FERMENTATIVE PRODUCTION OF PREBIOTICS

1. ABSTRACT: The production and applications of food-grade carbohydrates are increasing rapidly. Amongst them, Oligosaccharides represent one of the major classes in terms of production. They are relatively new functional food ingredients that have great potential as Prebiotics. They are having a number of desirable characteristics which are beneficial to the health of consumers. These are manufactured by transfructosylation of sucrose using fructosyltransferases in case fructooligosaccharides and by transgalactosylation of Lactose using -galactosidases in case of galactoosligosacchrides. This review focuses on the recent developments in the production of oligosaccharides, their purification and characterization. Keywords: Aspergillus, -galactosidases,Fructosyltransferase,Oligosaccharides, Prebiotic.

INTRODUCTION:

Prebiotics are digestion-resistant carbohydrates that selectively stimulate the growth and activity of health-promoting microorganisms such as bifidobacteria and lactobacilli. Major prebiotics include fructooligosacchrides,

galactooligosaccharides, inulin and lactulose. Prebiotics taken alone or with probiotics, as in a symbiotic supplement, contribute to the integrity of the gut barrier, help normalize colonic motility, improve nutrient bioavailability, enhance gastrointestinal and systemic immunity, and may favourably modulate blood sugar and lipid levels. Numerous studies in both animals and humans have demonstrated the health benefits of prebiotics. Prebiotic use in nutritional supplements and functional foods is rapidly gaining wide acceptance. (Klair Labs) Prebiotic offers following Health benefits:
Promotion of Normal Colon Transit Time Production of Short-Chain Fatty Acids Improved Gut Mucosal Barrier & Immune Function Enhancement of Mineral Absorption 1

 Favourable Modulation of Lipid Levels Reduction in Colon Cancer Risk

It is necessary to establish clear criteria for classifying a food ingredient as a prebiotic (Roberfroidet al., 2008b). Indeed, such classification requires a sciencebaseddemonstration that the ingredient: Resists gastric acidity Isnot hydrolysed by GIT enzymes Is not absorbed in the upper GIT Is fermented by intestinal microorganisms Induces selective stimulation of growth and/or activity of intestinal bacteria, potentially associated with health and well-being.

The daily dose of the prebiotic is not a determinantof the prebiotic effect, which is mainly influenced by the number of bifidobacteria per gram in faeces before supplementation of the diet with the prebiotic begins. The ingested prebiotic stimulates the whole indigenous population of bifidobacteria to growth, and the larger that population, the larger is the number of new bacterial cells appearing in faeces. In connection with this, a new concept of prebiotic index is proposed and is defined as the increase in the absolute number of bifidobacteria expressed divided by the daily dose of prebiotic ingested (Roberfroidet al., 2007). Prebiotics of various types are found as natural components in milk, honey, fruits, and vegetables, such as onion, Jerusalem artichoke, chicory, leek, garlic, artichoke, banana, rye, barley, and salsify (Mussattoet al., 2007). In most of these sources, concentrations of prebiotics range between 0.3% and 6% of fresh weight. Asparagus, sugar beet, garlic, chicory, onion, Jerusalem artichoke, wheat, honey, banana, barley, tomato, and rye are special sources of Fructooligosaccharides (Ziemeret al., 1998 and Sangeethaet al., 2005). Isomaltulose is a potential candidate as prebiotics and is naturally occurs in honey, sugarcane juice, and products derived thereof, such as treacle or food-grade molasses (Linaet al., 2002). XOS is also an emerging prebiotic that is found in bamboo shoots, fruits, vegetables, milk, and honey (Vazquez et al., 2000). Galactooligosaccharides are found naturally in human and bovine milk (Alanderet al., 2001). Seeds of legumes, lentils, peas, beans, chickpeas, and mustard are rich in raffinose oligosaccharides (Johansen et al., 1996 and Sanchez et al., 1998).
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A number of benefits can be ascribed to prebiotic intake and a few widely addressed areas of high relevance to human health are depicted in Figure 1.

Figure 1-Potential health benefits of prebiotics (Aachary and Prapulla 2009). Oligosaccharides are commercially important food ingredients. They act as prebiotics, selectively increasing the activity ofbeneficial, or probiotic intestinal microflora1 to impart health benefits (Roberfroid, M. Prebiotics,2007). Prebiotics although found naturally in wide variety of food products but its amount is very less as compared to the demand, therefore FERMENTATIVE APPROACH for the production of Prebiotics using microorganism is practised. Large no. of Oligosaccharides are used as a Prebiotics out of which Galactooligosaccharide and Fructooligosaccharides have been extensively studied. 3. RATIONALE: The estimated global retail market for prebiotic and probioticfoods grew from U.S. $13.7 billion in 2007 to U.S. $15.4 billion in 2008. Thus, research on the factors that improve the efficiencyof oligosaccharide manufacture is of academic and commercial interest.(Ohr. L. M.,2010). 4. OBJECTIVE: To perform efficient fermentative process for the production of Prebiotics and its recovery for the better economical and eco-friendly outcomes.
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5. LITERATURE SURVEY: 5.1 Galacto-Oligosaccharides: Galacto-oligosaccharides have been defined as a mixture of those substances produced from lactose, comprising between 2 and 8 saccharide units, with one of these units being a terminal glucose and the remaining saccharide units being galactose and disaccharides comprising2 units of galactose (Tzortziset al., 2009). Theglobal market size of GOS was recently estimated to be about20,000 tons with aCompound Annual Growth Rate(CAGR)of 10% to 20% (Affertsholt et al., 2007). Galacto-oligosaccharides (GOS) have now been definitely established as prebiotic ingredients after in vitro and animal and human in vivo studies. Currently, GOS are produced by glycoside hydrolases (GH) using lactose as substrate. Converting lactose into GOS by GH results in mixtures containing GOS of different degrees of polymerization (DP), unreacted lactose, and monomeric sugars (glucose and galactose).

5.1.1 Production of Galacto-oligosaccharides: It is well known that oligosaccharides can be formed from monosaccharides by the action of mineral acids (chemical synthesis).This process, known as reversion, explains the production of oligosaccharides during acidic hydrolysis of lactose, first observedin the 1950.(Aronsonet al., 1952). Probably due to the lack of product specificity andextreme conditions applied during acidic hydrolysis of lactose, thisGOS production process is not used on a large scale. The preferred mode for GOS synthesis is by enzymatic catalysisfrom lactose using glycosyltransferases (EC 2.4) or glycosidehydrolases (EC 3.2.1) (De Roodeet al., 2003). Glycosyltransferasesand glycoside hydrolases are enzymes that are responsiblefor the transfer of glycosyl moieties froma donor sugar to an acceptor(Lyet al., 1999). Glycosyltransferases use sugar donorscontaining a nucleoside phosphate or a lipid phosphate remaininggroup (Coutinhoet al., 2003 andLairsonet al., 2008). Althoughhighly regio-selective, stereo-selective, and efficient, theseenzymes are not used for industrial GOS production due to
4

theirunavailability, prohibitive prices of commercial enzyme preparations, and the need of specific sugar nucleotides as substrates (DeRoodeet al., 2003).Currently, GOS are industrially produced using the catalyticactivity of glycoside hydrolases(Figure2). These enzymes are more readilyavailable than glycosyltransferases but are generally less stereoselective(Tzortziset al., 2009).

Figure 2.Scheme of process steps involved in the industrial production of GOS. (Tzortziset al., 2009) Converting lactose into GOS by -galactosidases is a kinetically controlled reaction, by means of the competition between hydrolysis and transgalactosylation. Specifically, during this conversion, the thermodynamically favoured hydrolysis of lactose, which generates D-galactose and D-glucose, competes with the transferase activity that generates a complex mixture of various galactose-based di- and oligosaccharides of different structures (Tzortziset al., 2009). Hence, knowledge of the reaction time course or lactose conversion is required to determine the point of maximum yield of the desired product. Transgalactosylation involves both intermolecular and intramolecular reactions. Intramolecular or direct galactosyl transfer to D-glucose yields regio-isomers of lactose. Intermolecular or indirect transgalactosylation is the route by which disaccharides, trisaccharides, and

tetrasaccharides, and eventually longer GOS, are produced from lactose (Huber et al., 1976) (Figure 3).

Figure 3General model of lactose hydrolysis and GOS synthesis. (Duarte P.M.et al., 2010) a, b, and c indicates the glycosidic linkage position. X is galactosyldonor,Yis galactosyl acceptor. Inintramoleculartransgalactosylationgalactosyldonor X is also galactosyl acceptor, only linkageposition changes. Generally, lactose is the initialsubstrate (a = 4 and X = Glucose). During theprogress of the reaction, generated products arepotential substrates for the enzyme. Y can haveone of the following structures: Glucose, Gal,Gal-Gal, or [Gal]n-Glc (with 1 n 6). 5.1.2 Literature survey of Galacto-Oligosaccharides: this paper composition of the commercial -galactosidase derived

fromBacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availabilitywere assessed.

Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constantlactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose,while initial reaction rates for transfer increased but remained constant for hydrolysis. There were different effects from changing the initialconcentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be moreimportant for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosylacceptors.(Aaronet al., 2011)
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5.1.3Effect of the Lactose Concentration on GOS Yield: Commercial, food-grade -galactosidase preparations generally respond to raised lactose concentrations by producing moreGOSand Biolacta FN5 behaves in this way(Boon et al., 1999). This expectedphenomenon was reproduced with the maximum oligosaccharideconcentration increasing across the initial lactose range of 1-20% (w/v) (Table 1). The values reported here are higher thanthose reported by Boon et al., 1999 for the same enzymatic preparationand reaction temperature. It seems likely that this differencewas primarily due to Boon et al., 1999 quantifying only trimericoligosaccharides, while here all GOS species were measured,including species with a higher degree of polymerization (Aaronet al., 2011). Table1 Increasing GOS Yields with an Increasing Initial Lactose Concentration (Aaronet al., 2011): Initial lactose concentration (% w/v) 1 5 10 20 Maximum GOS concentration (% w/v) 0.2 1.5 4.2 8.4 GOS yield (% w/w)

18 29 42 42

The maximum oligosaccharide concentrationand oligosaccharide yield were measured at variedconcentrations of lactose and sucrose.The presence of excess sucrose (20%, w/v) was found tosignificantly increase the maximum oligosaccharide

concentrationachieved at all lactose concentrations examined (Fig.4)

Figure4 Maximum oligosaccharide concentration and yield observed at varied initial lactose concentrations. Lactose only, Lactose plus 20% (w/v) sucrose, 20%

(w/v) total saccharide(Aaron et al., 2011). The production of galactooligosaccharides (GOSs) by transgalactosylation using -galactosidase fromBifidobacteriumlongum BCRC 15708 was studied. Other than lactose, galactose, and glucose, twotypes of GOSs, tri- and tetrasaccharides, were formed after -galactosidase action on 40% lactose.Trisaccharides were the major type of GOS formed. Generally, an increase of the initial lactoseconcentration in the reaction mixture resulted in a higher GOS production. A maximum yield of 32.5%(w/w) GOSs could be achieved from 40% lactose solution at 45 C, pH 6.8, when the lactose conversion was 59.4%. The corresponding productivity of GOSs was 13.0 g/(L.hr). Transgalactosylationactivity of -galactosidase from a test organism showed a relatively lower sensitivity toward glucoseand galactose than that from other organisms. The addition of 5% or 10% glucose or galactose tothe reaction mixture did not significantly (p > 0.05) reduce the transgalactosylation reaction ofgalactosidase(Hsuet al., 2007). 5.1.4 Effects of Lactose Concentrations on GOS Production: Various investigators reported that the initial lactose concentration in the reaction mixture is the most significant factors affecting GOS formation. Figure 5shows thecarbohydrate yields of GOSs, glucose, and galactose in thereaction mixture after 10 hr. of catalysis of the reaction by-galactosidase of B. longumBCRC 15708. It was found thatthe production of GOSs increased with increasing initial lactoseconcentration from 5% to 40%. A maximum GOS productionwas reached when the initial lactose concentration was 40%,and further increases in lactose concentration resulted in the reduction of GOS production. It was also noted that thehydrolysis reaction dominates in reaction solutions containinga lower lactose concentration (5-30%), while GOS formationdominated in reaction mixtures having a higher lactose concentration(40-50%). Transgalactosylation is a process in whichgalactosidasehydrolyzes lactose, and instead of transferringthe galactose moiety to the hydroxyl group of water, it transfersthe galactose moiety of lactose to a hydroxylated compound,which could be galactose, lactose, or galactose-containingoligosaccharides. It follows that, at a low lactoseconcentration, transgalactosylation is inferior to

hydrolysis, sincethe amount of hydroxyl groups of carbohydrates is low, andthis results in a higher amount of glucose and galactose in thereaction solution. Therefore, to increase transgalactosylation,high concentrations of lactose are usually

required.(Hsuet al., 2007).

Figure5Effect of the initial lactose concentration on the GOS productioncatalyzed by -galactosidase from B.longum BCRC 15708. The reaction was performed at 45 C and pH 6.8 for 10 hr. (Hsuet al., 2007) -galactosidase (GOS)

recombinant

from by

Sulfolobussolfataricu transgalactosylation.

sproducedgalactooligosaccharides
1

fromlactose

Theoptimal amount of enzyme for effective GOS productionwas 3.6 U of enzyme ml . GOS production increasedwith increasing lactose concentration, whereas the yield from lactose was almost constant. The rates ofhydrolysis and

ofGOS

transgalactosylation reactions increasedwith increasing temperature but the final concentration ofGOS was maximal at 80oC. Under the conditions of pH6.0, 80oC, 600 g lactose l-1and 3.6 U enzyme ml-1,315 g GOS l-1 were obtained for 56 hr. with a yield of52.5% (w/w). The -galactosidase from S. solfataricusproduced GOS with the highest concentration and yieldamong thermostable-galactosidases reported to date. (Park et al.,2008). Table2 Comparison of GOS production by Sulfolobussolfataricus -galactosidase with other thermostable -galactosidases (Park et al.,2008).

(Park et al., 2010) described theenzymatic production of galactooligosaccharides usingmicrobial -galactosidases. Additionally, the productionof galacto-oligosaccharides by biocatalysts fromvarious sources is reviewed and the enhanced production ofgalacto-oligosaccharides via the reduction of productinhibition is suggested herein. (Table 3) from lactose by microbial -

Table3Galacto-oligosaccharides galactosidases (Park et al., 2010)

production

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5.1.5

Factors Affecting GOS Yield: The amount of GOS produced in a reaction varies widely, and itdepends on

the reaction conditions mainly lactose concentration,enzyme source, glucose and galactose concentration, and temperature. Some studies have been focused on sourcingthermostable-galactosidase because it seems that higher reactiontemperature favortransgalactosylation reaction. -galactosidasehas been isolated from various thermostable microorganisms likeS. solfataricus, P. furiosus, Thermusspp., T. caldophilus, C. saccharolyticus,T. maritimaby various research groups. -galactosidase fromthese organisms can be used for GOS production at a temperature around 80 C and higher (Sangwanet al., 2011). Various factorsaffecting GOS production are listed in Table 4. Table 4 Factors affecting GOS production (Sangwanet al., 2011).

The -galactosidasesof Lactobacillus reuteri L103 and L461 proved to be suitable biocatalystsfor the production of prebiotic galacto-oligosaccharides (GOS)
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from lactose. Maximum GOS yieldswere 38% when using an initial lactose concentration of 205 g/L and at 80% lactose conversion.The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance

anionexchangechromatography with pulsed amperometric detection (HPAECPAD).(Splechtnaet al., 2006) Three -galactosidases from Aspergillusoryzae, Kluyveromyceslactis and Bacillus sp. used for the production of low-content galactooligosaccharides(GOS) from lactose (Table 5)This study has produced high-content GOS by fermentation with Kluyveromycesmarxianus of the low-content GOS syrups, produced either by galactosidase alone or when mixed with glucose oxidase, to remove digestible sugars including glucose, galactose and lactose.(Cheng et al., 2006) Table 5 Comparison of low-content GOS produced by three -galactosidases under various conditions.(Cheng et al., 2006)

Table 6 Comparison of GOS produced by enzyme catalysis and succeeded yeast fermentationwith KluyveromycesMarxianus (Cheng et al., 2006)

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The individual contributions of four -galactosidases present in Bifidobacteriumbifidum NCIMB 41171 towardgalactooligosaccharide (GOS) synthesis were investigated. Although the -galactosidase activity of the whole cells significantly decreased as a function of temperature (4075 C), GOS yield was at its maximum at 65 C. Native-PAGE of the whole cells showed that the contributions of BbgIII and BbgIV to GOS synthesis increased as the temperature increased. Moreover, BbgIIIand BbgIV were found to be more temperature stable and to produce a higher GOS yield than BbgI and BbgII, when used in their free form. The GOS yield using BbgIV was 54.8% (percent of total carbohydrates) and 63.9% (percent lactose converted to GOS) at 65 C from 43% w/w lactose. It was shown that BbgIV is the most important -galactosidase in B. bifidumNCIMB 41171 and can be used for GOS synthesis at elevated temperatures. The maximum GOS yield (YP) obtained using the five biocatalysts in GOS synthesis reactions, performed at temperatures ranging from 40 to 75C. It can be observed that as the temperature increased from 40 to 55C, the GOS yield obtained using BbgIand BbgII gradually increased. This trend was similar when using BbgIII, BbgIV, and whole cells, although in these cases YP increased to a temperature of 65 C. (Osman et al., 2012)

5.1.6 Analysis of GOS: Liquid chromatography has been largely used depending on thematrix from which GOS is to be extracted and analysed. Suitable types of saccharide HPLC columns and detectors, usually refractive index (RID), have also been employed along with appropriate analytical conditions. However, when it comes to analysis of GOS in dairy products such as in milk, skim milk, and milk with high solids levels, very little has been accomplished due to the presence of casein and whey proteins. (Daniel 2010). It has been suggested that the use of carrez reagents (potassium hexacyanoferrate(II) 3-hydrate, potassium ferrocyanide, and zinc sulfate) and perchloric acid might be useful in protein clarification and precipitation from dairy based matrices to overcome inherent obstacles in GOS analysis from these products (Cab et al., 2004). However, methanol-chloroform extraction remains a more suitable alternative of protein removal thus precluding the need for the use of perchloric acid. Still then, the most practical and accurate analytical means of identifying and
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quantifying individual GOS synthesized in dairy products is the high-performance liquid chromatography (HPLC). Quemeneret al., (1997) developed a method based on high performance anionexchange chromatography with pulsed amperometricdetection (HPAE-PAD) to measure GOS in food and feed products. A few years later, De Slegteet al., (2002) organized a successful Assn. of Official Analytical Chemists (AOAC) collaborative study of this method in which galactose and other sugars were separated on a CarboPacTM PA1 column and detected by pulsed amperometric detection (PAD) using a triple potential waveform.HPAE-PAD has been found to be more superior in the detection of GOSs than high-performance liquid chromatography with RIdetection. However, in the event that HPAE-PAD is not available for use, HPLCRI can be reliably used instead.

5.2 Fructooligosaccharides: Fructo-oligosaccharides (FOS) have gained large commercial interestdue to their beneficial properties in the human health as prebiotics. FOS, namely, kestose (GF2), nystose (GF3) and fructofuranosylnystose(GF4), are nondigestible food ingredients that are selectively fermented in the colon, increasing the number of beneficial bacteria by modulation of the gut microflora (kolidaet al., 2007). Although FOS occur naturally in many common foods such asfruit, vegetables, milk and honey, they are present in low concentrations and are season-limited (Table 7).On a large scale FOS can be produced by fermentation using microorganisms (sangeethaet al., 2005).

Table 7 Concentration of FOS in natural foods (sangeethaet al., 2005).

FructosylTransferases (FTases) are the enzymesresponsible for the microbial production of FOS. FTaseproduces FOS (GFn) from sucrose (GF) in a disproportionate mode, thereby forming 1-kestose (GF2) initially, then 1-nystose (GF3), followed by 1-fructofuranosyl nystose(GF4) (Yun, 1996). Microbial FTasesare
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derived from bacterial andfungal sources. Several microorganisms capable of producingFTase have been screened (Sangeethaet al., 2003a).

5.2.1 Sources of FructosylTransferases (FTases): Table8Microbial and plant sources of FOS synthesizingenzymes(Sangeethaet al., 2005)

5.2.1.1Bacterial FructosylTransferases (FTases):

It produces FOS from sucrose. It has been isolated from Bacillus maceransEG-6 which, unlike other FTases, producedselectively GF5 and GF6 fructooligosaccharide. The finalyield of FOS was reported to be 33% when 50% sucrose wasused as substrate.(Park et al.,2001) The ethanol producing bacteria Zymomonasmobilis hasbeen reported to produce a levansucrase capable of producing FOS and levan. The extracellular levansucrasethat precipitated along with levan after ethanol treatment ofculture fluid has been used as a biocatalyst for FOS production in sugar syrup. The yield of FOS was found to be 2432%, which constituted a mixture of 1-kestose,6-kestose, neokestose and nystose. Glucose content was found to increase during all 24 h of reaction. The presenceof ethanol (7.0%) in sucrose syrup limited the enzymesFOS forming activity to 24% during the first 24 h ofincubation. Fructan syrup produced from sucrose by using levan-levansucrase sediment as biocatalyst was reported to
15

have satisfactory taste, reduced energetic value and therefore, may be used as source of prebiotics (Beker et al., 2002). Lactobacillus reutristrain 121 has been reported to produce 10 g L-1FOS (95% kestose and 5% nystose) in the supernatants when grown on sucrose containing medium. FTase isolated from the strain when incubated with sucrose, produced FOS as well as inulin. After 17 h of incubation with sucrose, 5.1 g L-1 FOS and 0.8 g L1

inulin were synthesized (Hijumet al., 2002).

5.2.1.2 Fungal FructosylTransferases (FTases): Several fungal strains, especially of Aspergillussp. are known to produce extracellular or intracellular FTase. Aspergillusniger AS 0023 has been reported to producean intracellular FTasewhich yielded 54% FOS using 50% sucrose as substrate (Hocineet al., 2000). Purification and partial characterization of fructosyltransferase and invertase from the cells of Penicilliumcitrinumhave been reported (Hocineet al., 2000) to produce a syrup containing neofructooligosaccharideswherein the efficiency of FOS production was more than 55 using 70% sucrose as substrate. The product mixture comprised of 1-kestose (22%), nystose (14%) and neokestose(11%) (Hayashi et al., 2000).The authors have reported Aspergillusoryzae as a novelsource of extracellular FTase (Sangeethaet al., 2003a). Thecultural conditions and reaction parameters have beenstandardized to get FOS yield of 58% (Sangeethaet al., 2002). Culture fluid, cells and culture brothhomogenate of A. oryzae CFR 202 and A. pullulansCFR 77 have also been used for FOS production to get up to 60%FOS (Sangeethaet al., 2004a). Figure 6illustrates a flow chart for the production of FOS using microbial FTase.

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Figure6Flow chart for producing FOS.(Saneethaet al., 2005)

5.2.2 Production of Fructooligosaccharides: FTases from different microorganisms have been reported to produce FOS with different linkages to form 6-kestose, 6-kestose and neokestose. Microbial production of oligosaccharides has been extensively reviewed by (Prapullaet al.,2000). The intracellular fructosyltransferase (FTase) of a novel strain of Aureobasidiumpullulans (CFR 77) capable of producing 59% of

fructooligosaccharides (FOS) within 9 hr. of reaction time was obtained by wetmilling, and then purified and characterized. The purified FTase revealed 2 bands of 147 and 170 KD; its activity was optimum at an approximate pH of 5.0 and temperature of 55C. The specific activity of the final purified material was 42, representing a purification factor of 79.44 and yield of 43%. The enzyme is very stable, retaining more than 80% of its original activity at the optimum reaction conditions after 12 hr. Using the crude intracellular FTase, 59% of FOS was produced within 9 hr. of reaction time, which is a considerable reduction in the reaction time of 12-25 hr. that has been reported in the literature. The purified FTase yielded 59% of FOS within 3 hr. of reaction time.(Lateefet al., 2006) Fructooligosaccharide production with the fructosyltransferasefrom free cells of the native strain Aspergillussp. N74 at laboratory level was evaluated. The biomass of the native strain Aspergillus sp. N74 was produced in a sucrose fermentation medium and was employed in the enzymatic reaction in solutions of sucrose and phosphate buffer, where pH, temperature, and initial sucrose concentration effectwere evaluated. Fructooligosaccharides and reaction
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subproductswere identified and quantified by HPLC. The enzyme produced by the strain Aspergillus sp. N74 possessed hydrolytic and transfructosylating activities that changed with process conditions. The best transfructosylating condition was obtained at 80 min reaction time at pH 5.5, 60C and initial sucrose concentrations higher than 550 g L1, with fructooligosaccharideproduction of about 50% w/w (based on initial sucrose concentration) and conversion selectivity higher than 90%. In

addition,transfructosylatingand hydrolytic activities ratio was of 20.(Oscar et al., 2008)

Fructooligosaccharide

(FOS)

production

was

carried

out

using

fructosyltransferase (FTase) produced by AspergillusoryzaeCFR 202under submerged fermentation conditions. The pellets of A. oryzaeCFR 202 obtained after 48 h of fermentation were supplemented with fresh media after every 24 h and fermentation was carried out to produce FTase. FTase so obtained was used to produce FOS using 60% sucrose as substrate at 55C at pH 5.15. FTase activity was maintained in the range of 15 2 U/ml/min up to six recycles. FOS yields were maintained at 53% up to 6th cycle. Recycling of pellets could not be carried out after 6th cycle due to disintegration.(Figure 7)The system is advantageous and economical in that it does not require supplementation of any additional nutrients nor it requires the development of fresh inoculum. It can be seen from table 3 that aconsistent yield of 53% (w/w) of FOS was obtained using the FTase produced for six sequential recycles.(Sangeethaet al., 2005)

Figure 7 Production of FOS using FTase obtained recycling cell culture of A. oryzaeCFR202 (Sangeethaet al., 2005).

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Batch fructooligosaccharides (FOS) production by fructosyltransferase from Aspergillussp. N74 immobilized in calcium alginate was studied. The used biomass forimmobilization was obtained in 250 ml shake flask from the culture of 106 Aspergillussp. N74 spores in 100 mL medium during 48 hr. After biomass immobilization, the sucrose bioconversion was carried out with a mean dry weight biomass: reaction volume ratio of 0.4:100. pH, temperature and initial sucrose concentration effect on FOS production was evaluated, obtaining the higher transfructosylating activity and hydrolytic activity relation (3.78 to 5.62) at pH 5.0, 55C and 55-80% initial sucrose concentration for 5 hr. at these conditions were obtained the greatest FOS productions (~ 50 % w/w in sucrose basis). (sanchezet al).

5.2.3 Fermentative methods of microbial production of FOS: There are two methods of FTase production by fermentation-Submerged Fermentation (SmF) and Solid State Fermentation (SSF). Production of enzymes by SSFhas potential advantages overSmF with respect to simplicity in operation, high productivity fermentation, less favourable for growth of contaminants and concentrated product formation. SSF requires less space capital and operating costs, simpler equipment and the downstream processing is easier compared to SmF. In addition, it permits the use of agro-industrial residues as substrates, which are converted in to bulk chemicals and fine products with high commercial value. (Prapullaet al., 2000) have discussed FTase production by SmF in detail. However, SSF has not been attempted for FTase production except for the report using apple pomace as substrate (Hang et al., 1995).

Despite most of all industrial enzymes are produced in SmF, SSF presents an interesting potential for small-scale units. Some advantages of this process are high volumetric productivity and product concentration, low capital cost and energy consumption. Moreover, the risk of contamination is reduced. On the other hand, the main disadvantages are related to mass and energy transport, difficulty in measuring and controlling pH, temperature, pO2, cell growth, and moisture. Therefore, it leads to severe engineering problems to scale up the process (Holkeret al. 2004; Pandey 2003). Generally, the most efficient culture media and strain to produce enzymes in SSF are not the same as those in SmF, and vice versa (Antieret al.1993).
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Optimization of fermentation medium for -fructofuranosidaseproduction by A. nigerNRRL 330 in SSF and SmF was carried out using a fractional factorial design (Balasubramaniemet al. 2001). Fructosyltransferase (FTase) production by AspergillusoryzaeCFR 202 was carried out by solidstatefermentation (SSF), using various agricultural byproductslike cereal bran, corn products, sugarcane bagasse,cassava bagasse (tippi) and by-products of coffee and tea processing. The FTase produced was used for the production of fructo-oligosaccharides (FOS), using 60% sucrose as substrate. Among the cereal bran used, rice bran and wheat bran were good substrates for FTase production by A. oryzae CFR 202. Among the various corn products used, corn germ supported maximum FTase production, whereas among the by-products of coffee and tea processing used, spent coffee and spent tea were good substrates, with supplementation of yeast extract and complete synthetic media. FTase had maximum activity at 60C and pH 6.0. FTase was stable up to 40C and in the pH range 5.07.0. Maximum FOS production was obtained with FTase after 8 hr. of reaction with 60% sucrose.(Sangeetha et al., 2004).

5.2.4 Continuous production of FOS: The production of fructooligosaccharides (FOSs) from sucrose catalyzed by -D-fructofuranosidase was achieved with the use of immobilized mycelia of Aspergillusjaponicusin gluten. When 1 g mycelia-immobilized particles having a cell content of 20% (w/w) were incubated with 100 ml of sucrose solution with an initial concentration of 400 g/litre, the total produced FOSs were determined to be about 61%, w/w of total sugars in the mixture after a batch reaction for 5 hr. (Fig. 8) The reaction velocity increased with the cell content in the gluten matrix and reached the maximum value when the cell content was as high as 20% (w/w). As the myceliaimmobilized gluten particles werepacked in to a column reactor for continuous production of FOSs, a productivity of 173 g per hour per litre of reactor volume was achieved at a flow-rate of 0.8 ml/min. The mass fraction of FOSs increased from 0.20 to 0.54 w/w as the flow-ratedecreased from 1 to 0.1 ml/min, corresponding to the residence time increasing from 0.35 to 3.5 hr. (Chienet al., 2001).

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Figure 8 Production of fructooligosaccharides employing 1 g ( ) and 0.2g () cell immobilized particles containing 20% (w/w) of mycelia, and 0.04 g lyophilized mycelia (), incubated with 100 ml of 40% (w/v) sucrose solution.(Chienet al., 2001)

Fructose oligosaccharide (FOS)produced by the immobilized mycelia (IM) of a strain of Aspergillusjaponicus, isolated from soil. The -

fructofiranosidaseactivity (Uf), transfictosylating activity (Ut), hydrolysing activity (Uh), and FOS production were analysed by high performance liquid

chromatography. FOS production was performed in a batch process in a 2 lit.jar fermenter by IM in calcium alginate beads. The optimum pH and temperature were 5.0-5.6 and 55oC, respectively. No loss of activity was observed when themycelium was maintained at 60C for 60 min. Maximum production was obtained using 5.75% (cellular weight/volume) of mycelia (122.4 Ut g-1) and 65% sucrose solution (w/v) for 4 h of reaction, when the final product reached 61.28% of total FOScontaining GF2(30.56%), GF3 (26.45%), GF4(4,27%), sucrose (9.6%) and glucose (29.10%). In the assay conditions, 23 batches were performed without loss of activity of the IM, showing that the microorganism and the process utilized have potential for industrial applications. (Cruz et al., 1998)

Sucrose biotransformation to fructooligosaccharides was carried out with biomass harvested after 24 or 48 h ofculture. For 6.21 0.33 or 9.66 0.62 g biomass

21

dry weight L-1, the highest FOS yields were obtained at batch operating 62.1 and 66.4% after 26 or 6 h of reaction, respectively. Reduction in fructooligosaccharides yield was observed for both biomass concentrations at semibatch operating, while a comparable yield was obtained during continuous operating (62.1% for 6.21 0.33 g L-1and dilution rate 0.016 s-1 and 62.8% for 9.66 0.62 g L-1 and a dilution rate 0.032 s-1). (Caicedoet al., 2009)

Table 9Mean FOS yield (YFOS) and composition, and remnant sucrose obtained during batch, semibatch and continuous process for biomass after 24 and 48 hr. culture, 6.21 0.33 and 9.66 0.62 g L-1, respectively. Operating conditions; pH 5.5, 60oC, and initial sucrose concentration 70%w/v. (Caicedoet al., 2009)

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF)module was employed to produce high-content

fructooligosaccharides (FOS) in acontinuous process initiated by a batch process. The system used mycelia of AspergillusjaponicusCCRC 93007 or
22

AureobasidiumpullulansATCC

9348

with

-fructofuranosidaseactivity

and

GluconobacteroxydansATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h-1, and the volumetric productivity for total FOS was more than 160 g L-1 h-1(Sheuet al., 2002).

Neo-fructooligosaccharides (neo-FOSs) were produced in a 500 ml continuous packed-bed reactor using whole cell immobilization
o

of

Penicilliumcitrinum KCCM 11663, the optimum reaction conditions were 50 C, pH 6 with 600 g sucrose L-1being fed as substrate at 1.3 ml min-1. Under these conditions, the maximum neo- FOSs production was 49 g.L-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production.(Park et al., 2005)

A forced-flow membrane reactor system for transfructosylation was investigated using several ceramic membranes having different pore sizes. Fructofuranosidasefrom Aspergillusniger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-coupling reagent. Sucrose solution was forced through the ceramic membrane by crossflow filtration while transfructosylationtook place. The saccharide composition of the product, which was a mixture of fructooligosaccharides (FOS), was a function of the permeate flux, which was easily controlled by pressure. Using 0.2 m pore size of symmetric ceramic membrane, the volumetric productivity obtained was 3.87 kgm-3s-1 which was 560 times higher than that in a reported batch system, with a short residence time of 11 s. The half-life of the immobilized enzyme in the membrane was estimated to be 35 days by a long-term operation.(Nishizawa et al., 2000)

23

5.2.5 Production of High Content FOS: High content FOS is produced by removing the liberated glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS. Industrial production of FOS carried out with microbial FTaseshas been found to give a maximum theoretical yield of 5560% based on the initial sucrose concentration. The FOS yield does not increase beyond this value because glucose liberated during the enzymatic reaction acts as a competitive inhibitor (Yun, 1996). To enhance the FOS conversion by removing the liberated glucose, the use of mixed enzyme systems has been recommended by many authors.

Studies were carried out on mixed enzyme systems using a commercial enzyme, with glucose oxidase andcatalase, and mycelia of A. japonicus CCRC 93007 and A.niger ATCC 20611 with -fructofuranosidase activity to produce high yields of FOS. The reaction was performed in an aerated stirred tank reactor maintained at pH 5.5 by a slurry of CaCO3. Glucose, an inhibitor of -fructofuranosidase, produced was converted by glucose oxidase to gluconic acid, which was then precipitated by slurry of CaCO3 to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dryweight basis, the remainder was glucose, sucrose and asmall amount of calcium gluconate(Sheuet al., 2001).

Nishizawaet al. (2001) have achieved higher yields of FOS with a simultaneous removal of glucose using a membrane reactor system with a nanofiltration membrane,through which glucose permeated but, not sucrose and FOS. FOS percentage of the reaction product was increased to above 90%, which was much higher than that of the batch reaction product (5560%).

Studies have been carried out by (Crittenden et al.,2002) to remove glucose, fructose and sucrose present in food grade oligosaccharide mixtures using immobilized cells of the bacterium Z. mobilis. Unpurifiedfructo, malto, isomalto, gentio and inulin oligosaccharides containing total carbohydrate concentrations of 300 g L-1were added to immobilized cells, in 100 ml batch reactors. Glucose, fructose, and sucrose present in the mixtures were completely fermented within 12 hr. without any pH control or nutrientaddition. The fermentation end products were ethanol and
24

carbon dioxide without anydegradation of the oligosaccharides in the mixtures. A minor amount of sorbitol was also produced as a fermentation by-product. The methods using mixed enzyme systems and mixed cultures have facilitated the removal of the residual sucrose as well as the inhibitory by-product glucose, thereby improving the final FOS yields.

The use of mixed enzyme system of fructosyltransferaseand glucose oxidase for the production of high content fructooligosaccharides has been investigated by Yunet al., 1993. They have reported that by using 10 units of fructosyltransferase of AureobasidiumpullansKFCC 10524 and 10 units of glucose oxidase (E.C.1.1.3.4) from A. nigerwith a stated activity of 25,000 units/g per gram of sucrose, highly concentrated FOS up to 90% was obtained.

Yun et al., 1994have reported the production of high content FOS using a mixed enzyme system of -fructofuranosidase and glucose oxidase. Under the optimized conditions, high content FOS up to 98% wasobtained. Complete consumption of released glucose and unreacted sucrose by the mixed enzyme system resulted in high content FOS. They have reported that there was significant difference in sugar composition in the FOSproduced by the mixed enzyme system when compared to that produced by fructosyltransferase/furanosidases. The content of nystose was higher in the former.

Barthemeufet

al.,

1995have

reported

the

use

of

crude

fructosyltransferasefrom a new strain of Pencilliumrigulosumisolated in their laboratory for the production of high content FOS. They have reported that the crude enzyme from Pencilliumrigulosum to be a mixed enzyme system of

fructosyltransferase and glycosidase. Under optimized conditions they were able obtain a yield of 80% FOS. FOS thus produced had a high concentration of fructofuranosylnystose.

Fernandeet al., 2004have reported the use of whole cells of Aspergillussp.27H, a soil isolate for the production of FOS. The organism was found to possess both hydrolytic and the transfructosylating activity. Under optimized conditions they were able to obtain a maximum concentration of FOS of 376 d m-3 corresponding to a value
25

of 600-620 g kg-1 of FOS solids in the reaction mixture by 6 h of reaction. A complex enzyme system in a bioreactor with a micro filtration facility using both the mycelia with -fructofuranosidase activity and bacterial cells with dehydrogenase activity has been reported by Duanet al.,2003.

5.2.6 Maximization of fructooligosaccharide production:

The production of Fructooligosaccharides (FOS) was carried out by applying two stage continuous process. In the first stage FructosylTransferase (FTase) from AspergillusoryzaeCFR 202 was grown in sucrose containing medium. In the second stage the enzyme was used to produce FOS with sucrose as the substrate. Though, the processes were two stage, the system was continuous as the enzyme prepared was immediately used to prepare the FOS. Yields of FOS production were maximized using response surface methodology (RSM) based on shell design. RSM was used to evaluate the important parameters that influence the production of FOS in both the stages. With this the feasibility of developing a maximization programme for a continuous two stage process was demonstrated. Fermentation time (36108 h), KH2PO4 concentration (0.21%) and sucrose concentration (124%) in the fermentation medium, reaction time (124 h) and pH of the reaction mixture (56) were chosen as process variables for the optimization. Among these parameters, reaction time and fermentation time had significant effects compared with KH2PO4concentration,sucrose concentration and pH of the reaction mixture. Optimum conditions for the production of maximum FOS yields were fermentation time-108 h,KH2PO4-0.723%, sucrose-6.455%, reaction time-18 h and pH of the reaction mixture-5.15. The maximum FOS yield predicted by the equation (58.9%, w/w) agreed well with the values obtained from the experimental verification (56.4%, w/w) at the optimum values based on stationary points. To have few more options for higher yields of above 50%, contour plots were also used to predict the experimental conditions. This maximized the FOS yields at 58% (w/w). These optimum conditions were then scaled up to 10 Llevel of FOS production and the results were matching the shake flask level studies.(Sangeethaet al., 2005).

5.2.7 Analysis of FOS:

26

5.2.7.1 High performance liquid chromatography High performance liquid chromatography (HPLC) has been the most popular technique for analysis of FOS. Both polar-bonded phase and resin-based HPLC columns are commonly used with Refractive Index Detector (RID) for separation of FOS of different Degree of Polymerization (DP) (Prapullaet al., 2000). The polar bonded phases are efficient, and carbohydrates elute in order of increasing monosaccharide chain length. On the other hand, components elute in order of decreasing molecular size from resin based columns. There have been many reports on the use of polar bonded phase columns like NH2column at 30oC with acetonitrile: water (75:25) as mobile phase at a flow rate of 1 or 1.5 mL/min (Vigantset al., 2000; Nishizawaet al., 2000; LHocineet al., 2000; Sheuet al., 2001; Chienet al., 2001). Gorrecet al., 1872) have used the resin based ion exchange KC column Aminex HPX-87 K column at 65oC with water as mobile phase at a flow rate of 0.6 mL/min whereas Kim et al., (2001) and Park et al. (2001) have used Aminex HPX 42 C column at 85oC using the same mobile phase and flow rate. Trujillo et al. (2001) have reported the use of Aminex HPX 87 N column at the same conditions with 10 mM Na2SO4 as mobile phase at 0.5 mL/min flow rate. Another resin based column, Aminex HPX 87C has also been widely used for FOS analysis using water as mobile phase (Crittenden &Playne, 2002). High Performance Anion Exchange Chromatography with Pulsed Amperometric Detector (HPAEC-PAD) is another widely used technique for analysis of FOS. LHommeet al. (2003) have used a Carbopac PA 100 analytical anion exchange column using a 20 min linear gradient from 0 to 40% of a 80 mMNaOH, 500 mMsodium acetate in 80 mMNaOH, 5 mM sodium acetate whereas Finke et al. (2002) have used a Dionex DX-300 chromatograph equipped with a pulsed electrochemical detector with gold electrode operating in the integrated amperometry mode. Fructans originally extracted from chicory roots were separated by continuos annular and fixed bed conventional gel chromatography. Both columns were packed with Toyopearl HW 40 (S) and eluted with deionized water. A multicomponent fractionation was established to obtain single oligosaccharides in a low molecular weight range up to a chain length of 90 monosaccharide units. The productivity and resolution ofthe continuos annular size exclusion chromatograph (40 cmbed height) were investigated and compared with those of the fixed bed counterpart (2x100 cm
27

bed height). The eluting fractions were analyzed by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The

productivity of the annular system was found 25-fold higher than the conventional system. Thus, annular chromatography exemplified for the fractionation of fructans is suggested to be a powerful method for the large scale and continuous fractionation of oligomericand polymeric carbohydrates (Finke et al., 2002).

5.2.7.2 Thin layer chromatography (TLC): Park et al. (2001) have reported the quantitative analysis of FOS by TLC using the solvent systems; isopropyl alcohol: ethyl acetate: water (2:2:1). The products were visualized by heating the plates after spraying phenol sulfuric acid. A routine method has been proposed by Vaccariet al., (2000) for the analysis of FOS utilizing modern instrumental thin layer chromatography, which meets most of the criteria and gives a rapid method for the detection and quantitative determination of the oligosaccharides in beet molasses and other products. Diol HPTLC plates were used and development was done using solvents like acetonitrile and acetone. A ninestep gradient was performed by mixing the two solvents using a Camag Automated Multiple Development apparatus. Derivatization was performed with 4-aminobenzoic acid reagent, glacial acetic acid, water, 85% phosphoric acid and acetone added to 4-aminobenzoic acid. The developed plates on heating at 115oC for 15 min showed yellowish to brown spots corresponding to FOS (Vaccariet al., 2000).

5.2.7.3 Gas chromatography-mass spectrometry (GC-MS): Hayashi et al. (2000) have reported the analysis of FOSusing GC-MS after methylation of the samples by methyl iodide and hydrolysis with 1 M H2SO4for 1 h. The samples were then reduced by the addition of NaBD4 and then alditolacetylated with acetic anhydride at 110oC for 3 h. GC-MS was performed on a Hitachi M-2000 AM instrument fitted with an OB 225 fused silicone column at 170-200oC using Helium as carrier gas with a temperature program of 1oC/min.

5.2.7.4 Nuclear magnetic resonance (NMR): Hayashi et al. (2000) have reported
13

C NMR analysis of FOS prepared from

sucrose using the cells of P. citrinum. They have identified the presence of 1-kestose, nystose andneokestose in the products.
28

5.3 Xylooligosaccharides(XOS): XOS are sugar oligomers made up of xylose units, which appearin bamboo shoots, fruits, vegetables, milk, and honey (Vazquez et al., 2000). However, there is no report available on the exact quantity of XOS present in these sources. Depending upon various xylan sources used for XOS production, the structures of XOS vary in degree of polymerization (DP), monomeric units, and types of linkages. Generally, XOS are mixtures of oligosaccharides formed by xylose residues linked through (14)-linkages (Aachary and Prapulla 2008). The number of xylose residues involved in their formation can vary from2 to 10 and they are known as xylobiose, xylotriose, and so on. (Vazquez et al., 2000) XOS are produced from xylan containing lignocellulosic

materials(LCMs) by chemical methods, direct enzymatic hydrolysis of a susceptible substrate (Katapodiset al., 2002; Christakopouloset al., 2003; Izumi et al., 2004a; Vardakou et al.,2004; Katapodis and Christakopoulos 2005) or a combination of chemical and enzymatic treatments (Izumi et al., 2004b; Kokubo and Ikemizu 2004; Yuan et al., 2004a; Yang et al.,2005). The production of XOS with chemical methods can be accomplished by steam, diluted solutions of mineral acids, or alkaline solutions. Extraction of xylan with steam or acid produces large amounts of monosaccharides and their dehydration products (Yuan et al., 2004a; Nabarlatz et al., 2005; Yang et al., 2005). To produce XOS with chemical and enzymatic methods, xylan is generally extracted with an alkali, such as KOH or NaOH, from suitable LCMs and extracted xylan is converted to XOS by xylanase enzyme having low exo-xylanase and/or xylosidase activity (Akpinaret al.,2007). In contrast to autohydrolysis, this method is more desirabledesirablebecause it does not produce undesirable by-products or high amounts of monosaccharides and does not require special equipment. Therefore, there are many reports describing the production of XOS by enzymatic hydrolysis of xylan, from oat spelt (Chen and others 1997), beech wood (Freixoet al., 2002), birch wood (Aachary and Prapulla 2008), corncob (Pellerin and others 1991; Ai and others 2005; Yoon and others 2006; Aachary and Prapulla 2009), wheat straw (Zilliox and Debeire
29

1998; Swennenet al., 2005), and hardwood (Nishimura et al., 1998). Acidic XOS were obtained from birch wood xylan by treatment with family-10 endoxylanases from Thermoascusaurantiacusand family-11 endoxylanases from Sporotrichum thermophile (Christakopouloset al., 2003). The main difference between the products liberated by these xylanases concerned the length of the products containing 4-Omethyl-D-glucuronic acid. The xylanasefrom T. aurantiacusliberated an

aldotetrauronic acid from glucuronoxylanas the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. The recombinant xylanase B (XynB) from a hyperthermophilicEubacterium, Thermotoga maritime, is not only an extremely thermostableenzyme but also stable in the neutral to alkaline region. Jiang et al (2004) demonstrated that XynB exhibited the highest activity towards beech woodxylan and low activity towards carboxy methyl cellulose. XynBhydrolyzed XOS and xylans to yield predominantly xylobiose as end product, suggesting that it was an endoxylanase. Therefore, the enzyme could be used for the large-scale production of xylobiose from xylans. The researchers concluded that the recombinant XynB from T. maritimacould be of future commercial interest for the large-scale production of xylobiose.

5.4 Inulinooligosaccharides:

Inulin is a fructose polymer which has been widely investigated as a source for the production of high fructose syrup through enzymatic hydrolysis by either the sole action of exoinulinase (EC 3.2.1.80; -D-fructanfructohydrolase) or the synergistic action of exoinulinase and endoinulinase (EC 3.2.1.7; 2,1--Dfructanfructanohydrolase) (Byun&Nahm1978, Nakamura et al. 1994). A novel inulinolytic microorganism, Xanthomonassp. produced an endoinulinase, to be used for inulooligosaccharide(IOS) formation from inulin, at an activity of 11 units ml-1 (1.2 mg protein ml-1). The endoinulinase was optimally active at 45oC and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l-1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.(Park et al., 1999) Continuous production of inulo-oligosaccbarides from pure inulin was conducted using an immobilizedendoinulinase reactor. The optimal operating conditions of the
30

reactor for maximizing the productivity were asfollows: 50 gL-1 of inulin feed concentration, flow rate assupeficial space velocity 1.1 h-l, and temperature55C. The enzyme reactor was run for 15 d at 55C achieving an oligosaccbaride yield of 83% without any significant loss of initial enzyme activity, during which the volumetric productivity was 55 g/Lh and half lifeof the immobilized enzyme indicated 35 d.(Yun et al., 1997) Inulo-oligosaccharides were produced from inulin by using high activities of an endo-acting inulinase. The total yields of oligosaccharide were slightly decreased as the concentration of inulin increased from 50 to 200 g/L. Under theoptimal reaction conditions, the products consist of inulo-oligosaccharides ranging from DP (degrees of polymerization) 2 to DP7, where the major oligosaccharides are 29.8% DP2, 21.4% DP3, and 8.1% DP4 oligomer, respectively. The maximum yield was 75.6% when 50 g inulin/L and 15 units/g substrate were used. (Kim et al., 1997)

5.5 In vitro evaluation prebiotics: Rycroft et al. (2001) evaluated Prebiotics by monitoring the growth of predominant gut bacterial groups over 24 h of batch culture through fluorescent in-situ hybridization. Short-chain fatty acid and gas production were also measured. All prebiotics increased the numbers of bifidobacteria and most decreased clostridia. Xylo-oligosaccharides and lactulose produced the highest increases in numbers of bifidobacteria whilst fructo-oligosaccharides produced the highest populations of lactobacilli. Galacto-oligosaccharides (GOS) resulted in the largest decreases in numbers of clostridia. Short-chain fatty acid generation was highest on lactulose and GOS. Gas production was lowest on isomalto-oligosaccharides and highest on inulin.

6) WORK PLAN: Decide, prebiotic to produce. Selection of strain of microorganism, as a source of enzyme. Fermentation of microorganism on a lab scale. Isolation of enzyme from Microorganism. Determination of Enzyme activity.

31

 Production of prebiotic on a lab scale by using isolated enzyme, substrate and other necessary ingredients. Purification of prebiotic from reaction mixture. Characterization of prebiotic by suitable techniques as mentioned in literature. Determination of yield of produced prebiotic.

7) HYPOTHESIS: The concepts in nutrition have changed in the recentyears. Presently, the focus is on the use of foods that promote a state of well-being, better health and reduction of the risk of diseases. These concepts have recently become popular as the consumer is becoming more and more health conscious. There is a growing awareness of the additional benefits and market potential for Prebiotics. The substantial market of Prebiotic as food ingredientssupports a wide scope of isolation of novel FTaseand-galactosidaseproducing strains. More emphasis should also be given to elaborative characterization of Oligosaccharides using sophisticated analytical techniques. Novel production techniques for Prebiotics using native/recombinant enzymes, highly efficient purification systems and/or new substrates should be explored. REFERENCES: Aachary A. &Prapulla SG., Corncob-induced endo-1, 4--D-xylanase of

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