**3. Prebiotics**

32 New Advances in the Basic and Clinical Gastroenterology

Progress towards elimination of use of organic solvents has been made with development of microencapsulation technique using supercritical technology (Moolman et al., 2006). This microencapsulation technique is based on the formation of an interpolymer complex between poly (vinyl pyrrolidone) (PVP) and poly (vinyl acetate-co-crotonic acid) (pVA-CA) in supercritical carbon dioxide (scCO2). A supercritical substance is neither a gas nor a liquid but possesses properties of both, making it unique (Moshashaee et al., 2000). Since supercritical fluids have a wide spectrum of solvent characteristics, they can be used as solvents in different techniques (Frederiksen et al., 1997). Microparticles produced using this method have suitable morphological characteristics, encapsulation efficiency and affords encapsulated probiotic cultures protection in simulated gastrointestinal fluids (Mamvura et

Although there are numerous advantages and health benefits associated with probiotics or probiotic food products, there are risks associated with probiotic therapy. These risks are mainly concerned with respect to safety in vulnerable target groups such as immunocompromised individuals (pregnant women, babies and the elderly) or critically ill

Probiotic cultures are resistant to some antibiotics. There is concern about the possible transfer of antimicrobial resistance from probiotic strains to pathogenic bacteria in the gut. For example, many *Lactobacillus* strains are naturally resistant to vancomycin, which poses a potential threat of transfer of this resistance to other pathogenic bacteria such as *Staphylococcus aureus*. However, these vancomycin-resistant genes in lactobacilli are

Another important area of concern is the risk of sepsis. There have been several reports of cases of *Lactobacillus* sepsis and other bacterial sepsis due to the intake of probiotic supplements (Boyle et al., 2006). One case included a 67 year-old man who was taking probiotic capsules daily for mitral regurgitation and developed *Lactobacillus rhamnosus* endocarditis after a dental procedure (Borriello et al., 2003; Mackay et al., 1999). In another case, a 4-month old infant with antibiotic-associated diarrhoea, who was given *Lactobacillus rhamnosus* after cardiac surgery, developed *Lactobacillus* endocarditis 3 weeks after *Lactobacillus rhamnosus* treatment (Boyle et al., 2006; Kunz et al., 2004). However, there have been no reports to date on the occurrence of *Bifidobacterium* sepsis. All cases of bacterial sepsis from the use of probiotics (*Lactobacillus* spp.) have occurred in immunocompromised individuals or patients who have a chronic disease or debilitation. No cases have been reported in healthy individuals (Boyle et al., 2006). There have also been several cases of fungemia associated with *Saccharomyces boulardii*. However, investigation of these cases revealed that the infection was due to contamination of inserted catheters. It is therefore now recommended that *Saccharomyces boulardii* probiotics be prepared in powdered form under stringent hygienic conditions to prevent contamination (Borriello et al., 2003; Salminen et al., 1998). There is a small risk of adverse metabolic effects from manipulation of the microbiota with the use of probiotics, although probiotic studies to date have not shown significant adverse effects on growth or nutrition (Boyle et al., 2006). A review of safety

or hospitalized patients (Boyle et al., 2006; Jankovic et al., 2010).

chromozomal and not readily transferred to other species.

assessments of probiotics was recently published (Sanders et al., 2010).

al., 2011; Thantsha et al., 2009).

**2.9 Concerns about probiotics** 

Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of colonic bacteria and thereby improve host health (Femia et al., 2002; Gibson & Roberfroid, 1995; Roberfroid, 1998; Theuer et al., 1998; Tuohy et al., 2003; Young, 1998). Gibson et al. (2004) redefined them as 'a selectively fermented ingredient that allows specific changes; both in the composition and/or activity in the gastrointestinal microflora that confers benefits to host well-being and health'. The refined definition takes into account both the microbial changes and the nutritional and physiological benefits attributed to prebiotics. Just like probiotics, they modulate the composition of the natural ecosystem though probiotics does so through introduction of exogenous bacteria into the colon (Bouhnik et al., 2004). Prebiotics pass through the upper GIT unfermented and are only utilized in the colon and are therefore called 'colonic food' (Roberfroid, 2000). Non-digestibility can be demonstrated *in vitro* by subjecting the carbohydrates to pancreatic and small intestinal enzymes. It can be shown *in vivo* on human subjects with an ileostomy (i.e people who have had their large intestine removed and have a stoma at the end of the ileum) (van Loo et al., 1999). Compounds that are not digested and absorbed by the host but are preferentially fermented by *Bifidobacterium* species in the colon are called 'bifidogenic' factors (Shah, 2007). Prebiotics are not readily digested by pathogenic bacteria (Annika et al., 2002; Farmer, 2002; Femia et al., 2002). They favour or promote growth of potentially health-promoting bacteria such as lactobacilli and bifidobacteria, thereby allowing them to be predominant (Bouhnik et al., 2004; Flamm et al., 2001; Gibson et al., 1999; Roberfroid, 1998; Wang, 2009). This subsequently leads to predominant numbers of the stimulated endogenous bacteria in faeces as well (Femia et al., 2002; Losada & Olleros, 2002). Scientific studies in Japan indicated that consumption of prebiotics increases the populations of bifidobacteria and other beneficial microorganisms even in the absence of probiotics in diet. The selective stimulation of growth of bifidobacteria by prebiotics is characterized by a substantial decrease in numbers of potentially pathogenic bacteria (Losada & Olleros, 2002).

The following criteria are used for selection of a carbohydrate as a prebiotic: It must not be absorbed in the stomach or small intestine; It must be selectively fermented by the beneficial gut microflora; It should also stimulate the growth and/or activity of beneficial bacteria; Its fermentation should induce the beneficial luminal or systemic effects within the host; It must be resistant to gastric acidity and mammalian enzyme hydrolysis (Kolida, et al., 2002; Manning and Gibson, 2004).

Classes of non-digestible oligosaccharides (NDOs) commercially available are cyclodextrins, fructooligosaccharides (FOS), gentiooligosaccharides, glycosylsucrose and isomaltulose (also known as palatinose). Other classes include lactulose, lactosucrose and maltooligosaccharides (Sako et al., 1999). NDOs such as inulin, fructooligosaccharide, lactulose and dietary fibre are common prebiotics (Davis & Milner, 2009). There are conflicting views on the prebiotic classification of resistant starch. Some researchers classify it as a prebiotic (Douglas & Sanders, 2008) while others (Shah, 2004) differ, arguing that resistant starch is not digested by some beneficial bacteria, and therefore cannot be classified as a prebiotic. Inulin and FOS are the only NDOs that have been sufficiently studied to give adequate data to analyze their functional properties (Roberfroid, 2000). Galacto-oligosaccharides (GOS), glucooligosaccharides, lactulose, isomalto-oligosaccharides, raffinose, transgalacto-oligosaccharides,

Probiotics – What They Are, Their Benefits and Challenges 35

intestine and act as a substrate for bifidobacteria (Frost & Sullivan, 2000). Soy oligosaccharides are extracted directly from soybean whey. Their bifidogenecity has been

In the last decade there have been numerous investigations on the health-promoting effects of prebiotics. Bifidogenic oligosaccharides increase the level of nutrient supplementation and enhance nutrient solubility (Farmer, 2002). Some of these effects include better mineral absorption, alleviation of constipation and irritable bowel syndrome, protection against colon cancer, enhancement of the immune system, anticarcinogenic effects and lowering of cholesterol (Davis & Milner, 2009; Manning & Gibson, 2004; Tuohy et al., 2003; Venter, 2007). Supplementation of diet with oligofructose and inulin-type oligosaccharides significantly lowers serum triglycerols and phospholipids. This hypotriacylyglycerolaemic effect could be caused by a decrease in the concentration of plasma very low-density lipoproteins (VLDL) (Delzenne et al., 1993; Fordaliso et al., 1995). Prebiotics acidifies colonic contents by increasing the concentration of short-chain carboxylic acids. They also aid in colonic absorption of minerals, particularly Mg2+ and Ca2+. Mineral absorption is promoted through establishment of an osmotic effect whereby entry of water into the colon increases

Ingestion of prebiotics is also associated with relief of constipation due to faecal bulking and possible effects on intestinal motility, aiming at daily defecation. They suppress diarrhoea when it is associated with intestinal infections. They also reduce the risk of osteoporosis when improved bioavailability of calcium due to use of inulin-type fructans is followed by significant increase in bone density and bone mineral content. Prebiotics reduce the risk of

Most of the studies on prebiotic effects on bone development have been done on animal models, particularly rats (Scholz-Ahrens, 2007). *In vivo* studies carried out on humans showed that inulin and oligofructose increase the absorption of calcium but not iron, zinc or magnesium (Coudray et al., 1997). More research needs to be done to substantiate these claims, including that of bowel cancer prevention (Ziemer & Gibson, 1996). Studies that have been carried out on animal models so far have shown promise, though human studies are required (Reddy et al., 1997; Rowland et al., 1998). It is vital for studies conducted *in vitro* on prebiotic effects to be carried out *in vivo* in well-designed and reproducible

Prebiotics, unlike probiotics, are not living organisms, and therefore they do not have survival problems both in the products and the gut (Frost & Sullivan, 2000). Prebiotics have an advantage over probiotics in that they are not affected by factors such as oxygen and heat in industrial applications. This attribute led to increased interest in their health issues (Kolita et al., 2002). Some commercially available prebiotic supplements are water-soluble. Solubility in water allows their possible incorporation in any type of food and also renders

However their excess levels can cause symptoms such as flatulence, bloating and diarrhoea. This may be caused by a change in osmotic potential or due to excessive fermentation.

confirmed in humans (Frost & Sullivan, 2000).

**3.1 Beneficial effects of prebiotics** 

dissolution of minerals (Roberfroid, 2000).

experiments.

obesity and possibly type-2 diabetes (van Loo et al., 1999).

them undetectable once dissolved (Douglas & Sanders, 2008).

xylo-oligosaccharides, soya bean oligosaccharides and oat β-glucans are considered as prebiotic candidates (Lomax & Calder, 2009). Other NGOs such as lactulose (Crittenden & Playne, 1996) and xylobiose (Vazquez et al., 2000) are also included in the prebiotic category. All the prebiotic candidates except maltooligosaccharides, glycosylsucrose and cyclodextrins are reported as being bifidogenic (Ziemer & Gibson, 1998).

Prebiotics are found naturally in a number of materials. Honey, fruits and vegetables such as artichoke, asparagus, banana, barley, chicory, garlic, leeks, oats, onion, rye, soybeans, tomatoes and wheat are sources of non-digestible oligosaccharides, especially inulin (Davis & Milner, 2009; Femia et al., 2002; Losada & Olleros, 2002; Manning & Gibson, 2004; Mussatto & Mancilha, 2007; Sangeetha et al., 2005). They are also present in burdock, Chinese chives, garlic, graminae (fodder grass), pine, even bacteria and yeasts (Bengmark et al., 2001; Ziemer & Gibson, 1998). Honey and bamboo shoots are natural sources of isomaltulose (Lina et al., 2002). Raffinose and stachyose can be found in soyabeans and other leguminous seeds and pulses (Voragen, 1998). Milk is a good source of glycoproteins and oligosaccharides (lacto-N-tetraose and lacto-N-neotetraose), including those believed to be prebiotic (Alander et al., 2001; Newburg, 2000; Petschow & Talbott, 1991). Human milk contains more galactooligosaccharides than cow's milk. Oligosaccharides can be found in levels as high as (12g/l), making them the third bulkiest constituent of human milk (Newburg et al., 2004). Taking foods containing prebiotic oligosaccharides is not enough for modulation of gut flora as they are present in only small concentrations in these foods. Instead, prebiotics are extracted from these foods and transferred into more commonly ingested foodstuffs like biscuits and other carbohydrate based materials (Taylor et al., 1999).

These natural compounds can also be produced commercially through enzyme hydrolysis and extraction processes (Losada & Olleros, 2002; Mussatto & Mancilha, 2007; Sako et al., 1999). The enzymes employed include β-D-fructofuranosidase or fructosyltransferase, which joins the fructose molecules by means of transfructosylation mechanisms (Losada & Olleros, 2002). These mechanisms are employed for production of oligosaccharides with the exception of raffinose, soybean oligosaccharides and lactulose (Mussatto & Mancilha, 2007; Sako et al., 1999). Raffinose and soybean oligosaccharides are extracted directly from plant materials using solvents such as water, aqueous methanol or ethanol (Johansen et al., 1996; Mussatto & Mancilha, 2007). Lactulose is produced by enzymatic action of β-galactosidases (E.C 3.2.1.23) on lactose. The glucose moiety is converted to a fructose residue by alkali isomerization and the process results in a lactulose disaccharide (Villamiel et al., 2002). Inulin can also be extracted from chicory (*Cichorium intybus*) and then partially hydrolyzed to short-chain fructans (oligofructose) using inulase (E.C 3.2.1.7) or to long chain fructans by applying an industrial separation technique (Roberfroid, 2000). Additionally, inulin type fructans can be manufactured through transfer of fructosyl residue to and between sucrose molecules using fungal fructosyl transferases (E.C 2.4.1.9) (Cummings & Roberfroid, 1997). FOSs are manufactured from sucrose using the trans-fructosylation activity of βfructofuranosidase (E.C 3.2.1.26). Alternatively, FOS can are produced by controlled enzymatic hydrolysis of inulin (Crittenden & Playne, 1996; Frost & Sullivan, 2000). Lactose is also used in the industrial production of GOS through the transgalactosylation activity of β-galactosidases (Sako et al., 1999). The production of FOS and GOS requires high concentrations of the starting material for efficient transglycosylation (Park & Almeida, 1991). GOS are synthesized from lactose syrup using the enzyme β-galactosidase (Frost & Sullivan, 2000; Gibson, 2004). They are neither hydrolyzed nor absorbed in the human intestine and act as a substrate for bifidobacteria (Frost & Sullivan, 2000). Soy oligosaccharides are extracted directly from soybean whey. Their bifidogenecity has been confirmed in humans (Frost & Sullivan, 2000).

#### **3.1 Beneficial effects of prebiotics**

34 New Advances in the Basic and Clinical Gastroenterology

xylo-oligosaccharides, soya bean oligosaccharides and oat β-glucans are considered as prebiotic candidates (Lomax & Calder, 2009). Other NGOs such as lactulose (Crittenden & Playne, 1996) and xylobiose (Vazquez et al., 2000) are also included in the prebiotic category. All the prebiotic candidates except maltooligosaccharides, glycosylsucrose and cyclodextrins

Prebiotics are found naturally in a number of materials. Honey, fruits and vegetables such as artichoke, asparagus, banana, barley, chicory, garlic, leeks, oats, onion, rye, soybeans, tomatoes and wheat are sources of non-digestible oligosaccharides, especially inulin (Davis & Milner, 2009; Femia et al., 2002; Losada & Olleros, 2002; Manning & Gibson, 2004; Mussatto & Mancilha, 2007; Sangeetha et al., 2005). They are also present in burdock, Chinese chives, garlic, graminae (fodder grass), pine, even bacteria and yeasts (Bengmark et al., 2001; Ziemer & Gibson, 1998). Honey and bamboo shoots are natural sources of isomaltulose (Lina et al., 2002). Raffinose and stachyose can be found in soyabeans and other leguminous seeds and pulses (Voragen, 1998). Milk is a good source of glycoproteins and oligosaccharides (lacto-N-tetraose and lacto-N-neotetraose), including those believed to be prebiotic (Alander et al., 2001; Newburg, 2000; Petschow & Talbott, 1991). Human milk contains more galactooligosaccharides than cow's milk. Oligosaccharides can be found in levels as high as (12g/l), making them the third bulkiest constituent of human milk (Newburg et al., 2004). Taking foods containing prebiotic oligosaccharides is not enough for modulation of gut flora as they are present in only small concentrations in these foods. Instead, prebiotics are extracted from these foods and transferred into more commonly ingested foodstuffs like biscuits and other carbohydrate based materials (Taylor et al., 1999). These natural compounds can also be produced commercially through enzyme hydrolysis and extraction processes (Losada & Olleros, 2002; Mussatto & Mancilha, 2007; Sako et al., 1999). The enzymes employed include β-D-fructofuranosidase or fructosyltransferase, which joins the fructose molecules by means of transfructosylation mechanisms (Losada & Olleros, 2002). These mechanisms are employed for production of oligosaccharides with the exception of raffinose, soybean oligosaccharides and lactulose (Mussatto & Mancilha, 2007; Sako et al., 1999). Raffinose and soybean oligosaccharides are extracted directly from plant materials using solvents such as water, aqueous methanol or ethanol (Johansen et al., 1996; Mussatto & Mancilha, 2007). Lactulose is produced by enzymatic action of β-galactosidases (E.C 3.2.1.23) on lactose. The glucose moiety is converted to a fructose residue by alkali isomerization and the process results in a lactulose disaccharide (Villamiel et al., 2002). Inulin can also be extracted from chicory (*Cichorium intybus*) and then partially hydrolyzed to short-chain fructans (oligofructose) using inulase (E.C 3.2.1.7) or to long chain fructans by applying an industrial separation technique (Roberfroid, 2000). Additionally, inulin type fructans can be manufactured through transfer of fructosyl residue to and between sucrose molecules using fungal fructosyl transferases (E.C 2.4.1.9) (Cummings & Roberfroid, 1997). FOSs are manufactured from sucrose using the trans-fructosylation activity of βfructofuranosidase (E.C 3.2.1.26). Alternatively, FOS can are produced by controlled enzymatic hydrolysis of inulin (Crittenden & Playne, 1996; Frost & Sullivan, 2000). Lactose is also used in the industrial production of GOS through the transgalactosylation activity of β-galactosidases (Sako et al., 1999). The production of FOS and GOS requires high concentrations of the starting material for efficient transglycosylation (Park & Almeida, 1991). GOS are synthesized from lactose syrup using the enzyme β-galactosidase (Frost & Sullivan, 2000; Gibson, 2004). They are neither hydrolyzed nor absorbed in the human

are reported as being bifidogenic (Ziemer & Gibson, 1998).

In the last decade there have been numerous investigations on the health-promoting effects of prebiotics. Bifidogenic oligosaccharides increase the level of nutrient supplementation and enhance nutrient solubility (Farmer, 2002). Some of these effects include better mineral absorption, alleviation of constipation and irritable bowel syndrome, protection against colon cancer, enhancement of the immune system, anticarcinogenic effects and lowering of cholesterol (Davis & Milner, 2009; Manning & Gibson, 2004; Tuohy et al., 2003; Venter, 2007). Supplementation of diet with oligofructose and inulin-type oligosaccharides significantly lowers serum triglycerols and phospholipids. This hypotriacylyglycerolaemic effect could be caused by a decrease in the concentration of plasma very low-density lipoproteins (VLDL) (Delzenne et al., 1993; Fordaliso et al., 1995). Prebiotics acidifies colonic contents by increasing the concentration of short-chain carboxylic acids. They also aid in colonic absorption of minerals, particularly Mg2+ and Ca2+. Mineral absorption is promoted through establishment of an osmotic effect whereby entry of water into the colon increases dissolution of minerals (Roberfroid, 2000).

Ingestion of prebiotics is also associated with relief of constipation due to faecal bulking and possible effects on intestinal motility, aiming at daily defecation. They suppress diarrhoea when it is associated with intestinal infections. They also reduce the risk of osteoporosis when improved bioavailability of calcium due to use of inulin-type fructans is followed by significant increase in bone density and bone mineral content. Prebiotics reduce the risk of obesity and possibly type-2 diabetes (van Loo et al., 1999).

Most of the studies on prebiotic effects on bone development have been done on animal models, particularly rats (Scholz-Ahrens, 2007). *In vivo* studies carried out on humans showed that inulin and oligofructose increase the absorption of calcium but not iron, zinc or magnesium (Coudray et al., 1997). More research needs to be done to substantiate these claims, including that of bowel cancer prevention (Ziemer & Gibson, 1996). Studies that have been carried out on animal models so far have shown promise, though human studies are required (Reddy et al., 1997; Rowland et al., 1998). It is vital for studies conducted *in vitro* on prebiotic effects to be carried out *in vivo* in well-designed and reproducible experiments.

Prebiotics, unlike probiotics, are not living organisms, and therefore they do not have survival problems both in the products and the gut (Frost & Sullivan, 2000). Prebiotics have an advantage over probiotics in that they are not affected by factors such as oxygen and heat in industrial applications. This attribute led to increased interest in their health issues (Kolita et al., 2002). Some commercially available prebiotic supplements are water-soluble. Solubility in water allows their possible incorporation in any type of food and also renders them undetectable once dissolved (Douglas & Sanders, 2008).

However their excess levels can cause symptoms such as flatulence, bloating and diarrhoea. This may be caused by a change in osmotic potential or due to excessive fermentation.

Probiotics – What They Are, Their Benefits and Challenges 37

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Undesirable effects occur when very high doses are ingested. This is advantageous as it allows a relatively broad "therapeutic window", i.e. the dose above the minimal effective level (Holzapfel & Schillinger, 2002). However, FOSs are slightly laxative and produce flatulence when taken in high doses (Losada & Olleros, 2002).
