Duncan CrowI. INTRODUCTION
A growing awareness of the complex interactions between diet, the intestinal microflora resident in the gastrointestinal tract and health has encouraged the development of dietary strategies promoting the growth of specific bacterial groups perceived to be beneficial. Even though these complex relationships are now recognized from clinical studies, they remain poorly understood. Of particular interest is the use of a new class of dietary foodstuffs termed nondigestible oligosaccharides (NDO). Due to their unique chemical structure some of these carbohydrates resist digestion by the human alimentary tract. Consequently, they reach the caeco-colon essentially as intact molecules, not providing the body with digestible monosaccharides. Because these carbohydrates enter the colon as intact compounds they elicit systemic physiological functions and act as fermentable substrates for colonic microflora-influencing the species composition and metabolic characteristics of the intestinal microflora, and therefore provide important health attributes. Inulin is a natural NDO extracted in industrial quantities from chicory with potential use in producing physiological functional foods and promoting human health.Evidence of the growing interest in inulin is the increased research in both short-chained fructooligosaccharides (FOS) and the longer chained inulin. A number of review papers have been published discussing various aspects of the health benefits of FOS and inulin. These include: Farnworth, 1993; Fuchs, 1992; Gibson and Roberfroid, 1995; Gibson et al., 1996; Roberfroid, 1993; Roberfroid, 1996; Roberfroid, et al., 1998; Van Loo et al., 1995; and Yuri, 1996.
II. IDENTITY
Inulin falls under the general class of carbohydrates called fructans, those polymers containing fructose. Fructans serve as storage polymers in many members of the CompositX such as Cichorium intybus (chicory), Inula helenium (elecampane), Taraxacum officinalis (dandelion), and Helianthus tuberosus (Jerusalem artichoke). Inulin extracted from chicory is a natural polydisperse carbohydrate (Phelps, 1965). It is known as a fructan consisting predominately of linear chains of 1,2-B-linked d-fructofuranose units bound by a (a1-B2) type linkage (as in sucrose) to a terminal glucose moiety. All fructans found in the dicotyledons, as well as some monocotyledons, are of this type. By comparison, fructans composed predominately of linear fructose units bound by a B(2-->6) glycosidic bond are typically levans that are produced by many soil and oral bacteria, yeasts and fungi.The gross molecular formula of inulin is GFn, with G being a terminal glucosyl unit, F representing the fructosyl units and "n" representing the number of fructosyl umits. The basic GF2 trimer in inulin and the shortest fructan of the inulin type is 1-kestose. The same bonds link the ensuing fructosyl units, i.e. P(2-->l) as that in 1-kestose, Figure 1.
Figure 1: Chemical Structure of sucrose (left), inulin (center), oligofructose (right)
Short chain fractions of fructooligosaccharides such as 1-kestose, the major GF, compound in chicory roots or Jerusalem artichoke, and neokestose in onion do not differ analytically (Van Loo et al., 1995). Further, fructan chains linked to either of these naturally occurring trisaccharides have the B(2-->1) configuration, implying that with the exception of one glycosidic linkage within the basic trisaccharide there is no difference between a fructan molecule based on 1-kestose or neokestose. The chain length or degree of polymerization (DP) in such molecules is n+ 1.
Pollock et al., (1993) reviewed the enzymology of fructan synthesis in vivo. The natural biosynthesis of inulin within plant cells begins by the cells using a vacuolar enzyme, sucrosesucrose fructosyltransferase (SST), to catalyze the reaction (Equation 1) that produces trisaccharides (Isokestose, kestose, and neokestose) and a glucose molecule from two sucrose molecules (Edelman & Dickerson, 1966).
Equation 1. GF + GF --> GFF + G
Sucrose-sucrose fructosyltransferase is only active at high concentrations of sucrose (6-15%) so fructan metabolism is an extension of the sucrose metabolism. Following trisaccharide production, the polymeric chain is lengthened by the action of another vacuolar enzyme, fructan-fructan fructosyl transferase (FFT), catalyzing the transfer of a unit of fructose from one donor polymer to another acceptor. The enzyme uses the trisaccharide formed by SST for further chain elongation of the fructan polymer, releasing sucrose (Equation 2). Therefore, SST and FFT have some overlapping activities. During this transfer sucrose molecules only act as acceptors and cannot function as donors. The balance mechanism between the various fructans with a different DP and in the absence of new syntheses appears to have a regulatory effect on the plant's physiology (Marchetti, 1993a).
Equation 2. G(F)n + G(F)m --> G(F)n-1 + G(F)m+1
An understanding of different terms used to describe fructose-containing polymers is important as more commercial products become available. Oligofructose was introduced as a synonym for fructo-oligosaccharides in 1989 (Coussement, 1999). The oligofructose product is a partial enzymatic hydrozlyate of native chicory inulin containing predominately molecules of the Fm-type (homopolymers of fructose bound by a B(2-->1) glycosidic linkage having no terminal glucose), Figure 1. Oligofructose has been defined by the IUB-IUPAC Joint Commission on Biochemical Nomenclature and the AOAC as fructose oligosaccharides containing 2-10 monosaccharide residues connected by glycosidic linkages (Niness, 1999). As previously stated, inulin, as extracted from chicory root, is a polydisperse fructan with chain lengths ranging from 2 to 60 units with a modal DP of ± 9. Commercial chicory inulin is composed of approximately 2% monosaccharides, 5% disaccharides and 93% inulin. Neosugar is a FOS mixture of kestose (n=2), nystose (n=3) and IF-B-fructofuranosyl nystose (n=4). Basically these are sucrose molecules to which one to three additional fructose units have been added. The development and isolation of neosugar was first reported in the Japanese literature in 1983 (Oku et al., 1984).
Neosugar can be isolated from brans of triticale, wheat and rye or artificially by the action of the fungal enzyme 0-fructofuranisidase on sucrose, the enzyme being a product of the fungus Aspergillus niger (Fishbein et al., 1988). Using the most widely available and accepted nomenclature, all FOS and inulins are fructans, all FOS are inulins, but not all inulins are FOS. Those inulin molecules having a degree of polymerization of < 10 fructose units generally are considered to represent FOS.
II. (a). Origin and historical human consumption. Inulin was discovered by Rose, a German scientist, who in 1804 found "a peculiar substance" from plant origin in a boiling water extract from the roots of Inula helenium, a genus of perennial herbs of the group Compositae, natives of the temperate regions of Europe, Asia, and Africa (Goudberg, 1913). The substance was named inulin but was also identified by other names such as helenin, alantin, meniantin, dahlin, sinanternin, and sinisterin. The biochemical production was elucidated around the middle of the 19th century.
Inulin belongs to the group of naturally-occurring carbohydrates known as non-digestible oligosaccharides (NDO). It is produced naturally in over 36,000 plants worldwide, including 1,200 native grasses belonging to 10 families. After starch, they are the most plentiful carbohydrates occurring in the plant kingdom (Carpita et al., 1989; Marchetti, 1993b). It has been estimated that as much as one third of the total vegetation on earth consists of plants that contain fructans. Inulin-type carbohydrates obtained from fungal fermentation have been reported in commercial use but the predominant commercial source for Inulin/FOS is of chicory root origin. Inulin has extensive documented historical human use through the consumption of edible plants and fruits - a variety of which are common foodstuffs (Table 1).
Table 1: Inulin content of foods consumed by Americans
Inulin is an energy-reserve carbohydrate and may act as an osmoprotectant in plants. Because inulin is readily soluble in water, it is osmotically active. By changing the DP of the molecule in the plant's vacuole, the plant can readily change the osmotic potential of its cells without altering the total amount of carbohydrate. The internal hydrolysis of inulin by endoinulinase to lower DP Fm, and GFn molecules allows plants to osmoregulate, surviving winter periods in cold to moderately cold and drought stricken regions (Edelman & Jefford, 1968).
Historically, several inulin-laden foods, especially chicory, dahlia, Jerusalem artichokes, munong, and yacon, have been used as staple food or as sustenance crops. Australian aborigines ate murnong, a tuberous plant, in the 19th century as their main vegetable food with a reported daily intake of 200-300 grams (Gott, 1984).
Chicory is indigenous to Europe and has been cultivated since the 16th century with the roots and greens (known as Belgian endive) being used for human consumption. Post-World War 11 populations in England and Germany roasted root of the chicory plant to use as an extender or substitute for coffee beans (Meijer et al., 1993). This concept is still in use in the southern United States, particularly Louisiana. A cup of chicory coffee may contain three grams inulin (Douglas & Poll, 1986; Van Loo et al., 1995).
In the South Pacific a variety of yacon was introduced to Japan from New Zealand and grew in popularity (Asami et al., 1989). Native Americans used the Jerusalem artichoke tuber, native to North America, for food. Jerusalem artichokes were found by Champlain at Cape Cod, Massachusetts and were introduced in France in 1605, becoming the main source of carbohydrate in Western Europe until it was superseded by the potato in the middle of the 18th century (Wyse & Wilfahrt, 1982). Jerusalem artichokes were again cultivated as a staple crop by the post-World War II French and Germans due to scarcity of the potato at the time.
II (b). Common Intakes in Diet. Historically, daily inulin intake was estimated to be approximately 25 to 32 grams. Today, the average daily intake of inulin and its hydrolysis products in Western Europe is estimated between 2-12 g/person/day (Roberfroid, Gibson, & Delzenne, 1993). The U.S. consumption, estimated at 2-8 g/person/day, is slightly less based on data from the U.S. Nationwide Food Consumption Survey 1987-88 (Roberfroid et al., 1993).
A more recent USDA study by Moshfegh et al., (1999) showed that American diets provide about 2.6 g of inulin and 2.5 g of oligofructose. Mean intakes varied by gender and age groups with a range from 1.3 grams for young children to 3.5 grams for teenage boys and adult males. Per 1000 calories, mean intake ranged from 0.9 to 1.5 grams in American diets. Significant differences exist between variable sociodemographic categories. Whites who make up 73% of the US population consume significantly more of these inulin-containing components than Blacks or Hispanics (Moshfegh et al., 1999). The primary sources of inulin in American diets are wheat and onions, Figure 2.
Figure 2: Primary vegetable sources of inulin in the American Diet
II. (c). Production. Until recently, inulin as a pure compound was not produced economically on an industrial scale and was not available as a food ingredient for human consumption. Inulin was produced on a pilot scale in Deutsche Kulorfabrik in the early 1920s (Schone 1920), and later was extracted on an industrial scale. In 1927 Belval reported several German sugar factories extracted inulin from chicory, similar to sugar extraction from sugar beets. Like sugar from sugar beets, the extract was high in impurities that were removed by lime-carbon dioxide purification. Chilling precipitation further purified the inulin. However, the production process was deemed uneconomical due to the recovery process using precipitation by chilling (Gibson et al., 1994). Today's process is significantly more efficient and economical.
The primary industrial source of pure inulin is the chicory root (Cichorium intybus L.). Chicory is a member of the Compositx family. Other significant inulin-containing members of this family are dandelion, lettuce, globe and Jerusalem artichoke, dahlia and yacon. Other inulin-containing plants belong to the Liliales family, e.g. leeks, onion, garlic and asparagus. Although chicory has been cultivated for centuries, it still remains quite wild, having only had very minor influence in its genetic basis since its early cultivation occurred in England in 1548. The ancestral form of chicory is a perennial plant (var. silvestre) that probably originated in East India and grew widespread in the Mediterranean areas of Europe and Near Asia. Cultivated root chicory is a biennial plant that is grown as a long season annual. The root is indigenous to Western Europe and was cultivated on a larger scale beginning in the 16th century before being exported to the United States in 1806. Chicory is now a common purplish-blue-flowering weed growing wild in roadway ditches in the United States and Canada.
A distinction is made in cultivation between the three basic forms of chicory. The slightly bitter, curled dandelion-like greens (called Italian dandelion) are grown and used as potherbs. Witloof chicory, Cichorium endivia Linn Endive (also called French endive or Belgian endive) is a perennial herb that is forced as a blanched vegetable and used as a salad delicacy. Root chicory varieties, Cichorium intybus Linn var. sativum, have been used as roasted roots in Europe and North America to impart additional color, body and sedative action as a coffee extender or coffee substitute. In the last ten years, European root chicory cultivation has provided a means to produce high purity fructose syrups and refined inulin products with various chain lengths.
Chicory root is a biennial plant requiring soil and climatic conditions resembling those for sugar beets: deep, fertile, permeable and cool soil with pH tending towards neutral; intolerance of droughts or poor drainage. The best conditions for high yields are mild maritimelike climates, rich light -clay to sandy- soils and long periods of daily illumination, similar to that in Western European countries like France, Holland and Belgium. The plant, being similar to the sugar beet root, shares similarities in agronomic practices and inulin production technologies. The inulin production process involves three general steps: extraction of raw inulin with hot water, purification of the raw inulin and spray drying of the purified juice to a pure inulin powder. Although inulin is spray dried, the molecule is quite flexible, and crystallizes easily (French, 1989). Further outline of the process is given in Figure 3.
Figure 3: Inulin production process
III. BIFIDOGENIC PROPERTIES
The large intestine is the most heavily colonized region of the digestive tract, with up to 1011 bacteria for every gram of intestinal content (Gibson & Roberfroid, 1995). Gut bacteria are comprised of one hundred different species that include both beneficial and potentially deleterious bacteria in a balance that affects how food is digested and energy is obtained, Figure 4, When the main types of generally recognized beneficial bacteria, bifidobacteria and lactobacilli, are at optimum levels they constitute approximately one-third of the bacterial population in the gastrointestinal tract. In some cases the numbers of beneficial bacteria may be so low they are undetectable. The numbers of bifidobacteria are regarded as a marker of the stability of the human intestinal microflora (Mutt & Tanaka, 1987).
Figure 4: Schematic overview of colonic microflora and their health significnce
The importance of an indigenous microflora population as a natural means to fight potential pathogenic microorganisms was recognized by Metchnikoff during research on cholera in the 19th century (Bibel, 1988). Decades later, the importance of a well-balanced gut microbial ecosystem is becoming more widely recognized for host health. Research by Mutai and Tanaka (1987) stimulated further efforts to identify the role of bifidobacteria (Mitsuoka, 1990a; Romond & Romond, 1987), and other lactic acid bacteria, mainly lactobacilli (Salminen et al., 1993a, 1993b; Tannock, 1990) with regard to host health. Miller-Catchpole (1996) noted that bifidobacteria, even though implicated in incidental opportunistic anaerobic infections (as in individuals with weakened immune systems), are generally regarded as safe. Stimulation of bifidobacteria numbers as well as the numbers of lactobacilli in the colon is beneficial to host health.
Populations of bifidobacteria can represent up to 95% of the total intestinal microflora in breast-fed infants, in comparison with about 25% in the adult (Gibson, 1995). The mothers delivery canal and fecal flora inoculate the gastrointestinal tract of the newborn during birth (Gibson & Roberfroid, 1995). Drasar and Roberts (1989) recognized that fecal flora of breast-fed infants is dominated by populations of bifidobacteria, with only one percent enterobacteria. By contrast, formula-fed infants have a more complex microflora, with bifidobacteria, bacteroides, clostridia and streptococci all prevalent (Gibson & Roberfroid 1995). Although bifidobacteria have been considered to be the most important organisms in infants, and lactobacilli and E. coli are more numerous bacteria for children and adults than bifidobacteria it has now become clear that bifidobacteria also constitute one of the major organisms in the colonic flora of healthy children and adults (Mitsuoka, 1990b). The fecal material of children and adults have bacteroidaceae, eubacteria and peptococcaceae that outnumber bifidobacteria, which constitute 5 to 10% of the total flora. The numbers of enterobacteriaceae and streptococci in children and adults are also less than bacteroides, eubacteria, peptocococceae and bifdobacteria decreasing to less than 108 CFU per gram of fecal material. lactobacilli and veillonellae are also present in children and adult fecal material, but in numbers that usually are less than 108 CFU per gram (Mitsuoka, 1990b). In elderly persons bilidobaderia decrease or completely disappear, putrefactive bacteria like clostridia including C. perfringens, significantly increase. In addition, lactobacilli, streptococci, and enterobacteriaceae also increase, Figure 5.
Figure 5, Changes in the fecal Flora with increased age (from Mitsuoka, 1990b).
Thus, it is thought that presence of these healthy microorganisms in breast-fed infants contributes to their alleged health advantages compared to formula-fed infants, and the modification of elderly and maintenance of weaned bifidobacteria microflora patterns may provide a means to maintain health and potentially prevent some gut-home diseases. Following weaning, the microflora pattern in breast-fed infants begins to resemble that of adults. As a consequence to findings by Mutai and Taankk (1987); Romond and Romond (1987); Drasar and Roberts (1989); and Mitsuoka (1990a) and a number of other researchers, scientists now generally ascribe to the beneficial health effects of bifidobacteria in the colon, Figure 6.
Figure 6: colonic bacteria and SCHFA related health effects of bifidobacteria
Predominant gut microflora generally recognized as producing health promoting inactions, are the Bacteroides, Bifidobacterium, lactobacilli, and Eubacterium (Gibson &. Roberfroid, 1995). These beneficial microflora particularly the bifidobacteria and lactobacilli, may play critical roles in all aspects of our immunological responses, by either helping to resist infection, or by creating conditions which reduce the number of pathogenic bacteria. These beneficial bacteria may act as wards regulating the activity of the other bacteria in the colon. The other bacteria, such as Salmonella, Shigella, Clostridia, Staphylococcus aureaus, Candida albicans, Campylobacter jejuni, Escherichia coli, Veillonella, and Klebsiella, have varying potential to cause disease and are much less numerous. However, these pathogenic bacteria and several strains of yeasts, most notably Candida albicans, can produce harmful local and systemic effects if they overgrow as a consequence of a gut microflora imbalance (Elmer et a1., 1996). Research has shown beneficial bacteria, particularly bifidobacteria and lactobacilli, keep these potential disease-causing organisms under control, preventing several disease-related dysfunctions related to an imbalances GI situation (Elmer et a1., 1996).
Bifidobacteria, while significantly influencing other colonic microflora, are also affected by many exogenous and endogenous factors. Factors affecting intestinal flora observed by Rasic (1983) were: animal species; habits; age; sex; climate; diet; stress; exogenous organisms; and immune mechanisms of the host. Other modifers of the gut microflora are surgery of the stomach or small intestine, kidney or liver disease, cancer, pernicious anemia, blind loop syndrome or a change in the acidity of gastric juices (Modler et al., 1990). In addition, other pathogenic disorders, such as liver cirrhosis and impaired intestinal motility may modify colonic microflora. During stages of acute infection, antibiotic therapy used to combat the bacterial invasion can also induce digestive disorders due to alteration of normal gut flora. Bifidobacteria have been shown to be resistant to streptomycin, but have only moderate resistance to penicillin, tetracycline, neomycin and novobiocin (Kurmann and Rasic, 1991). Bifidobacteria can be completely eradicated from the colon when antibiotics such as erythromycin, spiramycin and chloramphenicol are used to combat other bacterial infections.
Reducing or eliminating more of the healthy gut microflora, like bifidobacteria, has its consequences. When the human diet influences the species composition and metabolic characteristics of the intestinal microflora, toxic metabolite production is affected, such as the conversion of procarcinogens to active carcinogens (Perman, 1989; Roland et al., 1993; Rowland, 1988). E. coli and clostridia are known to produce ammonia and amines, both liver toxins, and carcinogens and cancer promoters such as nitrosamines, phenols, cresols, indole and skatole, estrogens, secondary bile acids and aglycones. Chadwick and coworkers (1992) reported reductive enzyme activities are lower in bifidobacteria and lactobacilli relative to E. coli and clostridia. Bacteroides are generally thought to be health promoting, while Enterococcus faecalis produce nitrosamines, aglycones and secondary bile acids, and Proteus produces ammonia, amines and indole (Drasar & Hill, 1974; Kanbe, 1988; Koizumi et al., 1980; Mitsuoka, 1982, 1990a). In addition to producing toxic metabolites, several harmful bacteria, such as Salmonella, Shigella, Listeria, Bacteroides, Proteus, E. coli, Clostridium perfringens and Vibrio cholerae also have association with diarrhea, infections, liver damage, carcinogenesis and intestinal putrefaction. It is possible the health promoting effects prompted by bifidobacteria and other healthful bacteria is due to the growth inhibition of harmful bacteria, stimulation of immune functions, lowering of gas distention problems, improved digestion/absorption of essential nutrients and synthesis of vitamins (Gibson, 1995; Gibson & Roberfroid, 1995; Roberfroid & Delzenne, 1993).
Pathogenic effects associated with harmful intestinal microflora such as E. coli, Clostridia, Bacteroides, Proteus, Salmonella, Shigella, and Vibrio cholerae not only include colonic disorders but also have implication with possible vaginal infections and systemic disorders. Intestinal pathologies include antibiotic-associated diarrhea (AAD), inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, colorectal cancer, necrotizing entercolitis, and ileocecitis. Vaginal infections include candidal vaginitis while systemic disorders include gut origin septicemia, pancreatitis and multiple organ failure syndrome (Gibson & Roberfroid, 1995), Figure 7. Major factors in the biology of these disorders are the overgrowth of pathogenic bacteria such as clostridia, E. coli, as well as parasites, viral infections, extensive bum injury, post-operative stress, and antibiotic therapy. These disorders are often associated with bacterial translocation due to intestinal barrier failure (Gardiner et al., 1993; Gibson & MacFarlane, 1994; Solomons, 1993).
Figure 7: Pathogenic microflora suppression, from Roberfroid et al. 1998 and Rolfe 1981
Mechanisms related to microflora modification to a more-healthy environment vary for individual microflora groups. However, the antagonistic effects of lactic acid bacteria have been attributed to favorable competition for active sites on the colonic epithelial wall, the production of primary metabolites, such as acetic, lactic, propionic, butyric and benzoic acids, and hydrogen peroxide and the secretion of specific bacteriocins, e.g. Lactacins B,F and Lactocin 27 (Gibson and Wang, 1994c; Modler et al., 1990). Numerous other investigators have also reported the ability of lactic acid bacteria to produce antibacterial substances, which are active against certain pathogenic and putrefactive organisms (Mehta et al., 1983). Some Streptococcus spp. have also been shown to produce bactericidal substances (Miller-Catchpole, 1989). L. acidophilus strains produce three antibiotic substances, namely acidolin, acidophilin and lactocidin; L. bulgaricus produces one antibiotic substance, bulgarican.
Although bifidobacteria do not produce hydrogen peroxide, observations by Gibson and Roberfroid (1995) hint that bifidobacteria might secrete a bacteriocin-type substance that is active against Clostridia, E, coli, and many other pathogenic bacteria, such as Listeria, Shigella, Salmonella, and Vibrio cholerae, or antimicrobal substances. Anand and coworkers (1985) tested six strains of B. bifidum for their antibacterial activity and reported that antibacterial activity differed among the strains with maximum inhibitory action shown by one strain against M. flavus followed by Staph. aureus, B. cereus, E. coli, Ps. flourescens, S. typhosa and Sh. dysenteriae.
Like lactobacilli, bifidobacteria produce strong acids, i.e. acetic and lactic acid (Scardovi, 1986). The production of these acids reduces intestinal pH. One effect of lowering the gastrointestinal pH might be the protonation of toxic ammomia (NH3) to produce ammonium ion (NH4+), which is non-diffusable and could result in lower blood ammonia levels and a reduced hepatic load (Levrat et al., 1993; Miller-Catchpole, 1989). An additional, and potentially more important effect is restriction or prohibition of the growth of many potential pathogens and putrefactive bacteria.
Acetic acid has been observed to exert a greater antimicrobial effect than lactic acid, most likely due to a greater amount of undissociated acid at intestinal pH values (5.8) common to bifidobacteria and lactobacilli (Modler et al., 1990). Because bifidobacteria produce almost two-fold more acetate than lactate, the undissociated acetic acid would be approximately 11 -fold greater than lactate. This is an important factor as the growth of many potential pathogenic bacteria is very sensitive to concentrations of undissociated acid (Modler et al., 1990).
Scardovi (1986) suggested the optimum pH for bifidobacteria is between 6.5 and 7.0 with little or no growth below the pH range of 4.5 to 5. 0 or above 8. 0 to 8.5. Wang and Gibson (1993) observed that specific growth rates of B. infantis, E. coli and Cl. perfringens were approximately equal at neutral pH. As the pH was lowered the bifidobacteria growth rate remained relatively unaffected while the growth of E. coli and Cl. Perfringens was completely inhibited at pH 5.0 and 4.5.
Bifidobacteria do not form aliphatic amines, hydrogen sulfide or nitrites (Bezkorovainy & Miller-Catchpole 1989). They produce vitamins, largely of the B-group, such as biotin, thiamine, riboflavin, niacin, pyridoxine, cyanocobalamin, and folic acid (Deguchi et al., 1985; Gibson et al., 1995; Hartemink et al., 1994). These bacteria also produce digestive enzymes such as lactase (B-galactosidase), casein phosphatase and lysozyme that may improve lactose tolerance and digestibility of dairy products (Hughes & Hoover, 1991).
III. (a). Probiotics and Prebiotics To minimize the potential for imbalances in gut microflora which could lead to intestinal, systemic and, possibly, vaginal infections, researchers have investigated various means of achieving a greater population of healthy gut microflora. There is growing evidence that live selected bacteria, such as bifidobacteria and lactobacilli, when added to food (such as fermented milk beverages or yogurt) or used as a dietary aid, are beneficial for these purposes. Probiotics are defined as living microbial feed supplements added to the diet, which have beneficial effects on the host by improving its intestinal microflora balance (Fuller, 1989). In humans, lactobacilli, either as single species or in mixed culture with other bacteria such as bifidobacteria and streptococci, are common probiotics (Gibson, 1995).
When probiotics are added to the diet as a large bowel target to bring about microflora balance, these organisms must reach their intended destination intact and become viable. There is evidence that some probiotics added in the diet are able to reach the colon and provide health benefits (Elmer et al., 1996; Kageyama et al., 1984; Pochart et al., 1992). However, due to fluctuating activities in response to substrate availability, redox potential, pH, oxygen tension and colonic distribution, the survivability and effectiveness of ingesting living microorganisms for purposes of targeting colon microflora modulation is variable.
In addition, incorporation of healthy viable bacteria into processed foods is also somewhat difficult because of their high susceptibility to oxygen, shear, heat, and pH sensitivity (Tomomatsu, 1994). Intake of food as a bolus and the development of species that are more oxygen and acid-resistant is probably of importance. Further, due to competition for nutrient sources and colonization sites with previously well established endogenous microflora, individual probiotic fixation and activities are strain-dependent. There is also evidence that probiotic effects are transient. When consumption of the probiotic product ceases, the added bacteria are excreted (Bouhnik et al., 1992). However, in order for bifidobacteria to benefit host health, it is necessary that these organisms be metabolically active in the lower gastrointestinal tract (Hashimoto, 1985; Tamura, 1983).
As a prerequisite to their survivability, these bacteria require a carbohydrate source to use for fermentation that has not been metabolized by the human digestive system before reaching the colon. Selective non-digestible carbohydrate food sources that promote the proliferation of bifidobacteria and lactobacilli have been defined as prebiotics (Gibson and Roberfroid, 1995). Prebiotics that would have potential use, as ingredients in foods should be non-digestible, very shelf stable, require no refrigeration, be easily and effectively incorporated into processed foods and nourish all endogenous beneficial bacteria. The combined form of a probiotic and prebiotic, as described by Gibson and Roberfroid (1995) is termed a synbiotic. In a review, Fuller and Gibson (1997) discuss a variety of mechanisms that may be responsible for the beneficial effects of pro- and prebiotic supplements. A comparison of the terms probiotic, prebiotic and synbiotic is presented in Table 2 (Gibson and McCartney, 1998).
Table 2: Gut microflora definitions & mechanisms of gut modulation, Gibson & McCartney 1998
Various in vitro and in vivo studies have shown that a diet supplemented with B(2-1) inulin/FOS provides an effective means to promote growth of bifidobacteria and lactobacilli, while selectively reducing the growth of pathogenic microorganisms and potentially treating intestinal dysfunctions (Cummings et al., 1997; Gibson & Roberfroid, 1995; Gibson & Wang, 1994a, 1994b, 1994c; Gibson et al., 1995; Hartemink et al., 1997; Kleessen et al., 1994; Kleessen et al., 1997; Mitsuoka et al., 1987; Roberfroid et al., 1998; Rowland & Tanaka, 1993; Terada et al., 1993; Wang & Gibson, 1993; Williams et al., 1994). As a consequence of a European Commission-funded project on non-digestible oligosaccharides, the ENDO project (DGXII AIRII-CT94-1095) (van Loo, et al., 1999) it was determined by consensus that there is now strong scientific evidence, based on observations with over 100 volunteers differing in sex, age, race and dietary habits, that B(2-->1)-type fructans are prebiotic (Roberfroid et al., 1998). In addition, inulin used as a general human gastrointestinal aid (Paul, 1996), as a method of treating ulcerative colitis (Garleb & Demichele, 1995) and to inhibit C. difficile infections (Garleb et al., 1997) are defined in United States patents.
Several researchers have reported the selective fermentation of inulin from a variety of sources and production processes as well as other NDO using in vitro and in vivo studies and pure cultures of human colon microflora. Results from many of these researchers are summarized in
In review, Modler et al. (1990) and Roberfroid (1993) defined inulin as a good substrate for most Bacteroides and Bifidobacterium species, except Bifidobacterium bifidum and some strains of B. longum. Chain length is an important determinant for some specific species, due to their inherent inulinase activity. McBain and Macfarlane (1997) used a three-stage fermentation system to reproduce several nutritional and environmental characteristics of the proximal large intestine and the distal colon. Bifidobacteria populations were stimulated within three hours of FOS addition increasing by approximately 10-fold with 12 hours. Total anaerobic populations and the B. fragilis group also increased as did peptostreptococci and enterococcal numbers.
