intestinal flora, but a strong influence on their metabolic activity. FIGURE 4.3 — Schematic Presentation Of Interactions Between Food, Intestinal Flora And Host

With regard to host-related factors it should be noted that the intestines have a role in addition to their digestive function, namely that of a barrier against invading bacteria. The following factors are relevant in this respect: • gastric acid secretion; • bile and pancreatic juice; • intestinal motility and peristalsis;

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• rejection of epithelial cells; • secretion of immunoglobulins; • lysosomal and macrophagic activities. The antagonistic interactions among bacteria can be classified into indirect interactions and direct interactions. Indirect interactions (host-related factors) include: • deconjugation of bile salts; • formation of secondary bile salts; • induction of immune response (immunoglobulin A secretion); • stimulation of intestinal peristalsis. Direct interactions include: • competition for substrates; • competition for sites of attachment; • formation of growth-inhibiting metabolites (volatile fatty acids organic acids, sulfuric acid, bacteriocins); • decrease in pH of the environment. Recent studies have shown that metabolic activities of the intestinal flora could form potential carcinogens under specific conditions. It is essential, therefore, to have a thorough knowledge of dietary factors with a favourable effect on the composition and activity of intestinal flora. These dietary factors can be classified into three groups, namely prebiotics, probiotics and symbiotics (synergetic combinations of probiotics and prebiotics) (Figure 4.4). FIGURE 4.4 — Schematic Presentation Of Interactions In The Gastrointestinal Tract Between Probiotics/Prebiotics And Intestinal Flora

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PREBIOTICS Prebiotics are non-digestible food ingredients which beneficially affect the host by selectively stimulating the growth of and/or activating the metabolism of one or a limited number of health promoting bacteria in the intestinal tract, thus improving the host’s intestinal balance (Gibson and Roberfroid, 1995). All prebiotics to date have been carbohydrates, ranging in size from small sugar alcohols and disaccharides, to oligosaccharides and large polysaccharides. The defining criteria of prebiotics is: (1) A prebiotic should neither be hydrolyzed nor absorbed in the upper part of the gastrointestinal tract. (2) It should be a selective substrate for one or more potentially beneficial commensal bacteria in the large intestine. Colonization by an exogenous probiotic could be enhanced and extended by simultaneous administration of a prebiotic that the probiotic could utilize in the intestinal tract. As such it should stimulate that bacteria to divide, become metabolically active, or both. (3) Alter the colonic microenvironment toward a healthier composition. (4) Induce luminal or systemic effects that are advantageous to the host. The most studied non-digestible oligomers are galactooligomers, such as soya-derived raffinose and stachyose, and the fructooligomers or fructans. Chemistry Of Fructans A fructan is any compound where one or more fructosyl-fructose linkages constitute a majority of linkages (Englyst et al., 1992). Fructan is used to name molecules that have a majority of fructose residues whatever the number is. It even includes the disaccharide composed exclusively of two fructose residues, specifically the fructosyl-fructose or inulobiose but not sucrose, isomaltulose, and galactosucrose, etc. In addition, fructan is also sometimes either a cyclic or a branched molecule. Fructan is also known as polyfructosylfructose. All natural (plant and microbial) fructans are a mixture of oligomers or polymers or both, which is best described by the mean (or average) and the maximum number of fructose units, residues, or moieties, known as the average and the maximum degree of polymerization (DPav and DPmax), respectively. More than 50 generic names of fructans have appeared in old literature including, inulin, levan, and phlein but also

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fructoholoside, fructosan, graminin, inu-lenin, lavulan, levulosan, levosin, and pseudo-inulin, etc. (Suzuki, 1993). A fructan is defined as a carbohydrate that consists mostly of fructose plus a molecule of glucose or a molecule that has a majority of fructose residues and a glucose. A fructan can be linear [Inulin (2, 1 fructosyl-fructose), levan (2, 6 fructosyl-fructose)] or branched (2, 1 or 2, 6 and 2, 6) or cyclic. From a chemical point of view, the linear chain of fructans is either a (β-D-glucopyranosyl-[-β-D-fructofuranosyl]nl-(β-D-fructofuranoside (G py F n ) or a β-D-fructopyranosyl-[-β-D-fructofuranosyl] nl -β-Dfructofuranoside (FpyFn). The fructosyl-glucose linkage is always β-(2↔l) as in sucrose (the numbers indicate the linkage’s position on the C atoms of the fructose or glucose rings and the arrow points away from the reducing C atom (C2, in fructose or C1, in glucose) but the fructosylfructose linkages are either β-(l➞2) or β-(6➞2). In branched fructans the branching linkages are usually β-(2➞6). Fructans are mainly of plant origin, but they are also found in fungi and bacteria. In plant fructans the number of fructose monomers does not exceed 200, whereas in bacterial fructans it can be as high as 100,000, and it is highly branched. Inulin, levan, graminan, phlein, and kestoses are the general terms to describe fructans. Inulin: is a material that has mostly, or exclusively, the β-(l➞2) fructosyl-fructose linkage, and glucose may be present at the terminal position in the chain but is not necessary. Until recently, inulin was considered to be a linear molecule with β-(l➞2) linkages exclusively. However, using optimized permethylation analysis, it has been possible to demonstrate that even native inulin has a very small degree (1-2%) of branching (De Leenheer and Hoebregs, 1994). All fructans in dicotyledons, are inulin-type fructans, but only part of the fructans in monocotyledons are inulin-type fructans (Suzuki, 1993). Inulin exists also in a cyclic form that contains 6, 7, or 8 fructofuranose rings. Levan: is a material that has mostly, or exclusively, the β-(6➞2) fructosyl-fructose linkage. Like in inulin, glucose may be present at the terminal position in the chain but is not necessary. Levans are found mostly in bacteria (high molecular weight) but to some extent in higher plants also (short polymers). The levans of higher plants are heavily branched molecules through the formation of β-(2➞l) linkages. Phlein: has substantially the same meaning as levan, but the name has commonly been used to describe plant- (and not bacteria-) based material which contains, most exclusively, the β-(6➞2) fructosyl-fructose linkage; a glucose is allowed at position 1 in the chain but is not necessary. In general, plant-based fructans are of lower molecular

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weight (DP < 100) than those derived from bacteria, and thus this distinction has been useful. Phlein-type fructans occur mainly in monocotyledons, which represent the most frequently identified fructans. Graminan: is a material that has both β-(l➞2) and β-(6➞2) fructosylfructose linkages in significant proportions; glucose is allowed at position 1 in the chain but is not necessary. Kestoses or kesto-n-oses: are trimeric or oligomeric fructans containing one glucose and two or more fructose units linked by β-(l➞2) and/or β-(6➞2) fructosyl-fructose linkages. Bifurcose:
-D-glucopyranosyl-(l↔2)-β-D-fructofuranosyl-(6➞2)-βD-fructo-furanosyl-(1➞2)-p-D-fructofuranoside. Inulo-n-ose: Oligomeric fructofuranosyl-only fructans that have all(1➞2) linkages like inulobiose and inulotriose. Fructooligosaccharides, oligofructan, and oligofructose: These are oligomeric linear fructans with β-(l➞2) linkages. They can be of both (GpyFn) and (FpyFn) types. Among others, these terms include 1kestose, neokestose, and nystose. • 1-Kestose:
-D-glucopyranosyl-(l↔2)-β-D-fructofuranosyl-(l➞2)β-D-fructo-furanoside. • 6-Kestose:
-D-glucopyranosyl-(1↔2)-β-D-fructofuranosyl-(6➞2)β-D-fructo-furanoside. • Levan-n-ose: oligomeric fructofuranosyl-only fructans that have all β-(6➞2) linkages like levanbiose, levantriose, etc. • Neokestose: β-D-fructofuranosyl-(2➞6)-β-D-glucopyranosyl-(l↔2)β-D-fructofuranoside. • Nystose:
-D-glucopyranosyl-(1↔2)-β-D-fructofuranosyl-(1➞2)-βD-fructofuranosyl-(1➞2)-β-D-fructofuranoside. Natural Occurrence Of Fructans Fructans are reserve carbohydrates in at least 10 families of higher plants that store them in a soluble form in vacuoles in crowns, leaves, roots, stems, tubers, or kernels. The fructans and their linkage type and length differ greatly, depending on the plant and the plant organ. Moreover, the chain length of plant fructans can be modulated through changes in DP as a means to modulate osmotic pressure. Occurrence Of Fructans In Plants Fructan-containing plants are mainly angiosperms. The fructancontaining species belong to both mono- and dicotyledonous families. Some of these plants are eaten as vegetables such as artichoke, asparagus, chicory, garlic, Jerusalem artichoke, leek, onion, salsify, etc.

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In monocotyledons, fructans are widely present in the aerial parts of young seedings of Gramineae but significant concentration is found only in northern grasses (Pooideae), oat (Avena sativa), barley (Hordeum vulgare), rye (Secale sativa), and wheat (Triticum aestivum and Triticum durum). It is also present in the order Liliaceaes. Indeed, the bulbs, tuber, and tuberous roots of Amaryllidaceae, Agavaceae, Haemodoraceae, Iridaceae, Liliaceae, and Xanthorrhoeaceae produce and store fructans. Especially, fructans have been found in the family of Liliaceae, in the leaf and bulb of leek (Allium ampeloprasum), the bulb of onion, shallot (Allium cepa) and garlic (Allium sativum), and the tuber of asparagus (Asparagus officinalis and Asparagus racemosus) and in the family of Agavaceae in the tuber of palm lily (Cordyline terminalis) and Dracaena australis. In dicotyledons, the fructans-containing orders are the Asterales, the Campanulales, the Dipsacales, the Polemoniaceae and the Ericales. As far as is known, all members of the major family Compositae (Asterales order) store significant amounts of fructans in their underground storage organs such as tap roots and tubers but not in their leaves. This is the case for chicory (Cichorium intybus), elecampane (Inula hellenum), dandelion (Taraxacum officinale), Jerusalem artichoke (Helianthus tuberosus), murnong (Microseris lanceolata), salsify (Tragopogon porrifolius), and yacon (Polymnia sonchifolia). Occurrence Of Fructans In Fungi Fructans accumulate in various species of aspergillus, but some species also synthesize it extracellularly from sucrose. Specifically, it has been reported that Aspergillus sydowi synthesizes an inulin that has a molecular weight greater than that of plant inulin. However, fructan has not been demonstrated in penicillium, pestalotiopsis, myrothecium, or trichoderma. This observation correlates well with the fact that sucrose has not been confirmed as a fungal carbohydrate. Indeed, the most characteristic endogenous disaccharide of all fungal groups is trehalose (1-1-di-glucose). Occurrence Of Fructans In Bacteria With the exception of certain strains of Streptococcus mutans (a major component of dental plaque) that produce inulin-type fructans, the bacterial fructans are essentially of the levan type. Fructans or the genes for their synthesis appear essentially in five orders or families of bacteria, namely the Gram-negative aerobic (Pseudomonadaceae) and facultative, anaerobic (Enterobacteraceae) rods and cocci, the Gram-

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positive cocci (Streptococcaceae), endospore-forming rods and cocci (Bacillaceae), and Actinomycetaceae (Hendry and Wallace, 1993). INULIN The use of miscellaneous fructan- (but mostly inulin-) containing plants as food seems to be quite old, dating back to at least 5000 years, and one of the most commonly consumed vegetables in ancient times was onion (Allium cepa). It was in the 19th century that a German scientist discovered inulin after he had isolated a "peculiar substance of plant origin" from the boiling-water extract of Inula helenium.” That substance was called inulin. The first scientific report on the health benefits of inulin for humans also dates back to the last quarter of 19th century. Indeed, referring specifically to inulin, Kulz reported as early as 1874 that no sugar appears in the urine of diabetics who eat 50 to 120 g of inulin per day (Roberfroid, 2004). Inulin, structurally can be considered as a polyoxyethylene backbone to which fructose moieties are attached, as are steps to a winding stair. The degree of polymerization (DP) of inulin and the presence of branches are important properties that influence its functionality strikingly. Therefore, a strict distinction must be made between inulin of plant and bacterial origin. The DPmax of plant inulin is rather low (maximal DP < 200), but DPmax and DPav vary according to plant species, weather conditions, and the physiological age of the plant. Inulin is present in significant amounts in several fruits and vegetables (Figure 4.5). Chicory inulin Chicory (Cichorium intybus) is used today as an industrial crop and its fructan is known as chicory inulin. Native chicory inulin is a nonfractionated inulin, extracted from fresh roots, taking precautions to inhibit the plant’s own inulinase activity as well as acid hydrolysis. It always contains glucose, fructose, sucrose, and small oligosaccharides. Because of the beta configuration of the anomeric C2 in its fructose monomers, inulin is resistant to hydrolysis by human small intestinal digestive enzymes, which are specific for
-glycosidic bonds. It has thus been classified as “nondigestible” oligosaccharide (NDO). Production Of Inulin And Oligofructose And Related Products The production process involves extracting naturally occurring inulin from chicory roots by diffusion in hot water. The raw extract is then

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FIGURE 4.5 — Inulin Content And Chain Length Of Miscellaneous Plants Plant

Inulin (g/100g)

Asparagus Raw Boiled

2.0-3.0 1.4-2.0

Globe Artichoke (Cynara scolymus)

2.0-6.8

Banana (Musa cavendishii) Raw Raw-dried Canned

0.3-0.7 0.9-2.0 0.1-0.3

Barley (Hordeum vulgare) Raw Cooked

0.5-1.0 0.1-0.2

DP > 5 = 95% / DP > 40 = 87% DP < 5 = 100%

Chicory (Cichorium intybus) root

35.7-47.6

Dandelion greens (Taraxacum officinale) Raw Cooked

12.0-15.0 8.1-10.1

Garlic (Aliium sativum) Raw Dried

9.0-16.0 20.3-36.1

DP < 40 = 83% (DP 2-65) DP > 40 = 17%

DP > 5 = 75%

Jerusalem Artichoke (Helianthus tuberosus) 16.0-20.0

Leek (Allium ampeloprasum) Raw

Chain Length

DP < 40 = 94% (DP 2-50) DP > 40 = 6% DP 12 is most frequent

3-10

Onion (Allium cepa) Raw Raw-dried Cooked

1.1-7.5 4.7-31.9 0.8-5.3

DP 2-12

Wheat (Triticum aestivum) Bran – raw Flour – baked Flour – boiled

1.0-4.0 1.0-3.8 0.2-0.6

Rye - Baked

0.5-0.9

DP < 5 = 50%

DP ➞ Degree of polymerization. Adapted from Van Loo et al. (1995).

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refined by using technologies from the sugar and starch industries (e.g., ion exchangers), and then evaporated and spray dried (Figure 4.6). Chicory oligofructose is obtained by partial enzymatic hydrolysis of inulin, eventually followed by spray drying. Hydrolysis is catalyzed either by exo-inulinase (EC 3.2.1.80), by the combined action of exoand endo-inulinases, or solely by endoinulinase (EC 3.2.1.7). Although the best source of these enzymes is Kluyveromyces fragilis that produces only an exo-inulinase, most inulin-hydrolyzing enzymes of yeast origin have both exo- and endoinulinase activity (Uchiyama, 1993). The enzymes used for the commercial production of fructose and oligofructose come from Aspergillus niger or Aspergillus ficuum. The long-chain inulin or inulin HP is produced by using physical separation techniques to eliminate all oligomers with a DP < 10. The product known as Synergy 1 is obtained by mixing 30:70 (w/w) oligofructose and inulin HP. Other products are also made from inulin by intermolecular (depolymerizing) fructosyl-transferases (from Arthobacter globiformis, Arthobacter urefaciens, and pseudomonas) like DFA’s (difructose dianhydrides) and cyclic forms of difructose. Cyclofructans are also produced using an extracellular enzyme of Bacillus circulans. This enzyme forms mainly cycloinulohexaose (CFR-6), but also small amounts of cycloinuloheptaose and -octaose by an intramolecular transfructosylation reaction. Physicochemical and technological properties of chicory inulin, oligofructose, and their derivatives in powder form are presented in Figure 4.7 and their food applications are presented in Figure 4.8. Fructooligosaccharides are classified as prebiotics since they have the ability to selectively promote the growth of healthy intestinal bacteria (such as Bifidobacteria and Lactobacilli) at the expense of the putrefactive bacteria (such as bacteroides, clostridia, and other coliforms). Bifidobacteria produce acetic and lactic acids, which inhibit the growth of pathogenic bacteria and stimulate intestinal peristalsis. FOS facilitates the absorption of calcium, and possibly magnesium also, and may lower the risk of osteoporosis. They also suppress the activity of cancer causing enzymes in the large bowel. Because of these health benefits, these carbohydrates are being added to many processed foods. Sources Of Prebiotics Common food sources of prebiotics include whole grains, oatmeal, flaxseed, barley, dandelion greens, spinach, collard greens, chard, kale, mustard greens, berries, fruits and legumes (lentils, kidney beans, chickpeas, navy beans, white beans, black beans, etc), chicory, onion, leek, garlic, artichoke and asparagus. Yacon, which looks like a potato,

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FIGURE 4.6 — Inulin Production Process

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FIGURE 4.7 — Physicochemical And Technological Properties Of Chicory Inulin, Oligofructose, And Their Derivatives In Powder Form Inulin

Inulin HP Oligofructose

Chemistry

GpyFn DP 2-60

GpyFn DP 10-60

GpyFn and FpyFn DP 2-7

DP av Content (% dry matter) Dry matter (%) Sugars (% dry matter) pH (10% in H2O) Ash (% dry matter) Heavy metals (% dry matter) Color Taste

12 92 95 8 5-7 <0.2 <0.2 White Neutral

25 99.5 95 <0.5 5-7 <0.2 <0.2 White Neutral

Sweetness vs sucrose Water solubility (% at 25°C) Water viscosity (5% at 10oC) Food application (specific)

10% 12 1.6 mPa Fat replacers +Gelling agent

None 2.5 2.4 mPa Fat replacers +Gelling agent

4 95 95 5 5-7 <0.2 <0.2 White Moderately sweet 35% >75 <1 mPa Fat replacers +Intense sweetener

Food application (synergism)

Synergy 1 GpyFn and FpyFn DP 2-7 DP 10-60 95 95 5-7 <0.2 <0.2 White Moderately sweet

Adapted from Roberfroid (2004).

FIGURE 4.8 — Typical Examples Of Food Applications Of Chicory Inulin, Oligofructose, And Their Derivatives Food Products

Applications

Dairy products

Body and mouth feel, Foam stability, Sugar and fat replacement, Synergy with sweeteners

Frozen desserts

Sugar and fat replacement, Synergy with sweeteners Texture and melting

Table spreads

Fat replacement, Texture and spreadability, Emulsion stability

Baked goods and breads

Sugar replacement, Moisture retention

Breakfast cereals

Crispness and expansion

Fruit preparations

Sugar replacement, Synergy with sweeteners, Body and mouth feel

Meat products

Fat replacement, Texture and stability

Chocolate

Sugar replacement, Heat resistance

Adapted from Roberfroid (2004).

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is a root vegetable from Peru. It has a sweet, juicy taste. It is low in calories and rich in FOS. Onion and wheat are the major sources of FOS in American diet. TYPES OF PREBIOTICS OTHER THAN INULIN AND FOS Fiber gums are often used in such foods as yogurt to give the product a thicker consistency. They can be used as a prebiotic or as thickening material. Obviously, processing varies according to the desired outcome. Fiber gums are water-soluble and derived from such plants as acacia, carrageenan, guar, locust bean, and xanthan. Usually containing about 85% fiber, these gums help promote the production of large quantities of short-chain fatty acids, which are known to play several beneficial roles, including the development of such intestinal bacteria as Lactobacillus and Bifidobacteria. Isomalto-oligosaccharides are a mixture of glucose and other saccharide molecules. Produced by various enzyme processes, isomaltooligosacharides ultimately form several sugar molecules including isomaltose, panose, isomaltotetraose, isomaltopentaose, nigerose, kojibiose, isopanose and other higher branched oligosaccharides. They act to stimulate the growth of Bifidobacterium and Lactobacillus species in the large intestine. They are marketed in Japan as dietary supplements and used in functional foods. They are being developed in the United States for similar commercial uses. Lactilol is a disaccharide alcohol analogue of lactulose. Lactilol is used in many countries for treating constipation and hepatic encephalopathy, but not in the United States. In Japan, lactilol is also used as a prebiotic because it is resistant to digestion in the upper gastrointestinal tract and is fermented by a limited number of colonic bacteria. However, it is not approved as a prebiotic in the United States. In Europe, it is used as a food sweetener. Lactosucrose is a trisaccharide comprised of galactose, glucose, and fructose molecules. It is produced through enzyme action that results in sucrose. Resistant to digestion in the stomach and small intestine, lactosucrose acts on the intestinal microflora to increase significantly the growth of the Bifidobacterium species. Lactosucrose is widely used in Japan as a dietary supplement and in functional foods, including yogurt and is being developed in the United States for similar uses. Lactulose is a semisynthetic disaccharide comprised of galactose and fructose. Lactulose is resistant to human digestive enzymes and can be fermented by a limited number of bacteria in the colon, especially Lactobacilli and Bifidobacteria. Currently, lactulose is a prescribed drug

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in the United States for the treatment of constipation and hepatic encephalopathy, but it has not been proven to be a prebiotic substance. In Japan, it is marketed as a dietary supplement and used in functional foods. Lactulose has exhibited some ability to reduce infectious inflammatory bowel disorders, as well as some colonic tumors. Since it has some ability to improve glucose tolerance and is showing other improvements in carbohydrate metabolism, it is speculated that lactulose may be helpful in treating Diabetes Mellitus. In addition, it has significantly stimulated calcium absorption in postmenopausal women in preliminary clinical work. Oligofructose is a sweet product derived from native inulin and is approximately 30-60% as sweet as sugar. It is found on the market as an oligosaccharide because it consists mainly of fructose units with some glucose-terminated chains. It is also available as a mixture with inulin to reduce the amount of non-glucose terminated chains. The unbound fructose chains have prebiotic properties, but with a different fermentation profile than either inulin or FOS. However, it is fermented by a wider variety of probiotic bacteria than inulin. Unlike inulin, FOS has the ability to brown, making it a valuable addition to baked products. Pyrodextrins are a mixture of glucose-containing oligosaccharides derived from starch. Pyrodextrins are resistant to digestion in the upper gastrointestinal tract and have been found to promote the growth of Bifidobacteria in the large intestine and are being developed for the nutritional supplement market place. Soy oligosaccharides are those found mainly in soybeans, but can also be found in other beans and peas. There are two principal soy oligosaccharides: the trisaccharide raffinose and the tetrasaccharide stachyose. Raffinose is comprised of one molecule each of galactose, glucose and fructose. Stachyose is comprised of two molecules of galactose, one molecule of glucose and one molecule of fructose. Soy oligosaccharides act to stimulate the growth of Bifidobacterium species in the large intestine. They are marketed in Japan as dietary supplements and in functional foods and are being developed in the US for similar uses. Transgalacto-oligosaccharides (TOS) are a mixture of glucose and galactose oligosaccharides. They are produced from lactose via enzyme action obtained from Aspergillus oryzae, which can also be a pathogen. TOS are resistant to digestion in the upper gastrointestinal tract, and therefore able to stimulate the growth of bifidobacteria in the large intestine. TOS are marketed in Japan and Europe as dietary supplements and used in functional foods. They are being developed for similar use in the United States. TOS have demonstrated positive

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effects on calcium absorption and have prevented bone loss in some animal models. In preliminary studies, TOS have shown some ability to lower triglycerides. Xylo-oligosaccharides are comprised of oligosaccharides containing beta-linked xylose residues and obtained from enzymatic action. They are marketed in Japan as prebiotics and are being developed for similar use in the United States. Since xylo-oligosaccharides resist digestion in the upper gastrointestinal tract, they are able to function in the large intestine to increase the growth of Bifidobacterium species, thus improving gastric function. According to preliminary research, xylooligosaccharides have the potential to improve blood sugar levels and fat metabolism, restore normal intestinal flora following antibiotic, chemo, or radiation therapies, increase mineral absorption and vitamin B production, and reduce intestinal putrification. Beneficial Effects Of Prebiotics On Health Inulin, oligofructose, lactulose, galactooligosaccharides and synthetic FOS are probably the only prebiotics for which available scientific evidence would indicate limited and defined health benefits. The chemical structure of these prebiotics prevents their digestion in the small gut. Consequently, they reach the large bowel undigested and are fermented by bacteria. This fermentation stimulates the growth of Bifidobacteria, a species used as probiotics. The ability of these oligosaccharides to alter the gut microbial population towards a more beneficial composition has been consistently shown in human studies. The greatest benefit appears to be in those individuals with low levels of bifidobacteria. It must be pointed out that the daily intake of prebiotics can be increased by dietary means, which includes the regular consumption of leeks, artichokes, garlic, onions, wheat and wheat products, asparagus and bananas. The average daily intake of these prebiotics from food ranges from 1-4 g in the U.S.A. to 3-11 g in Europe. Although there is no daily recommendation for prebiotics, doses of 4-20 g per day have shown efficacy. Many other potential prebiotics are currently under investigation, including xylooligosaccharides, lactitol, soyoligosaccharides, pecticoligosaccharides, glucooligosaccharides, isomaltooligosaccharides and gentiooligosaccharides. Prebiotics have also been associated with a reduction in the risk for diarrhea, constipation, colon cancer, osteoporosis and heart disease. Their effect in improving constipation is largely attributed to increasing fecal bulk and improving gut motility.

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Mechanism Of Action Of Prebiotics In Reducing The Risk Of Colon Cancer The β 2-1 osidic bond of FOS, including the first glucose-fructose bond, is not readily hydrolyzed by mammalian digestive systems. However, they are food for Bifidobacteria, which possess β-fructosidases that can digest these compounds. As a result of this, short chain fatty acids acetic, butyric and lactic acids are produced which inhibit the growth of pathogenic bacteria and help to prevent colon carcinogenesis. Prebiotics stimulate the growth of endogenous Bifidobacteria, which after a short feeding period become predominant in human feces (Gibson et al., 1995). It has been suggested that the mechanisms by which fructooligosaccharides modulate human colon cancer incidence may involve multiple actions in the lumen and target tissue (Klurfeld, 1997). Reddy et al (1997) fed 10% oligofructose or inulin to rats given azoxymethane (AOM), a substance known to produce preneoplastic aberrant crypt foci (ACF) in the rat colon. At week 7 after the last dose of AOM, there were fewer ACF in the study groups (inulin, 78 ACF; oligofructose, 92 ACF) compared with the placebo group (120 ACF). The result may be due to the fact that Bifidobacteria contain a relatively small amount of enzymes βglucuronidase, azoreductase and nitroreductase that can convert precancerous substances into carcinogens (Hughes and Rowland, 2001). Indeed, trans-galactosylated oligosaccharides and oligofructose were found to suppress fecal activities of carcinogen metabolizing enzymes in humans and rats. Buddington et al (1996) noted significantly reduced nitroreductase activity while using 4 g/day of FOS. In addition, the study showed that reductive enzymes β-glucuronidase and glycoholic acid hydroxylase were decreased to 75% and 90%, respectively. β-Glucoronidase has implications in carcinogenesis through the release of aglycones from glycosides, while glycoholic acid hydroxylase is involved with the production of secondary bile acids, potentially linking it to the increased risk of cancer associated with high fat diets. A high fat, low fiber Western diet is responsible for reduced number of colonic apoptotic cells and is associated with tumorgenesis (Risio et al., 1996). In a study conducted by Hughes and Rowland (2001), rats were fed either a high fat diet alone, with oligofructans, or with inulin for 3 weeks. They were exposed to 1,2-dimethyl-hydrazine, and it was found that the mean number of apoptotic bodies was higher in the oligofructans and inulin groups than control. It is speculated that the bulking effects of these prebiotics contribute to their antineoplastic effect by decreasing exposure to carcinogenesis.

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Inulin and oligofructose are fermented by colonic microflora and behave as soluble fibers and selectively stimulate the growth of Bifidobacteria at the expense of Bacteroides, clostridia and coliforms. Bacterial fermentation of these prebiotics in the colon produces short chain fatty acids (SCFA) such as butyric acid which have been shown to increase apoptosis in human colonic cell lines (Campbell et al., 1997). Inulin is also found to inhibit preneoplastic lesions of the colon but the mechanisms involved are not fully known. Gibson et al (1995) have suggested that the effect of inulin proceeds through the modulation of microflora and production of SCFA in the colon. High butyrate levels following fermentation of soluble fibers may inhibit events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade. Verghese et al (2002) tested inulin, a known suppressor of azoxymethane (AOM)-induced aberrant crypt foci (ACF), for its ability to suppress preneoplastic ACF formation in mature rats. The authors found out that long-chain inulin dose dependently reduced ACF incidence in the colon (P<0.01). Compared with rats fed the control diet, the percentage of reductions of ACF in rats fed 2.5, 5.0 and 10 g inulin /100 g diets were 25, 51 and 65%, respectively. Because the long-chain oligosaccharides are fermented at a slower rate than short-chain oligosaccharides, they indeed may reach the more distal part of the colon where they can stimulate microbial metabolism. Altering the metabolic activity of the colonic microflora by inulin, which is bifidogenic reduction in cecal pH and stimulation of immune activity, may be the mechanisms by which the anticarcinogenic effect is exerted. Hsu et al (2004) evaluated the effects of xylooligosaccharides (XOS) and FOS on the precancerous colon lesions in male Sprague-Dawley rats. Both XOS and FOS markedly increased the total cecal weight and Bifidobacteria population. XOS had a greater effect on the bacterial population than did FOS. Moreover, both XOS and FOS markedly reduced the number of aberrant crypt foci in the colon of 1, 2dimethylhydrazine (DMH) treated rats. These results suggest that XOS and FOS dietary supplementation may be beneficial to gastrointestinal health. Taper and Roberfroid (1999) studied the influence of inulin and oligofructose on breast cancer and tumor growth. In a preliminary study on methylnitrosourea-induced mammary carcinogenesis in SpragueDawley female rats, 15% oligofructose added to the basal diet modulated this carcinogenesis in a negative manner. There was a lower number of

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tumor bearing rats and a lower total number of mammary tumors in oligofructose-fed rats than in the group fed the basal diet alone. The effect of dietary nondigestible carbohydrates (15% oligofructose, inulin or pectin incorporated into the basal diet) on the growth of intramuscularly transplanted mouse tumors, belonging to two tumor lines (TLT and EMT6), was also investigated. The results were evaluated by regular tumor measurements with a vernier caliper. The mean tumor surface in the experimental groups was compared with that in animals of the control group. Such nontoxic dietary treatment appears to be easy and risk free for patients, applicable as an adjuvant factor in the classical protocols of human cancer therapy. Prebiotics induce changes in the population and metabolic characteristics of the gastrointestinal bacteria, modulate enteric and systemic immune functions, and provide laboratory rodents with resistance to carcinogens that promote colorectal cancer. There is less known about protection from other challenges. Therefore, Buddington et al (2002) conducted a study in which mice of the B6CF1 strain were fed for 6 weeks a control diet with 100 g/kg cellulose or one of two experimental diets with the cellulose replaced entirely by the nondigestible oligosaccharides, oligofructose and inulin. From each diet, 25 mice were challenged by a promoter of colorectal cancer (1,2-dimethylhydrazine), B16F10 tumor cells, the enteric pathogen Candida albicans (enterically), or were infected systemically with L. monocytogenes or S. typhimurium. The incidences of ACF in the distal colon after exposure to dimethylhydrazine for mice fed inulin (53%) and oligofructose (54%) were much lower than in control mice (76%, P<0.05), but the fructans did not reduce the incidence of lung tumors after injection of the B16F10 tumor cells. Mice fed the diets with fructans had 50% lower densities of C. albicans in the small intestine (P<0.05). A systemic infection with L. monocytogenes caused nearly 30% mortality among control mice, but none of the mice fed inulin died, with survival intermediate for mice fed oligofructose. Mortality was higher for the systemic infection of S. typhimurium (>80% for control mice), but fewer of the mice fed inulin died (60%; P<0.05), with mice fed oligofructose again intermediate. The mechanistic basis for the increased resistance provided by dietary nondigestible oligosaccharide was not elucidated, but the findings are consistent with enhanced immune functions in response to changes in the composition and metabolic characteristics of the bacteria resident in the gastrointestinal tract.

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The Effect Of Fructan-Type Oligosaccharide Prebiotics On Lipid Metabolism In Humans In recent years, there has been increasing interest in the important nutritional roles of prebiotics as functional food ingredients. This interest has been derived from animal studies which have shown marked reductions in triacylglycerols (TAG) and, to a lesser extent, cholesterol levels when diets containing significant amounts of the prebiotic (FOS) were fed. However, studies conducted in humans examining the effects of prebiotics on plasma lipid levels have generated inconsistent findings. Prebiotics have been shown to be an ideal substrate for health promoting bacteria in the colon, notably bifidobacteria and lactobacilli (Gibson and McCartney, 1998). During the fermentation process, a number of byproducts are produced, including gases (hydrogen sulphide, carbon dioxide, hydrogen and methane), lactate and short chain fatty acids (acetate, butyrate and propionate). The short chain fatty acids acetate and propionate enter the portal blood stream where they are utilized by the liver. Acetate is converted to acetyl CoA in the liver and acts as a lipogenic substrate for de novo lipogenesis, whereas propionate has been reported to inhibit lipid synthesis (Demigne et al., 1995). Butyrate, on the other hand, is taken up by the large intestinal cells (colonocytes) and has been shown to protect against tumour formation in the gut. The type of short chain fatty acids which are produced during fermentation is dependent on the gut microflora that is stimulated by the prebiotic. Inulin, for example, has been shown to increase both acetate and butyrate levels (Van Loo et al., 1999). Inulin and FOS have been extensively studied to determine the mechanism of action of prebiotics in animals. Early in vitro studies using isolated rat hepatocytes suggested that the hypolipaemic action of FOS was associated with an inhibition of de novo cholesterol synthesis by propionate, following impairment of acetate utilization by the liver for de novo lipogenesis (Demigne et al., 1995). Fiordaliso et al (1995) demonstrated significant reductions in plasma TAGs, phospholipids and cholesterol in normolipidaemic rats fed a chow diet containing 10% (w/ v) FOS. The TAG-lowering effect was demonstrated after only 1 week of FOS and was associated with a reduction in very low density lipoprotein (VLDL) secretion. TAGs and phospholipids are synthesised in the liver by esterification of fatty acids and glycerol-3-phosphate before being made available for assembly into VLDL, suggesting that the hypolipidaemic effect of FOS may be occurring in the liver. The

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reduction observed in cholesterol levels in the rats was only demonstrated after long term feeding (16 wks) of FOS. Recent evidence has suggested that the TAG-lowering effect of FOS occurs via a reduction in VLDL TAG secretion from the liver due to a reduction in the activity of all lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, ATP citrate lyase and glucose-6- phosphate dehydrogenase), and, in the case of fatty acid synthase, via modification of lipogenic gene expression (Delzenne and Kok, 1998). However, further work is required to determine the mechanisms whereby short chain fatty acids lower cholesterol levels in humans. The Effect Of Prebiotics On Glucose And Insulin Levels It has been suggested that the mechanism of action of prebiotics on the lowering of glucose and insulin levels is associated with short chain fatty acids, especially propionate. A significant reduction in postprandial glucose concentration was observed following both acute and chronic intakes of propionate-enriched bread (Todesco et al., 1991). The effect of propionate intake on postprandial insulin levels was not investigated. A recent animal study has shown an attenuation of both postprandial insulin and glucose levels following 4 wks of feeding with FOS. These effects were attributed to the actions of FOS on the secretion of the gut hormones glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1). These hormones are secreted from the small intestine (GIP) and the terminal ileum and colon (GLP-1), and contribute towards the secretion of insulin following a meal in the presence of raised glucose levels (Kok et al., 1998). Prebiotics In Infant Health And Nutrition Oligosaccharides are the third most abundant solid constituent of human milk in which these are believed to play two major roles, i.e., defense agents by acting as receptor analogues to inhibit the binding of enteropathogens to the host cell receptors and bifidogenic factors. At least 21 different kinds of these oligosaccharides have already been identified that are either linear or branched, composed of simple sugars like galactose, or sugar derivatives like uronic acids or uronic esters, some being acidic and others being neutral. The oligosaccharide secretion in mother’s milk is a complex, variable and dynamic process. The amount of oligosaccharides in human milk change during lactation and also the composition of their mixture vary among different samples, being influenced by many factors one of which is the mother’s diet. The highest amount of oligosaccharides is reached on day four after birth.

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At days 30 and 120 of lactation, the content decreases by 20 and 40%, respectively, and is compensated by an increase in lactose content. Cow’s milk is very poor in oligosaccharides and infant formulas made with cow’s milk are deficient in oligosaccharides. It has been hypothesized that supplementing infant formulas with oligosaccharides could improve the nutritional value of formula and help mimic some of the effect of mother’s milk, especially the bifidogenic effect. Studies have shown that the gut microflora of breast-fed infants is dominated by bifidobacteria, whereas the gut microflora of infants fed infant formula have a diverse composition (higher numbers of Bacteroides spp., Clostridium spp. and Enterobacteriaceae). The high proportion of bifidobacteria present in the gut of breast-fed infants is associated with lower risk of intestinal infection. There is evidence that human milk oligosaccharides may promote the proliferation of intestinal bifidobacteria and lactobacilli, thus contributing to the natural defense against infection. Since the composition and structure of human milk oligosaccharides cannot be entirely reproduced by the food industry, prebiotics are being considered for fortification of infant formulas. In South Africa, there are formulas fortified with prebiotics for the infants older than 6 months. Preliminary studies have reported that infants fed a cow’s milk formula supplemented with fructo-oligosaccharides and galacto-oligosaccharides had a significantly increased number of faecal bifidobacteria after 28 days of feeding. In addition to this, stool characteristics of the babies fed the supplemented formula were similar to those of the breast fed babies (Roberfroid, 2004). Breast feeding must remain the gold-standard and the common recommendation. But to help in improving the intestinal health and well-being of babies who are not breast fed at all, breast-fed only for a short period, or are mixed-fed, supplementing infant formulas with inulin-type fructans and other prebiotics is a promising approach. At present, some infant formulas are being fortified with prebiotics in China. Probiotics – Friendly Creatures The health benefits of bacteria in food were known as early as the Persian version of the Old Testament (Genesis 18:8), which states “Abraham owed his longevity to the consumption of sour milk.” The Russian Nobel prizewinner Elie Metchnikoff, in the beginning of the 20th century observed high life expectancy in Bulgarians who consumed large amounts of fermented-milk products. It was these observations that led to the concept of “probiotic,” derived from the Greek, meaning

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“for life” which evolved to apply to those bacteria that “contribute to intestinal balance.” The term probiotic, as an antonym to the term antibiotic, was originally proposed in 1965 by Lilley and Stilwell. The first probiotic species introduced into research were Lactobacillus acidophilus by Hull et al in 1984 and Bifidobacterium bifidum by Holcombh et al in 1991 (Tanboga et al., 2003). Probiotic food is defined as a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora by implantation or colonization in a compartment of the host and by doing that exert beneficial health effects on the host (Schrezenmeir and deVrese, 2001). The probiotic microorganisms should be : • of human origin, • nonpathogenic in nature, • resistant to destruction during processing, • resistant to destruction by gastric acid and bile, • able to adhere to intestinal epithelial tissue, • able to colonize in the gastrointestinal tract, • able to produce antimicrobial substances, • able to modulate immune responses and • able to influence human metabolic activities (cholesterol assimilation, vitamin production, etc). The complex gut microflora, consists of >1 X 1011-13 living bacteria/g colon content and the bacteria with such beneficial effects are lactic acid bacteria (LAB). Probiotics And General Health The human body is a natural habitat for microorganisms and symbiosis with these microorganisms seems to be a condition for survival. A human individual has more prokaryotic organisms associated with skin, lung, and gut surfaces than human eukaryotic cells. A logical management approach to situations that alter our microbial ecology (e.g., diet, environment, antibiotics) would be to deliberately increase our association with specific non-pathogenic organisms to counter that alteration. Probiotics exert a wide spectrum of different effects ranging from direct antagonism against pathogens to influence upon intestinal epithelium and immune system of the host. Thus the use of probiotics constitutes a purposeful attempt to modify the relationship with our immediate microbial environment in ways that may benefit general health. Probiotic bacteria have been shown to influence the immune system through several molecular mechanisms (Gibson, 1998). A

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number of potential benefits arising from the use of probiotics have been proposed, including: • increased resistance to infectious diseases, • alleviating lactose intolerance, • prevention from gut, vaginal and urogenital infections, diarrhea and gastritis, • reduction in blood pressure and regulation of hypertension and serum cholesterol concentration, • reduction in allergy, respiratory infections and • resistance to cancer chemotherapy and decreasing risk of colon cancer. Today, research regarding probiotics concentrates essentially on L. acidophilus, L. casei, L. reuteri and B. bifidum. The growth in the production of probiotics by the dairy industry in some countries means that it is now increasingly difficult to purchase yogurts that do not contain probiotic bacteria such as L. acidophilus. Culture manufacturers recommend formulation of these products at 106 probiotic bacteria per gram or milliliter of dairy products, but viable counts may fall below these levels, especially at the end of shelf life. While defined as ‘medical probiotics’ (microbial preparation) and ‘other probiotics’ (functional food), probiotics are provided in products in one of four basic ways: • as a culture concentrate added to a beverage or a food • inoculated into prebiotic fibers • inoculated into a milk-based food (dairy products such as milk, milk drink, yogurt, yogurt drink, cheese, kefir, biodrink) and • as concentrated and dried cells packaged as dietary supplements (non-dairy products such as powder, capsule, gelatin tablets). Probiotics And Gastrointestinal Health Gastrointestinal infections and their consequences remain a major clinical problem despite numerous therapeutic improvements, especially in the field of antibiotics. In addition, there has been a dramatic increase in the incidence of antibiotic-resistant microbial pathogens. There is a concern that industry will no longer be able to develop effective antibiotics at a rate sufficient to compete with the development of microbial resistance to existing antibiotics. These factors have renewed interest in the possibility of deliberately feeding beneficial microorganisms to humans as an alternative to antibiotic therapy in gastrointestinal disorders. Probiotics are also an attractive treatment alternative because antibiotics further delay recolonization by normal colonic flora which can be avoided.

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Probiotics are usually targeted for use in intestinal disorders in which specific factors (such as antibiotics, medication, diet or surgery) disrupt the normal flora of the gastrointestinal tract, making the host animal susceptible to disease. Examples of such diseases include antibioticinduced diarrhea, pseudomembranous colitis and small bowel bacterial overgrowth. The goal of probiotic therapy is to increase the numbers and activities of those microorganisms suggested to possess healthpromoting properties until such time that the normal flora can be established. These diseases include traveler’s diarrhea, Helicobacter pylori gastroenteritis and rotavirus diarrhea. Intestinal Disorders Treated With Probiotics Antibiotic-induced diarrheal disease: Diarrhea is the most common side effect of antibiotic therapy. The pathogenesis of antibioticinduced diarrhea is not understood but is undoubtedly related to quantitative and qualitative changes in the intestinal flora. Several probiotics have been used in an attempt to prevent antibiotic associated diarrhea. These agents include Saccharomyces, Lactobacillus, Bifidobacterium, and Streptococcus. However, only S. boulardii, E. faecium and Lactobacillus have been shown to be clinically effective in preventing antibiotic-associated diarrhea. In a prospective, double blind, placebo-controlled study, treated 180 hospitalized patients were receiving antibiotic therapy concurrently with either placebo or S. boulardii. The overall incidence of diarrhea in these patients was 26%. There was significant difference between the placebo group and the S. boulardii group. Twenty two percent of the placebo group developed diarrhea whereas only 9% of the patients receiving S. boulardii treatment (Surawicz et al., 1989). In another study of 193 patients receiving at least one broad-spectrum β-lactam antibiotic, 97 patients received S. boulardii and 96 patients received placebo. Only 7.2% of the S. boulardii group developed antibiotic-associated diarrhea compared with 14.6% of the placebo group (McFarland et al., 1995). Clostridium difficile-associated intestinal disease. C. difficile is a classic example of opportunistic proliferation of an intestinal pathogen after breakdown of colonization resistance due to antibiotic administration. After antibiotic intake by animals and humans, C. difficile colonizes the intestine and releases two protein exotoxins, toxins A and B, which mediate the diarrhea and colitis caused by this microbe. Toxigenic C. difficile is the cause of ~20-40% of cases of antibioticassociated diarrhea (Fekety and Shah, 1993). In fact, this microorganism is the major identifiable cause of nosocomial diarrhea in the US,

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infecting 15-25% of adult hospitalized patients. This bacterium (C. difficile) can cause serious consequences, particularly in the elderly and debilitated; these include pseudomembranous colitis, toxic megacolon, intestinal perforation and even death. Standard treatment of C. difficile–associated intestinal disease, which involves either vancomycin or metronidazole, can be expensive and difficult. In addition, 25% of patients relapse with disease once treatment is discontinued (Fekety et al., 1989). Multiple relapses can occur and the relapses can be more severe than the original disease. The mechanism of relapse is unknown but is probably due to the survival of C. difficile spores in the intestinal tract until the antibiotic is discontinued. The spores then germinate and produce toxin. The antibiotic therapy prevents the normal flora from reestablishing itself. There is no uniform effective therapy to prevent further C. difficile recurrences in intractable patients. An attractive alternative to antibiotic therapy is to use probiotics to restore intestinal homeostasis. S. boulardii has demonstrated the most promise for use in C. difficile– associated intestinal disease. In a placebo-controlled study, McFarland et al (1994) examined standard antibiotic therapy (metronidazole or vancomycin) with concurrent S. boulardii or placebo in 124 adult patients, 64 patients with an initial episode of C. difficile disease and 60 patients with a history of at least one prior episode of C. difficile disease. The investigators found that in patients with an initial episode of C. difficile, there was no significant difference in the recurrence of C. difficile disease in the placebo or S. boulardii groups. However, in patients with prior C. difficile disease, S. boulardii significantly inhibited further recurrences of disease. The investigators concluded that in combination with standard antibiotics, S. boulardii is an effective and safe therapy for patients with recurrent C. difficile. Probiotic Treatment Of Infectious Diarrhea The two more common types of infectious diarrheal diseases are traveler’s diarrhea and rotavirus diarrhea. Traveler’s diarrhea: The incidence of diarrhea in travelers to foreign countries varies from 20 to 50% depending on the origin and the destination of the traveler, as well as the mode of travel. Although various infectious agents can cause traveler’s diarrhea, enterotoxigenic E. coli is the most common. Even small attacks can interrupt a holiday, and the traveling public has a great interest in medications that could be used to prevent traveler’s diarrhea. Thus, a safe, inexpensive and effective drug against traveler’s diarrhea would have important public

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health implications. Several probiotics have been examined for their ability to prevent traveler ’s diarrhea, including Lactobacillus, Bifidobacterium, Streptococcus and Saccharomyces (Hilton et al., 1997). These studies have involved several different groups of travelers such as Finnish travelers to Turkey, American travelers to Mexico, British soldiers to Belize and European travelers to Egypt. Rotavirus diarrhea: Rotaviruses are a significant cause of infant morbidity and mortality, particularly in developing countries. The principal means of treatment is oral rehydration, although an effective vaccine that should decrease dramatically the health impact of rotavirus infections has recently become available. Lactobacillus has demonstrated some promise as a treatment for rotavirus infection. Isolauri et al (1991) treated 74 children (ages 4–45 mo) with diarrhea with either Lactobacillus GG or the placebo. Approximately 80% of the children with diarrhea were positive for rotavirus. The investigators demonstrated that the duration of diarrhea was significantly shortened (from 2.4 to 1.4 d) in patients receiving Lactobacillus GG. The effect was even more significant when only the rotavirus-positive patients were analyzed. Helicobacter pylori gastroenteritis: H. pylori has recently been shown to be an important etiologic agent of chronic gastritis as well as gastric and duodenal ulcers. It has also been postulated that chronic H. pylori infection leads to stomach carcinoma. Lactobacillus has been shown to be antagonistic to H. pylori both in vitro and in a gnotobiotic murine model (Aiba et al., 1998). Hepatic encephalopathy: Hepatic encephalopathy is a neurologic disorder caused by increased blood levels of ammonia. The ammonia is produced in the intestine by the action of bacterial ureases. The ammonia is absorbed and, in healthy individuals, is detoxified by the liver. However, in patients with liver failure, the blood concentration of ammonia can reach toxic levels. Investigators have postulated that it may be possible to use probiotics to decrease intestinal urease activity. For example, patients treated with L. acidophilus and neomycin show a greater decrease in fecal urease activity than patients treated with neomycin alone (Scevola et al., 1989). The decreased fecal urease activities corresponded to lower serum ammonia levels and improvements in the clinical status of patients. HIV/AIDS diarrhea: Diarrhea is a very serious consequence of human immunodeficiency virus (HIV) infection. The etiology of this diarrhea is frequently unknown and there are no effective treatment modalities. However, S. boulardii was recently used to treat 33 HIV

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patients with chronic diarrhea (Born et al., 1993). In these double-blind studies, 56% of patients receiving S. boulardii had resolution of diarrhea compared with only 9% of patients receiving placebo. Sucrase-isomaltase deficiency: Sucrase-isomaltase deficiency is the most frequent primary disaccharidase deficiency seen in humans. It is an inherited condition that leads to malabsorption of sucrose. The resulting bacterial fermentation of the sucrose leads to an accumulation of hydrogen in the colon, producing diarrhea, abdominal cramps and bloating. A sucrose-free diet will lead to a disappearance of symptoms. However, not all patients will follow such a diet. Harms et al (1987) used Saccharomyces cerevisiae to treat eight children with sucraseisomaltase deficiency. These investigators demonstrated that in children given sucrose followed by S. cerevisiae, there was an improvement in both their hydrogen breath test and gastrointestinal symptoms. The investigators postulated that S. cerevisiae was supplying the missing enzymes. Lactose intolerance: People throughout the world suffer from a congenital deficiency of the enzyme β-galactosidase. This deficiency results in an inability to digest and absorb lactose. Bacteria in the gastrointestinal tract metabolize the lactose and the resulting by-products cause abdominal cramping, bloating, diarrhea and nausea. Lactasepositive strains of bacteria (e.g., Lactobacillus, Bifidobacterium and Streptococcus) are commonly added to pasteurized dairy products to increase digestibility of the lactose present in the dairy product. There are two probable mechanisms by which the addition of these bacteria is beneficial, i.e., the reduction of lactose in the dairy product through fermentation and the replication of the probiotic in the gastrointestinal tract, which releases lactase. Pouchitis: Pouchitis is a complication of ileal reservoir surgery occurring in 10–20% of the patients who undergo surgical treatment for chronic ulcerative colitis. Bacteria overgrow in the pouch, resulting in degradation of the mucus overlaying the epithelial cells. This results in inflammation and symptoms that include bloody diarrhea, lower abdominal pain and fever. Investigators have postulated that Lactobacillus GG may be an effective therapeutic agent for pouchitis because it does not demonstrate mucus-degrading properties (RuselerVan Embden et al., 1995). Irritable bowel syndrome: Irritable bowel syndrome is characterized by chronic, recurrent pain that occurs primarily during childhood. There is no specific treatment of this condition. However, a small, double blind, placebo-controlled, crossover study in Poland demonstrated a slight but

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significant reduction in the severity of abdominal pain in individuals receiving L. plantarum (Niedzielin and Kordecki, 1996). Small bowel bacterial overgrowth: Overgrowth of bacteria in the small intestine can have many causes, including blind loops, stenosis of the intestine, diverticula and motility disorders. Symptoms of small bowel overgrowth are frequently chronic and relapsing. Response to antibiotic treatment is often inadequate or incomplete. Surgical treatment is occasionally possible, but in many cases the underlying cause is not accessible for permanent treatment. Limited studies have suggested that L. plantarum and Lactobacillus GG may be helpful in eliminating the symptoms of small bowel bacterial overgrowth. Enteral feeding–associated diarrhea: Patients receiving nasogastric tube feeding frequently develop diarrhea. The mechanism of the diarrhea is not known, but investigators postulate that enteral feeding causes changes in normal flora that result in altered carbohydrate metabolism and subsequent diarrhea. Two separate studies (both placebo-controlled and double blind) demonstrated a significant reduction in diarrhea in these patients when they were given S. boulardii (Bleichner et al., 1997). Probiotics For Cancer Patients LAB play an important role in retarding colon carcinogenesis possibly by influencing metabolic, immunologic, and protective functions in the colon. Probiotics have anticarcinogenic-antimutagenic effects in vivo (Wollowski et al., 2001). In fact, Bifidobacterium longum supplementation reduces colon and liver carcinogenesis by 2-amino-3methylimidazo [4,5-f]quinoline as well as azoxymethane (AOM)-induced colon cancer in rats (Singh et al., 1997). Dietary supplements of Lactobacilli also increase the latency of induction of experimental colon cancer in rats, suggesting that Lactobacilli and Bifidobacteria may inhibit precancerous lesions and tumour development in animal models (Brady et al., 2000). Mechanism Of Action The mechanism by which probiotics exert the anticancer effect is unclear. However, potential mechanisms of anti-carcinogenicity of probiotics may include: • Alteration of the metabolic activities of intestinal microflora, • Alteration of physicochemical conditions in the colon, • Binding and degrading potential carcinogens, • Quantitative and qualitative alterations in the intestinal microflora,

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• Production of antitumorigenic or antimutagenic compounds, • Enhancing the host’s immune response, • Effects on the physiology of the host, • Fermentation of undigested food and the formation of metabolites Alteration Of The Metabolic Activities Of Intestinal Microflora Many foreign compounds which may be carcinogenic are detoxified in the liver which converts them to inactive glucuronides of procarcinogens. Subsequently those in bile enter the bowel and are excreted out of the body. But some intestinal flora possess enzymes such as β-glucuronidase, nitroreductase, azoreductase and choloylglycine hydrolase which hydrolyse glucuronides and liberate carcinogenic aglycones in the intestinal lumen. So when the diet is supplemented with probiotics, which have relatively low paucity of these enzymes, they may overtake the growth of the intestinal flora possessing higher activity of these enzymes. This alters the metabolic activities of intestinal flora resulting in inhibition of colon cancer (Rafter, 2002). Oral supplementation of the diet with viable L. acidophilus of human origin, which is bile resistant, caused a significant decline in three different fecal bacterial enzymes. The decline in fecal enzyme activity was noted in humans and rats. The bacterial enzymes that were affected included β-glucuronidase, azoreductase and nitroreductase. The effect of feeding of L. acidophilus strains NCFM and N-2 on the activity of three bacterial enzymes, i.e., β-glucuronidase, nitroreductase, azoreductase was studied in 21 healthy volunteers. Both strains had similar effects and caused a significant decline in the specific activity of the three enzymes in all subjects after 10 days of feeding. A reversal of the effect was observed within 10-30 days of ceasing L. acidophilus feeding, suggesting that continuous consumption of these bacteria was necessary to maintain the effect (Goldin and Gorbach, 1984). Consumption of milk with L. casei Shirota for 4 weeks temporarily decreased β-glucuronidase in 10 healthy subjects compared with 10 healthy controls (Rolfe, 2000). Another study using L. acidophilus, B. bifidum, S. lactis, and S. cremoris for 3 weeks demonstrated reduction of nitroreductase (Marteau et al., 1990). Thus, animal and human studies indicate that feeding certain lactic cultures can result in a decrease of fecal enzymes that may be involved in formation of carcinogens. Alteration Of Physico-chemical Conditions In The Colon Moddler et al (1990) have suggested that large bowel cancer could be influenced directly by reducing intestinal pH, thereby preventing the growth of putrefactive bacteria. In rats given inulin-containing diets

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with Bifidobacterium longum, an increase in fecal weight and a decrease in fecal pH were observed (Rowland et al., 1998). In a study by Biasco et al (1991), patients with colonic adenomas participated in a 3-month study, where L. acidophilus was administered together with B. bifidum. During this period, fecal pH and proliferative activity were reduced significantly after therapy with lactic acid bacteria. Binding And Degrading Potential Carcinogens One potential risk factor of colon cancer that is related to high meat consumption is the formation of heterocyclic amines formed during the cooking of meat. There are several reports of binding of carcinogens, such as heterocyclic amines, Aflatoxin B1 and benzopyrene, in vitro by LAB and other intestinal bacteria. Depending on the pH of the culture medium, LAB can bind to heterocyclic amines (Orrhage et al., 1994). When the dose of trypsin and bile acids was increased in a medium to simulate an in vivo situation in the intestine, the binding capacity of LAB decreased linearly and the negative influence of bile acids was more pronounced (Tanabe et al., 1994). Administration of L. acidophilus to healthy volunteers consuming a fried meat diet, known to increase fecal mutagenicity, resulted in a greater decrease in fecal mutagenic activity after 3 days than administration of ordinary fermented milk (Lidbeck et al., 1992). During L. acidophilus administration, the urinary mutagenic activity on days 2 and 3 was significantly lower compared to the ordinary fermented milk period. In most cases, an increase in the number of fecal Lactobacilli corresponded to a lower mutagen excretion, particularly in urine. Hayatsu and Hayatsu (1993) also demonstrated a marked suppressing effect of orally administered L. casei Shirota (LcS) on the urinary mutagenicity arising from ingestion of fried ground beef in man. It was estimated that the binding of mutagens could be attributed to the cell wall of bacteria and in view of the results of in vitro studies, it is possible that the lactic acid bacteria supplements are influencing excretion of mutagens by simply binding them in the intestine. Quantitative And Qualitative Alterations In The Intestinal Microflora The diet supplemented with products containing L. acidophilus has a beneficial effect on the intestinal microecology by suppressing the putrefactive organisms that are possibly involved in the production of tumor promoters and putative procarcinogens. Consumption of fermented milk containing L. acidophilus has been shown to significantly reduce the counts of fecal putrefactive bacteria such as

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coliforms and increase the levels of Lactobacilli in the intestine (Ayebo et al., 1980). Production Of Antitumorigenic Or Antimutagenic Compounds The antitumor activities of LAB may be due to the presence of an ex novo soluble compound produced by them during fermentation of milk or the microbial transformation of some milk components in a biologically active form. Although the protective activity of Lactobacilli observed in the colon may be explained by a local and direct effect on intestinal mucosa, some epidemiologic studies have also indicated a reduced risk of breast cancer in women who consume fermented milk products and/or the fermentative bacteria themselves may have chemoprotective effects. This was evident from studies using preimplanted cancer cells in animal models. Bogdanov et al (1978) observed that L. bulgaricus possessed a potent antitumour activity. They isolated three glycopeptides which had biological activity against sarcoma-180 and solid Ehrlich ascites tumour. Sekine et al (1994) reported that a single subcutaneous injection of whole peptidoglycan isolated from Bifidobacterium infantis strain ATCC 15697 significantly suppressed tumor growth. LAB significantly reduced the growth and viability of the human colon cancer cell line HT-29 in culture and dipeptidyl peptidase IV and brush border enzymes were significantly increased, suggesting that these cells may have entered a differentiation process (Baricault et al., 1995). L. bulgaricus prevented 1,2-dimethylhydrazine (DMH) induced DNA breaks in rats in vivo whereas S. thermophilus did not. However, both strains prevented DNA damage in vitro when rats were exposed to N-methyl-N-nitro-N-nitrosoguanidine (MNNG). Indeed extracts from the S. thermophilus were also effective in deactivating MNNG (Wollowski et al., 1999). The authors hypothesized that it is thiol-containing breakdown products of proteins created by bacterial proteases that deactivate various colonic mutagens. Milk fermented by B. infantis, B. bifidum, B. animalis, L. acidophilus and L. paracasei inhibited the growth of the MCF7 breast cancer cell line and the antiproliferative effect was not related to the presence of bacteria but due to the presence of an ex novo soluble compound produced by LAB (Biffi et al., 1997). It is expected that LAB or metabolites may prevent the carcinogens from inducing genotoxic effects. These preventive properties may be due to a scavenging of reactive carcinogen intermediates (by LAB or by their metabolites). Alternatively, LAB or LAB metabolites may affect carcinogen-activating and carcinogen-deactivating enzymes. Acetone

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extracts prepared from nonfermented milk, fermented milk, or L. acidophilus grown in De Man Rogosa Sharpe broth were investigated for their antigenotoxic activity in freshly isolated colon cells of rats treated with MNNG for 30 min. It was shown that fermentation resulted in short-lived metabolites that prevent DNA damage in these cells. The identity of these metabolites has not yet been characterized; however, protection by these metabolites was more pronounced than was protection observed by cellular components of LAB, eg., peptidoglycan or cytoplasma fractions (Wollowski et al., 2001). Enhancing The Host’s Immune Response The lactic acid bacteria are thought to suppress tumor formation by enhancing an immune response of the host. Sekine et al (1985) suggested that B. infantis stimulates the host-mediated response, leading to tumor suppression or regression. In addition there are studies to suggest that LAB play an important role in the host’s immunoprotective system by increasing specific and non specific mechanisms to have an antitumor effect (Schiffrin et al., 1995). Lactobacillus casei Shirota has been shown to have potent antitumor and antimetastatic effects on transplantable tumor cells and to suppress chemically induced carcinogenesis in rodents. Also, intrapleural administration of L. casei Shirota into tumorbearing mice has been shown to induce the production of several cytokines, such as interferon-, interleukin-1β and tumor-necrosis factor-
, in the thoracic cavity of mice resulting in the inhibition of tumor growth and increased survival. These findings suggest that treatment with L. casei Shirota has the potential to ameliorate or prevent tumorigenesis through modulation of the host’s immune system, specifically cellular immune responses. It has also been demonstrated that B. longum and B. animalis promote the induction of inflammatory cytokines in mouse peritoneal cells (Matsuzaki, 1998). Effects On The Physiology Of The Host Lactobacilli are one of the dominant species in the small intestine, and they presumably affect metabolic reactions occurring in this part of the gastrointestinal tract. They have been shown to increase colonic NADPH-cytochrome P-450 reductase activity (Pool-Zobel et al., 1996) and glutathione S-transferase levels (Challa et al., 1997) and to reduce hepatic uridine disphosphoglucuronyl transferase activity (Abdelali et al., 1995), enzymes which are involved in the metabolism of carcinogens in rats. It has been demonstrated that dietary administration of lyophilized cultures of B. longum strongly suppressed azoxymethane-

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induced colonic tumor development and that this effect was associated with a decrease in colonic mucosal and tumor ornithine decarboxylase and ras-p21 activities (Reddy, 1998). Fermentation Of Undigested Food And The Formation Of Metabolites A common characteristic of the microflora is fermentation. The anaerobic breakdown of substrates, such as undigested polysaccharides, resistant starch, and fiber, enhances the formation of short-chain fatty acids as fermentation products. Increased production of short-chain fatty acids leads to a decrease in the pH of colon content. A low pH in feces was associated with a reduced incidence of colon cancer in various populations (Segal et al., 1995). Depending on the nature, quantity, and fermentability of undigestible polysaccharides reaching the colon, the relation of the short-chain fatty acids acetate, propionate, and butyrate can vary. Resistant starch and wheat bran favor the production of butyrate, whereas pectin leads to a higher formation of acetate. Butyrate is associated with many biological properties in the colon (PoolZobel et al., 1996). One of the first observed effects of butyrate on the degree of methylation is probably associated with modified gene expression. Butyrate may also directly enhance cell proliferation in normal cells and suppress proliferation in transformed cells. In addition, apoptosis may be increased in transformed cells but inhibited in normal cells when butyrate is present (Marchetti et al., 1997). Butyrate is an important fuel for colon cells, which may explain the higher resistance of cells pretreated with butyrate to oxidative damage induced by hydrogen peroxide in comparison with cells not pretreated with butyrate. Butyrate has also been shown to increase glutathione transferase  in colon cells and may be a responsible factor for enhanced glutathione transferase  expression in colon tissue (Treptow-van Lishaut et al., 1999). Glutathione transferase  is the most abundant glutathione transferase species in colon cells and is an important enzyme involved in the detoxification of both electrophilic products and compounds associated with oxidative stress. Thus, enzyme induction by butyrate, or by the microflora and increased activity by prebiotics may be an important mechanism of protection against carcinogeninduced cancer. Probiotics For Oral Health Oral infections constitute some of the most common and costly forms of infections in humans. Dental caries and periodontal diseases occur in nearly 95% of the general public. Although fluoride and other

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preventive efforts have led to a dramatic decline in dental caries, the ability to control the actual infection has been limited (Caglar et al., 2005). The concept of microbial ecological change as a mechanism for preventing dental problems is an important one. The oral cavity is a complex ecosystem in which a rich and diverse microbiota has evolved. The wide range in pH, nutrient availability, shedding and non-shedding surfaces, salivary and crevicular fluids select localized, discrete microbial climax communities to fluctuate in composition and metabolic activity but reach a kind of homeostasis in balance with the host. Changes in the environment whether imposed by illness, debility, behavior, diet, or medications disturb the homeostasis and lead to endogenous infections or susceptibility to exogenous infections. The resident oral microflora is diverse, being comprised of species with differing nutritional (saccharolytic, proteolytic, secondary feeders), atmospheric (aerobic, anaerobic, facultative, micro-aerophilic, capnophilic) and physico-chemical (pH, co-factors) requirements. Dental disease may be a consequence of changes in the ecology stated above. If the local environment is perturbed, then potential pathogens may gain a competitive advantage and, under appropriate conditions, reach numbers that predispose a site to disease. Regarding elimination of pathogenic members of the oral cavity a new method such as probiotic approach (i.e., whole bacteria replacement therapy) is reported (Caglar et al., 2005). Possible Mechanisms Of Action Of Probiotics In Maintaining Oral Health As a result of cariogenic properties, lactobacilli have been of great interest to dental researchers for several decades. They are associated more with carious dentine and the advancing front of caries lesions rather than with the initiation of the dental caries process. Lactobacilli are the most common probiotic bacteria associated with the human gastrointestinal tract; therefore it may also play an important role in the eco physiology of oral microbiota. Various lactobacilli species (L. paracasei, L. gasseri, L. fermentum L. salivarius, L. plantarum, L. crispatus, and L. rhamnosus isolated) inhabit healthy mouths, although no species is specific to the mouths of healthy subjects. Development of new ways to block the pathogenesis of oral infections can reduce tissue destruction associated with oral infection and chronic inflammation. It is thought that probiotics particularly lactobacilli that hydrolyse proteins to amino acids and dipeptides, stimulate growth of streptococci which produce low pH conditions in the oral environment.

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Conversely, in recent studies, it was stated that probiotics might reduce the risk of the high level of Streptococcus mutans (Ahola et al., 2002) which are responsible for dental caries. Recently it was evaluated whether the oral administration of lactobacilli could change the salivary counts of these bacteria compared with placebo. Lactobacilli were administered in liquid and in capsule form to volunteer subjects to determine the role of direct contact with the oral cavity. It was found that the oral administration of probiotics, both in capsules and in liquid form, significantly increases salivary counts of lactobacilli while S. mutans levels were not modified (Montalto et al., 2004). There is a concept where these beneficial microorganisms can inhabit a bio-film and actually protect oral tissue from disease. It is possible that one of these biofilm’s mechanisms to keep pathogens out is to occupy a space that might otherwise be occupied by a pathogen. An in vitro study suggests that L. rhamnosus GG (LGG) can inhibit the colonization of streptococci caries pathogens, thus reducing the incidence of caries in children (Meurman et al., 1995). In a Swiss study, bacterial strains with potential properties as oral probiotics, were studied for the prevention of dental caries. From 23 dairy microorganisms studied, two were identified; which were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrins OMZ176. Streptococcus thermophilus NCC1561 and Lactobacillus lactis NCC2211, were successfully incorporated into a bio-film mimicking the dental plaque. Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque. In this system, Lactococcus lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Streptococcus sobrinus OMZ176 (Comelli et al., 2002). From a periodontal view, a Russian study examined probiotic tablets in complex treatment of gingivitis and different degrees of periodontitis. The treatment of the patients of the control group was provided by drug ‘Tantum Verde’. The effect of probiotics to the normalization of microflora was found to be higher in comparison with Tantum Verde, particularly in the cases of gingivitis and periodontitis (Grudianov et al., 2002). There is no research regarding relationship between dental restorative materials and probiotics. However, in the larynx, the second barrier after the oropharynx, probiotics strongly reduce the occurrence of pathogenic bacteria in voice prosthetic bio-films (Free et al., 2001).

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Installation Of Probiotics In The Oral Cavity Probiotics should adhere to dental tissue for them to establish a cariostatic effect and thus should be a part of the bio-film to fight with cariogenic bacteria. For this action, installation of probiotics in the oral environment seems important. However, the contact time between probiotics and plaque would be short, that the activity is not sufficient to stop the growth of cariogenic bacteria. This activity increases if probiotics could be installed in the oral environment for a longer duration. At this point, ideal vehicles of probiotic installation should be determined. Effects Of Probiotics On Blood Cholesterol Agerbaek et al (1995) tested the effect of commercially available yogurt GAIO® (containing a specific bacterial culture, CAUSIDO® [consisting of Enterococcus faecium and Streptococcus thermophilus, and has been shown to have hypocholesterolemic properties when tested on animals]) against identical yogurt that had been chemically fermented with an organic acid (-glucolactone). Fifty-eight middleaged men with moderately raised cholesterol levels (5.0-6.5 mmol/l) were fed 200 ml per day of yogurt for a 6-wk period. They observed a 9.8% reduction in LDL cholesterol levels (P < 0.001) for the live yogurt group. The mechanism of action of probiotics on cholesterol reduction is unclear, although a number of possible mechanisms have been proposed. These include the physiological actions of fermentation end products (short chain fatty acids), deconjugation of bile acids (which could reduce cholesterol by co-precipitation at acidic pH or by increasing excretion of bile acids, thereby increasing the amount of cholesterol required for de novo synthesis in the liver, or a combination of both these mechanisms), cholesterol assimilation, and cholesterol binding to bacterial cell walls. It has been well documented that microbial metabolism of bile acid is a peculiar probiotic effect involved in the therapeutic role of some bacteria. The deconjugation reaction is catalysed by a conjugated bile acid hydrolase enzyme, which is produced exclusively by bacteria. Deconjugation is widely found in many intestinal bacteria including genera such as Enterococcus, Peptostreptococcus, Bifidobacterium, Fusobacterium, Clostridium, Bacteroides and Lactobacillus. This reaction liberates an amino acid moiety and a deconjugated bile acid, thereby reducing cholesterol re-absorption by increasing fecal excretion of the deconjugated bile acids. Many in vitro studies have investigated the ability of various bacteria to deconjugate a variety of different bile

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acids. Grill et al. (1995) reported Bifidobacterium longum as being the most efficient bacterium when tested against six different bile salts. Studies performed on in vitro responses are useful, although in vivo studies in animals and humans are required to more fully determine the contribution of bile acid deconjugation to cholesterol reduction. Intervention studies on animals and ileostomy patients have shown that oral administration of certain bacterial species can lead to an increased excretion of free and secondary bile salts (De Smet et al. 1998). There is also in vitro evidence to support the hypothesis that some bacteria can assimilate (take up) cholesterol. It has been reported that Lactobacillus acidophilus (Gilliland et al., 1985) and Bifidobacterium bifidum (Rasic et al., 1992) have the ability to assimilate cholesterol during in vitro studies, but only in the presence of bile salts and under anaerobic conditions. However, despite such reports, there is uncertainty about whether the bacteria are assimilating cholesterol or whether cholesterol is co-precipitating with the bile salts. Studies have been performed to address this question. Klaver and Van der Meer (1993) concluded that removal of cholesterol from the medium in which Lactobacillus acidophilus and Bifidobacterium were growing was not due to assimilation, but due to bacterial bile salt deconjugase activity. Cholesterol binding to bacterial cell walls has also been suggested as a possible mechanism for the hypocholesterolemic effects of probiotics. Hosono and Tono-oka (1995) reported that Lactococcus lactis subsp. lactis biovar. diacetylactis R-43 had the highest binding capacity for cholesterol for a range of bacteria tested. It was speculated that differences in binding of the bacteria were due to chemical and structural properties of their cell walls, and that even non-viable cells may have the ability to bind cholesterol in the host intestinal tract. The mechanism of action of probiotics on cholesterol reduction could include one or all of the above mechanisms, with an ability of different bacterial species to have varying effects on cholesterol lowering. Sources Of Probiotics Kefir, a traditional fermented milk drink originating from the Bulkans, cultured buttermilks, yogurt products, fermented whey-based drinks, some cheeses, fermented juices, some fermented vegetable products, symbiotic beverages, and fermented soy products are the major sources of probiotics.

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SYMBIOTICS Recently it has been suggested that a combination of prebiotics and probiotics, the so-called symbiotics might be more active than the individual components on the colon. Symbiotic is defined as “a mixture of prebiotics and probiotics that beneficially effects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of healthpromoting bacteria, and thus improving the health of the host” (Gibson and Roberfroid, 1995). Accordingly, Rowland et al (1998) showed that concomitant administration of inulin and Bifidobacteria to rats resulted in a more potent inhibition of AOM-induced ACF than the administration of the two separately. Research in the author’s laboratory is going on to develop symbiotic foods and to study their effects on health. There are studies where oats and probiotics are combined to prepare symbiotic products (Gokavi et al., 2005). But human feeding trials are required to prove their health promoting effects. Beneficial Effects Of Symbiotics On Lipid Metabolism The use of symbiotics as functional food ingredients is a new and developing area; very few human studies have been performed which look at their effect on risk factors for CHD. In one study, the effect of a fermented milk product with and without the addition of Lactobacillus acidophilus and fructooligosaccharides was examined in healthy men (Schaafsma et al., 1998). The design of the study was a randomized placebo controlled crossover form, in which there were two treatment periods of 3 weeks, with a 1 week washout period. The authors reported a significant reduction in total and LDL cholesterol following ingestion of the fermented milk product containing both the probiotic and prebiotic, compared to the placebo fermented milk. Other research has concentrated on the composition of the gut microflora. In one study of healthy subjects, a fermented milk product containing Bifidobacterium spp. with or without 18 g of inulin was given daily for 12 days. The authors concluded that administration of the fermented milk product (probiotic) substantially increased the proportion of bifidobacteria in the gut, but that this increase was not enhanced with the addition of inulin. The composition of the gut microflora was then assessed 2 weeks

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after completion of the supplementation period; it was found that subjects who received the fermented milk product with inulin maintained their gut bifidobacterial population compared to subjects receiving the fermented milk product only. Although a synergistic effect on the bifidobacterial population in the gut was not observed with the symbiotic, these results suggest that either there was better implantation of the probiotic and/or there was a separate prebiotic effect on bifidobacteria already present in the gut. The maintenance of high numbers of bifidobacteria in the gut flora may be beneficial in terms of maintaining healthy intestinal function, however, its effect on blood lipid reduction remains to be determined. A more recent study has shown that a lower dose of prebiotic (2.75 g) added to a Lactobacillus-fermented milk was able to significantly increase numbers of bifidobacteria when fed over a 7 week period in healthy human subjects (Roberfroid, 1998). If this effect was a result of the symbiotic product used in the study, the use of lower doses of prebiotics in symbiotic preparations will help to reduce the gastrointestinal complaints observed with prebiotics alone and will improve the acceptability of these types of products by the general public. Examples Of Symbiotic Foods Commonly available symbiotic foods include yogurt and yogurt beverage made with cow milk, goat milk and soy milk. The process of yogurt making is an ancient craft which dates back thousands of years. Fortunately, the process has still survived through the ages which can be attributed to the fact that the scale of manufacture is very small which was handed down from parents to children. Yogurt is made by fermenting milk with lactic cultures which belong to a category of microorganisms that can digest the milk sugar lactose and convert it into lactic acid. For the cells to utilize lactose, deriving carbon and energy from it, they must also possess the enzymes needed to break lactose into two simple sugars: glucose and galactose. Some representative strains are Streptococcus lactis, S. cremoris, thermophilus, Lactobacillus bulgaricus, L. acidophilus, and L. plantarum. Yogurt is defined as the product resulting from the culturing of a mixture of milk and cream products with the lactic acid producing bacteria L. bulgaricus and S. thermophilus. Yogurt contains not less than 3.25 percent milk fat and 8.25 percent solids-not-fat. Commercial yogurt production is composed of the following steps: pretreatment of milk, homogenization, heat treatment, cooling to incubation temperature, inoculation with starter, fermentation, cooling,

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FIGURE 4.9 — Process For The Preparation Of Symbiotic Yogurt And Yogurt Beverage

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post-fermentation treatment (flavoring, fruit addition, pasteurization), refrigeration, and packaging. For set yogurt, the packaging into individual containers is carried out before fermentation. A good strain of starter culture not only affects the flavor and aroma, it can also speed up the process and thus reduces the production costs. Yogurt containing prebiotics and probiotics is a good synergistic fit and a true functional food. Research on health benefits of prebiotics and probiotics has driven attention towards development of products containing both. Thus developed products are symbiotic foods. Yogurt is the most suitable and common vehicle for this purpose. Most common symbiotic foods found in the market are milk based and soy based. Milk based foods include those made from cow milk, goat milk and buffalo milk. For soy based products, soy milk is the base. Soy based foods are good for people who are lactose intolerant. The process of making these products is outlined in Figure 4.9. SUMMARY This chapter is a review of the scientific data on beneficial effects of prebiotics, probiotics and symbiotics on human health which may lead them to be classified as functional foods in the near future. Intestinal flora is comprised of different types of bacteria living in symbiosis with the host. The stable composition of the flora is one of the factors responsible for a balanced ecosystem and good health. The composition and the activity of intestinal flora are influenced by some dietary factors which are nothing but prebiotics, probiotics and symbiotics. All prebiotics are mostly carbohydrates including sugar alcohols, disaccharides, oligosaccharides and polysaccharides which are neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract and are selective substrate for beneficial bacteria in the large intestine. Types of prebiotics include fructans, fiber gums, isomaltooligosaccharides, lactitol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto oligosaccharides, and xylooligosaccharides. Probiotics are live microorganisms which when consumed in sufficient numbers influence the microbial environment of the host in a beneficial way. The commonly known probiotics include L. acidophilus, B. bifidum, L. casei, and many others. Symbiotics are foods that contain both. The best strategy to cure chronic diseases is to prevent them, and to prevent them it requires modification of day-to-day diet. The scientific research so far indicates that if a sufficient amount of prebiotics, probiotics and symbitoics are included in the diet, incidence of diseases

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could be reduced to a great extent.The consumer of today is health conscious and demands foods which are tasty as well as low in fat and calories, with additional health benefits. In present day society, the leading health concerns are heart disease, cancer, high cholesterol and diabetes. The chapter discusses how prebiotics and probiotics could be a possible dietary treatment for these chronic diseases. With the limited information available from scientific studies, the use of prebiotics, probiotics and symbiotics for improving human health holds promise. References Abdelali, H., Cassand, P., Soussotte, V., Daubeze, M., Bouley, C. and Narbonne, J. F. 1995. Effect of dairy products on initiation of precursor lesions of colon cancer in rats. Nutr. Cancer 24:121-132. Agerbaek, M., Gerdes, L.U. and Richelsen, B. 1995. Hypocholesterolaemic effects of a new product in healthy middle-aged men. Eur. J. Clin. Nutr. 49:346-352. Ahola, A. J., Yli-Knuuttila, H., Suomalainen, T., Ahlström, A., Meurman, J. and Korpela, R. 2002. Short term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch. Oral Biol. 47:799–804. Aiba, Y., Suzuki, N., Kabir, A. M., Takagi, A., Koga, Y. 1998. Lactic acidmediated 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. Ayebo, A. D., Angelo, I. A. and Shahani, K. M. 1980. Effect of ingesting Lactobacillus acidophilus milk upon fecal flora and enzyme activity in humans. Milchwissen. 35:730-733. Baricault, L., Denariaz, G., Houri, J. J., Bouley, C., Sapin, C. and Trugnan, G. 1995. Use of HT-29, a cultured human colon cancer cell line, to study the effect of fermented milks on colon cancer cell growth and differentiation. Carcinogeneis 16(2):245-252. Biasco, G., Paganelli, G., Brandi, G., Brillianti, S. and Lami, F. 1991. Effect of Lactobacillus acidophilus and Bifidobacterium bifidum on rectal cell kinetics and fecal pH. Italian J. Gastroenterol. 23:142. Biffi, A., Coradini, D., Larsen, R., Riva, L. and Di Fronzo, G. 1997. Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line. Nutr. Cancer 28:93-99. Bleichner, G., Blehaut, H., Mentec, H. and Moyse, D. 1997. Saccharomyces boulardii prevents diarrhea in critically ill tube-fed patients. A multicenter, randomized, doubleblind placebo-controlled trial. Intensive Care Med. 23:517–523. Bogdanov, I. G., Velichkov, V.T., Gurevich, A. I., Dalev, P. G., Kolosov, A. M. N., Mal’kova, V. P., Sorokina, I. B., Khristova, L. N. 1978. Antitumor action of glycopeptides from the cell wall of Lactobacillus bulgaricus. Bull. Exptl. Biol. Med. 84:1750-1753. Born, P., Lersch, C., Zimmerhackl, B. and Classen, M. 1993. The Saccharomyces boulardii therapy of HIV-associated diarrhea (letter). Dtsch. Med. Wochenschr. 118:765. Brady, L. J., Gallaher, D. D. and Busta, F. K. 2000. The role of probiotic cultures in the prevention of colon cancer. J. Nutr. 130:S410-S414. Buddington, K. K., Donahoo, J. B. and Buddington, R. K. 2002. Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumor inducers. J. Nutr. 132:472-477.

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