R.S. SINGH and R.P. SINGH: Fructooligosaccharides from Inulin as Prebiotics, Food Technol. Biotechnol.

48 (4) 435–450 (2010)


ISSN 1330-9862 (FTB-2540)

Production of Fructooligosaccharides from Inulin by Endoinulinases and Their Prebiotic Potential
Ram S. Singh* and Rupinder P. Singh
Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147 002, Punjab, India Received: March 24, 2010 Accepted: April 29, 2010 Summary The production and applications of food-grade oligosaccharides are increasing rapidly. Amongst them, fructooligosaccharides represent one of their major classes in terms of production. They are relatively new functional food ingredients that have great potential as prebiotics, apart from having a number of desirable characteristics which are beneficial to the health of consumers. These are manufactured either by transfructosylation of sucrose using b-fructofuranosidases or hydrolysis of inulin by endoinulinases. Inulin, a polyfructan, occurs as a reserve carbohydrate in many plant families, representing more than 30 000 species. It is a potent substrate both for the production of inulinases and fructooligosaccharides. The review focuses on the recent developments in the production of fructooligosaccharides from inulin by endoinulinases, their prebiotic potential, functionalities and applications in food industry, and future perspectives. Key words: endoinulinase, inulin, fructooligosaccharides, fructans, prebiotic, probiotic, bifidogenic effect, dietary fibre, food industry

Inulinases have been characterized from inulin-storing tissue of plants and a wide variety of microorganisms (1). However, their quantity in plant material is not enough to be exploited for commercial use (2). Microorganisms are the best source of inulinases for commercial production because of their easy cultivation, rapid multiplication and high production yields (3). Microbial inulinases are an important class of industrial enzymes, which are usually inducible and extracellular. A number of fungal, yeast and bacterial strains have been reported for the production of inulinases (3–6). Amongst the filamentous fungi, Aspergillus sp. and Penicillium sp. are high inulinase producers, while among the yeasts this is Kluyveromyces sp. (1). From bacteria, Bacillus sp., Pseudomonas sp. and Streptomyces sp. have been reported as high-yielding inulinase strains (1). Inulinases have attracted researchers because of their usage both for the production of high fructose syrup (1,3,7–9) and

fructooligosaccharides (4,5). Apart from these, inulinases have various other applications such as in the production of ethanol (10,11), acetone and butanol (12), pullulan (13), gluconic acid and sorbitol (14). Inulinases can be produced by growing various microorganisms in an inulin-based medium (6). Inulin, a polyfructan, occurs as a reserve carbohydrate in many families of plants, representing more than 30 000 species (15,16). It consists of linear chains of b-2,1-linked fructosyl units terminating at the reducing end with a glucose residue attached through a sucrose-type linkage as illustrated in Fig. 1. Apart from its use for the production of inulinases, inulin has received a great interest as a renewable raw material both for the production of high fructose syrup (1) and fructooligosaccharides (17). Inulinases target the b-2,1 linkage of inulin and are both exo and endo acting. Exo-acting inulinases produce fructose as the main end product, whereas endo-acting ones produce mainly fructooligosaccharides and monosaccha-

*Corresponding author; Phone: ++91 175 304 6262; Fax: ++91 175 228 3073; E-mail: rssingh11@lycos.com Special issue: Probiotics, Prebiotics and Synbiotics

1. weather conditions and physiological age of the plant (16). while surprisingly A.3). releasing fructose with a molecule of glucose. their distinction is difficult and controversial. niger strain 12 has been reported to produce both exo.26) and have specific characteristics. SINGH: Fructooligosaccharides from Inulin as Prebiotics. Fructose has many superior technical and functional properties compared to sucrose.2. EII) and one extracellular endoinulinase (Eexo) have been investigated from Kluyveromyces sp.18) that inulinases are classified as exo and endo acting on the basis of cleavage of b-2. in the early 1800s first isolated it as a carbohydrate substance from the roots of Inula helenium (33) and the substance was later named 'inulin' by Thomson (34). because it influences the functionalities of the fructans (16). Jerusalem artichoke. chicory. it is the second most abundant storage carbohydrate after starch (16). Inulinases from fungi are generally exo acting (4). Rose. Inulin is also the most commonly used substrate for the production of inulinases (6). 1).P. inulinases are distinguished from invertases on the basis of . a German scientist.80) cleave b-2.and endoinulinases. asparagus. Nowadays. Interestingly. Two forms of exo-acting (EI and EII) extracellular inulinases have been reported from Penicillium sp. but some fungal strains like Aspergillus ficuum (24).1-glycosidic bonds and terminated with a D-glucose moiety linked to fructose by a-1. Inulin content of some plants is given in Table 1 (35–39). onion. five exoinulinases (Exo I.2. 19–22). whereas endoinulinases (EC 3. naturally occurring inulin-rich plant materials have been used for the production of inulinases (3.18). it is considered an invertase (23). The property of having an exo or endo action depends upon the microbial origin of the enzyme.4. II. III. and future perspectives. Fructooligosaccharides constitute one of the most popular functional food components because of their bifidogenic and health-promoting properties (17. Some inulin-containing plants commonly used in human nutrition are leek. but a small amount is also found in some monocotyledonous plants from the Poaceae. Inulin and its partially hydrolyzed products (fructooligosaccharides) may significantly improve organoleptic characteristics such as both taste and mouth- Action Pattern of Inulinases It is clear from the literature (1. belonging to the Asteraceae and Campanulaceae families. the enzyme is referred to as an inulinases. (31).1.2 bonds as in sucrose (Fig.18). A Potent Substrate rides in a minor amount. Most of the strains of Aspergillus niger have been reported to produce only endoinulinases extracellularly (27–29).17).1 linkage in inulin (Fig. Exoinulinases (EC 3. If the I/S ratio is >10–2. these are synthesized enzymatically either from sucrose by fructosyltransferases or from inulin by endoinulinases. sucrose hydrolytic enzymes are called invertases (EC 3.7) act randomly and hydrolyze internal linkages of inulin to yield fructooligosaccharides.4.436 R. Earlier. Y-85 (32). (35). dahlia. Inulin consists of linear chains of D-fructofuranose molecules linked by b -2. fructose H HO OH O H H HO OH O H H OH H HO H HO H O endoinulinase CH2 O n OH exoinulinase Fig. Chrysosporium pannorum (25) and Penicillium rugulosum (26) produce both exo. 48 (4) 435–450 (2010) H HO HO HO OH H H O H HO CH2 H O H OH O glucose I/S (inulinase activity/invertase activity) ratio (1. In another interesting case. high fructose syrup (1. Inulin and its hydrolyzed products are legally classified as food or food ingredients in all the countries where they are used (21). Action pattern of inulinases on inulin Inulin. their prebiotic potential. Inulin is a potent substrate for the production of inulinases (3.15) and fructooligosaccharides (3. functionalities and applications in food industry. SINGH and R. Generally. However. most of the commercially available fructooligosaccharides were either synthesized from sucrose (19) or extracted from edible parts of some plants (22).2. etc.6).1 linkages sequentially starting from the non-reducing end of inulin and split off terminal fructosyl units.1. yacon. The DP of plant inulin is rather low (DP<200) and varies according to the plant species.S. The mode of action of inulinases from different microbial sources has been described recently (1. Because of the overlapping substrate specificity of invertases and inulinases. In nature. Apart from pure inulin.6. IV and V) and three endoinulinases (Endo I. Liliaceae and Amaryllidaceae families (15). one invertase (Inv). garlic. two intracellular (EI.and endoinulinases intracellularly (30). 1). Most of the inulin-containing plants are dicotyledonous.3. Food Technol. and also has a GRAS (Generally Recognized as Safe) status (1. II and III) were isolated from a commercial preparation derived from Aspergillus ficuum (23). and if I/S ratio is <10–4. Inulinases often show a certain activity towards sucrose (1). This review focuses on the recent developments in the production of fructooligosaccharides from inulin by endoinulinases. 1. The degree of polymerization (DP) is one of the important properties of inulin. Biotechnol.4).

beef extract. its mode of action and yield depend upon the kind of microorganism and the substrate used during fermentation (1). Inulinases are both extra. inulin is a promising substrate for the enzymatic synthesis of fructooligosaccharides and high fructose syrup.3–0. mixed and natural substrates (4). otherwise it may exhibit an inhibitory effect on inulinase production (4.3–6). fructose. SINGH and R. fructosan.15–10.64). 41–51.55–60. meat extract.5 8–13 0. Ca2+ (70–72). their complete hydrolyzed product. NH4H2PO4.32. Although inulin is the most commonly used substrate. All the endoinulinase-expressing microorganisms are from bacteria and fungi except for two yeast strains of Yarrowia lipolytica (60) and Kluyveromyces sp. Nowadays. sucrose may serve as a better source for enzyme production.41–60). Ammonium salts usually cause acidic conditions.S.0 10–15 10–15 12–22 15–20 3–10 15–20 14–19 0. Microbial Production of Endoinulinases Fungi. NaNO3. 24. sucrose. Jerusalem artichoke tubers. Mg2+. all the strains are reported to produce extracellular endoinulinases. etc. maltose. lactose. produced by exoinulinase is emerging as a safe sweetener in food industry. fructooligosaccharides produced from inulin by endoinulinases are used as potent prebiotics and dietary fibres and also have many more beneficial functionalities (10.) have also been used for different microorganisms (4. have been widely used in inulinase-producing media (65). The complex N-sources have been reported to be better than inorganic N-sources (24. except for Aspergillus niger strain 12 (30) and Kluyvero- myces sp. If the microorganism exhibits inulinase activity coupled with invertase activity.0 3–19 12–15 15–20 8.26–30.7 0.52. dahlia tubers and chicory roots are the most commonly used inulin-rich substrates for endoinulinase production.26. fructan.5–4.5–1.37 35 37 35. (NH4)2HPO4. Asparagus officinalis Asparagus racemosus Camassia sp. corn steep liquor. Y-85 (32).18). because acid is liberated in the medium after the utilization of ammonium ions and highly acidic conditions may inhibit the growth and synthesis of inulinase (18).24–30. Apart from this. Pure inulin from various sources and many naturally occurring inulin-rich substrates has been used for most of the endoinulinase-producing microorganisms (Table 3.30. yeast and bacteria are all capable of producing inulinases and many of them have been successfully used for enzyme production (1. Y-85 (32).37 35 35 35 35 35 35 37 38 39 feel in a wide range of food applications (21). Biotechnol. KNO3.61).R. 48 (4) 435–450 (2010) 437 Table 1. and inorganic N-sources like (NH4)2SO4. urea. Furthermore. etc. Complex N-sources such as yeast extract.40). inulinases are both exo and endo acting but many microorganisms are known to produce mixed forms (23–26.32. porrum Allium cepa Allium sativum Arctium sp. peptone. . Cichorium intybus Cynara cardunculus Dahlia sp. Furthermore. Scorzonera hispanica Saussurea lappa *percentage of fresh mass Common name agave leek onion garlic burdock shatwaar safed musli/shatwaar camas chicory artichoke dahlia Jerusalem artichoke barley murnong banana rye yacon dandelion salsify Spanish salsify kuth Plant part lobes bulb bulb bulb roots root tubers root tubers bulb roots leaves/heart root tubers tubers grains roots fruit grains roots leaves roots roots roots Inulin/%* 7–10 3–10 2–6 9–16 3. NH4Cl.62).63.and intracellular. SINGH: Fructooligosaccharides from Inulin as Prebiotics. which are known to produce endoinulinase intracellularly. etc. The positive influence of inorganic salts like Mn2+.P. a variety of other substrates (glucose.17). Microorganisms expressing endoinulinases and their enzyme activities in submerged fermentation are listed in Table 2 (5. Food Technol.75 18–20 Reference 36 35 35 35 35 37 37 35 35. Inulin content of some plants Botanical name Agave americana Allium ampeloprasum var. but some microorganisms have the ability to express this enzyme in both ways (4. Similarly. Fe2+ and K+ (73) on inulinase synthesis has also been reported.18. The localization of an enzyme.5–1. Helianthus tuberosus Hordeum vulgare Microseris lanceolata Musa acuminate Secale cereale Smallanthus sonchifolius Taraxacum officinale Tragopogon sp. The raw material used for the production of inulinases includes a wide range of pure.66– 71).

Bacillus smithii Paenibacillus sp. Agitation. 1.0 2.2 NS NS NS 3 1 9. Penicillium purpurogenum Penicillium rugulosum Penicillium sp. Agitation not only affects the O2 availability.0 1. 41 42 27 5 43.0 2. Y-85a.0 2.0 1. and from bacterial strains between 4.0 0.S. Trichoderma harzianum Trichoderma viride Yeast Kluyveromyces sp. Food Technol. 0.b A. phaseoli Xanthomonas oryzae Xanthomonas sp. 49 U/mgc 11 15 Ref. 108 U/mgc 40 NS 115 3.85 c Rhizoctonia solani Rhizopus sp. Yarrowia lipolytica 26 27 55 56 57 58 59 32 60 produces both exo.438 R. SINGH: Fructooligosaccharides from Inulin as Prebiotics.0 (65). 0.5 and 7. Rhizoctonia solani Rhizopus sp.0.0 3.53 NS A.44 45 46 47 48. but it also exerts influence on the availability of other nutrients in the medium. The . Generally.0 40 1.0 1. Biotechnol. aeration and shear stress are the key factors influencing inulinase yield in bioreactors. 48 (4) 435–450 (2010) Table 2.75). niger mutant strain Chaetomium sp. Pseudomonas sp.0 3.5 2.44 45 56 57 46 58 59 47 48. rubisclerotium Penicillium rugulosum Penicillium sp.and endoinulinases intracellular enzyme localization *natural substrates were used while in others commercial inulin was used for the production of endoinulinase NS – not specified specific activity (units/mg of protein) of enzyme in the crude extract NS – not specified Inulinase production is also influenced by many other factors like pH of the medium. Chrysosporium pannoruma Penicillium purpurogenum var.5 2.P.0 2.74 NS. while yeasts have the optima in the mesophilic range (65).0 2.0 1. NS NS NS NS dahlia tubers NS NS dahlia tubers chicory root powder* Jerusalem artichoke powder* dahlia tubers chicory roots Jerusalem artichoke powder* Jerusalem artichoke powder* chicory roots chicory root extract* dahlia tubers chicory root powder NS w/% 1.5 1.0 0. Rheological properties are defined for broth viscosity during the fermentation process. from yeast strains between 4.0 Ref. Streptomyces rochei Trichoderma harzianum Trichoderma viride Xanthomonas campestris pv.0 4.0 1. whereas the shear rate is a function of impeller geometry and impeller rotational speed (74. SINGH and R.4 and 6.9 NS.5 1. niger strain 12a. Shear stress in a bioreactor is related to rheological properties of broth and shear rate.49 50 45 60 Enzyme activity U/mL NS.0 2.b Yarrowia lipolytica a b c a Table 3.84 U/mgc 135. Microbial sources producing extracellular endoinulinases in submerged fermentation Source Bacteria Arthrobacter sp.24 NS.0 3. Inulin from different sources used for the production of endoinulinases Microorganism Arthrobacter sp.0 3. The higher agitation in bioreactors causes shear stress on the producer organism as well on the enzyme structure (70). The rate of oxygen transfer also plays an important role in the overall microbial metabolism and different microorganisms behave differently under different conditions of oxygen supply. Streptomyces rochei Xanthomonas campestris pv. aeration and temperature of fermentation.4 U/mgc 0. Pseudomonas mucidolens Pseudomonas sp.5.0 2.75 94 NS 62.6 U/mg NS 9. Inulin source Jerusalem artichoke extract* chicory roots Jerusalem artichoke extract* dandelion tap root extract* NS dahlia tubers NS dahlia tubers Bacillus smithii Chrysosporium pannorum Paenibacillus sp.25 U/mgc NS. 22. Fungal and bacterial inulinases show temperature optima in the mesophilic and thermophilic range.49 50 45 24 51 40 5 29 28 27 30 52 53 25 54 Aspergillus ficuum Aspergillus niger Aspergillus fumigatus Aspergillus niger NS. Fungi Aspergillus ficuuma NS NS NS NS 55 33. phaseoli mutant KM 24 Xanthomonas oryzae Xanthomonas sp. 41 24 51 29 28 52 27 30 42 63 27 64 26 27 55 43.0 0. inulinase from fungal strains shows pH optima between 4.8 and 7.

All of them were glycoproteins. respectively. attempts have also been made to use solid-state fermentation (Table 4.5 and 5. sugar cane molasses wheat bran.S. hydrophobic interaction chromatography (41.5-fold higher and apparent Km for inulin was much lower than for the wild-type endoinulinase (III). wheat bran.49. Y-85 (32). a hybrid neural network approach has been described to model inulinase production in a batch bioreactor using agroindustrial residues as a substrate (87). three endoinulinases (Endo I. ficuum and Penicillum purpurogenum endoinulinases. endoinulinases localized intracellularly need cell disruption first and then followed by similar techniques. SINGH: Fructooligosaccharides from Inulin as Prebiotics. Two forms (P-1A and P-1B) of endoinulinases have been purified from Aspergillus niger mutant 817 (52). IV and V).40).9 % similarity between A. Two intracellular (EI and EII) and one extracellular endoinulinase (Exo) were purified from Kluyveromyces Y-85 (32). All the exoinulinases showed the same molecular mass (74 kDa). Endoinulinases from Aspergillus niger (52.5. corn flour Ref.0 and 45–55 °C.9 250 Characteristics of Endoinulinases Purified from Various Microbial Sources Most of the reports on purification of extracellular endoinulinases from various microorganisms deal with the conventional methods of centrifugation.R. corn steep liquor. Several metal ions have a stimulatory or inhibitory effect on endoinulinase activity. Their specific activities were 3.40–42.53. There is not much difference in pH and temperature optima. Fungal strains show pH and temperature optima of 4. corn steep liquor. From a commercial inulinase preparation from Aspergillus ficuum.64. bacterial strains show higher temperature optima.55. coconut oil cake. and molecular mass of endoinulinases purified from different strains. The mathematical framework developed has been proved a useful tool for simulation of this process. generally act in antagonism. The two mechanisms. Exo-II and Exo-III) and two endoinulinases (Endo-I and Endo-II) have been purified from the culture broth of A. corn steep liquor. 49. respectively. 77–86).P. The stimulatory effect of Mn2+ (30. However. some endoinulinases are reported to be inactivated by Microorganism Cryptococcus aureus Kluyveromyces marxianus Substrate wheat bran. corn steep liquor. and 50 and 70 °C.57) and Ca2+ (57. Generally. Recently. 23. Inulinase production in solid-state fermentation Enzyme activity U/gds* 436 391.92) were strongly inactivated by p-chloromercuribenzoate (p-CMB). soybean bran sugarcane bagasse. soybean bran sugarcane bagasse. Inulinases purified from all the bacterial strains are either exo. Most of the reports on purification of endoinulinases are from fungi (Table 5). coconut oil cake. .52. rice bran. while from some fungal strains both forms are purified (24– 26. it is imperative to improve the genetic characteristics of the producer organism. ficuum JNSP5-06 (40). Biotechnol. Their pH optima were 4. Food Technol.91) have also been used. etc.63. The former contributes to biomass increase and the latter acts against better enzyme yield (76). An endoinulinase-encoding gene INU2 from Aspergillus ficuum has been cloned and sequenced (88). 77 78 79 199 80 436. rice husk sugarcane bagasse sugarcane bagasse.92) and preparative electrophoresis (40.30. Conversely. Most of the research reports on inulinase production are on submerged fermentation. II and III) and one invertase (Inv) were purified by conventional techniques (23). Furthermore. corn flour Xanthomonas campestris pv. wheat bran Staphylococcus sp. which has 139 kDa. garlic peel 409. salt/organic precipitation or ultrafiltration followed by ion-exchange and gel filtration chromatography (Table 5.52.5–7. Table 4. p-CMB reacts with thiol groups forming mercaptides (a reversible process) and serves as a specific reagent for SH groups (94).5 and 7. SINGH and R. The molecular mass ranged from 31 to 75 kDa for all the purified endoinulinases except Xanthomonas oryzae. the enhanced production of A. corn steep liquor. III. three exoinulinases (Exo-I. Endoinulinases purified from most of the fungal and bacterial strains are extracellular. Alignment of amino acid sequence revealed 73. respectively. rice bran. All the inulinases were stable below 50 °C with optimum activity at 45 °C. five exoinulinases (Exo I.91) on endoinulinases from different microorganisms is well established. while for bacterial strains they are between 4.0 for exoinulinase and endoinulinase.64 85 84 117 101 86 *units/gram of dry substrate To make the process economically feasible. 48 (4) 435–450 (2010) 439 supply of oxygen to microorganisms poses many problems specific to it like foam formation. This indicates that the active site of inulinases contains the sulfhydryl (SH) groups (93). soybean bran sugarcane bagasse. soybean bran sugarcane bagasse. The characteristics of some purified endoinulinases from different microorganisms are also summarized in Table 5. phaseoli onion peel. In some cases fast protein liquid chromatography (23).55. Their intracellular nature has also been reported only from Aspergillus niger strain 12 (30) and Kluyveromyces sp.7 81 463 82 445 83 122. namely oxygen supply and shear stress. The endoinulinase INU2 gene has been successfully expressed in Saccharomyces cerevisiae (89).92). while endoinulinases were of 64 kDa.57.91.30. II.88 84 Kluyveromyces sp. Recently. ficuum endoinulinase in Saccharomyces cerevisiae was achieved by SUC2 deletion mutation (90).32.or endoinulinase.8 107. however.

GFC (NH)2SO4 PPT. IEC Km 1. GFC – gel filtration chromatography.80 mM NS 56 5. GFC.3 5.440 R.3 6. GFC isopropanol PPT.92) and Ca2+ (40). PREP PAGE – preparative polyacrylamide gel electrophoresis. ficuum Endo I Endo II Endo III A.8 mg/mL 40. niger 22. PREP PAGE (NH)2SO4 PPT. GFC. IEC.0 mM 16. 52 NS NS 352 338 1105. Cu2+.3 IU/mg 47 5. They are important primarily because of their functional properties rather than sweetness.1 64.1 mM vmax NS 773 U/mg 12. UF.0 50 4. GFC.5 5–7 5–8 6–9 NS NS NS <55 <40 <30 <45 32 64 55 57 49 106 µmol/(min·mg) 68 NS 12.48 mM 0.3 45 5–7 <60 30 A.6 mg/mL 53. °C 5–10.7 mM 8. ethanol PPT. GFC dialysis. Xanthomonas oryzae NS 106. IEC UF. °C 50 50 Stability pH Temp. Characteristics of some purified endoinulinases from microbial sources Purifi. GFC (NH)2SO4 PPT. HIC. IEC.55.0 Temp.41.9 NS NS 61. UF. 40.6 4.54 1158. dialysis. HIC – hydrophobic interaction chromatography.74 0. Ref.91.5 4.8 0.0–7. Fe3+ and Al3+ (23. IEC.2 <60 41 91 Source Arthrobacter sp. FPLC – fast protein liquid chromatography.7 g/L 0. Rhizopus sp. FPLC (NH)2SO4 PPT.2 6. As a consequence. IEC. GFC IEC.3 NS NS NS NS NS NS NS NS 64 64 64 34 31 NS NS NS 5.0 7. SINGH: Fructooligosaccharides from Inulin as Prebiotics.S. IEC. PPT – precipitation.5 30–40 4.5–9 4–7 NS <50 <50 <70 NS 42 53 (NH)2SO4 PPT. IEC.17 mM 109 µmol/(min·mg) 70 139 µmol/(min·mg) 68 833. the outstanding stereo.4 NS 0.199 mmol/L 115 µmol/(min·mg) 66 Chrysosporium pannorum Kluyveromyces sp.6 328 50.5 9.92) on endoinulinases from various sources are also well known.0 NS NS NS 45 45 NS NS NS 4–8 4–8 NS NS NS <50 <50 23 14.91. The synthesis . IEC.5 6. Inhibitory effects of Ag+. ficuum Endo I Endo II Purification technique (NH)2SO4 PPT. NS – not specified both Mn2+ (40. HIC IEC-PREP PAGE (NH)2SO4 PPT. GFC dialysis.5 5.52.8–5.30. HIC 31.Specific cation activity fold U/mg 63 NS 53 348 Mr kD 75 66 Optimum pH 7. This makes it more important to develop efficient synthesis reactions for the production of oligosaccharides.57.3 4.0476 mg/(mL·min) 69 5.5 52 52 55 55 50 40 50 NS NS NS 5–7.5–8.50 mM 4. Zn2+. GFC (NH)2SO4 PPT. IEC.0 55 4–7 <55 92 A.8 mg/(mL·min) 25.4 30. They can be extracted from some biological materials or synthesized enzymatically from a variety of substrates (95).21 mM 0. IEC. IEC.0 50 50–55 70 55 5–7 3. Food Technol. Co2+.and regioselectivity of enzyme catalysis can be considered as a complementary tool to the chemical synthesis which necessitates the complex successive protection and deprotection steps (96).7 NS NS NS 82. niger mutant 817 form P-1A form P-1B Bacillus smithii Chaetomium sp. it has become more and more important to develop efficient synthesis routes that are easy to scale-up.8 mg/(mL·min) 40 Aspergillus niger 146 0. 48 (4) 435–450 (2010) Table 5. Aspergillius ficuum A.P.8 105 17 1407 NS NS NS 0. SINGH and R. HIC (NH)2SO4 PPT.0 5. Enzymatic Production of Food-Grade Oligosaccharides Oligosaccharides are attracting increasing interest as prebiotic and functional food ingredients.2 NS NS 58 6.1 5. Hg2+.1 g/(L·h) 83 139 IEC – ion exchange chromatography. At this level. Biotechnol. UF – ultrafiltration. EI EII Eexo Penicillium purpurogenum Penicillium sp.6 5.5 <45 63 NS NS NS 394 45 12 28.7 g/L NS NS NS NS 42 65 57 64 4. Ni2+. IEC.20 mM 9.

Current status of the production of food-grade oligosaccharides. China Orafti Active Food Ingredients. 4% 11 % 10 % 17 % 20 % 12 % 4% 5% 17 % lactulose palatinose oligosaccharides maltooligosaccharides cyclodextrins galactooligosaccharides fructooligosaccharides glucosyl sucrose isomaltooligosaccharides others Fig..98). Belgium Cosucra Groupe Warcoing. The enzymatic synthesis of various oligosaccharides has been extensively reviewed (19. 3. Japan Victory Biology Engineering Co. Lactulose is unusual among prebiotics as it is the only one manufactured by chemical synthesis. Food Technol.R.4-b-xylanase enzymatic transglucosylation of glucose syrup extracted directly from soybean whey by removal of proteins and salts debranching of starch by pullulanase and a-amylase. Biotechnol. 2). Various enzymatic routes currently used for the commercial production of oligosaccharides are summarized in Table 6. On industrial scale. The companies that commercially manufacture FOSs from sucrose or inulin and their trade names are listed in Table 7. isomaltooligosaccharides and lectulose are the major classes of food-grade oligosaccharides (Fig. and then transglucosylation by cyclomaltodextrin glucanotransferase transfructosylation of mixture of lactose and sucrose by b-fructofuranosidase alkali-isomerization of lactose F 2 b -fructofuranosidase O 1 2 b-D-Fru-(2 a-D-Glu-(1 Fig. Commercially produced food-grade fructooligosaccharides* Substrate Manufacturer Sucrose Beghin-Meiji Industries.S. Belgium Jarrow Formulas. and transglucosylation of maltose by a-glucosidase which produce slightly different end products (Fig. SINGH: Fructooligosaccharides from Inulin as Prebiotics. fructo-. Korea GTC Nutrition. A survey of commercial oligosaccharide manufacturers reveals that galacto-. France Cheil Foods and Chemicals Inc. USA Meiji Seika Kaisha Ltd. The developments in industrial enzymology enabled the large scale production of FOSs by enzymatic synthesis.P. 2.. 3). followed by intermolecular dehydration of palatinose hydrolysis of xylan by endoacting 1. SINGH and R. USA Trade name Actilight Oligo-Sugar NutraFlora Meioligo Prebiovis scFOS Raftilose Orafti Fibrulose Inulin FOS de gr ad ati on F O 1 F 2 O 1 G sucrose 1)-[b-D-Fru-(2 and 1)-]n where n=1-9 1)-]n where n=2-9 2)-[b-D-Fru-(2 fructooligosaccharides *manufactured by physicochemical process **manufactured by chemical synthesis Production of fructooligosaccharides (FOSs) FOSs represent one of the major classes of bifidogenic oligosaccharides (oligosaccharides selectively stimulate the growth and/or activity of bifidobacteria and lactobacilli in the colon). Schematic presentation of the production of fructooligosaccharides from inulin by endoinulinase and sucrose by b-fructofuranosidase Lactosucrose Lactulose** Table 7. a-D-Glu-(1 2)-[b-D -Fru-(1 2)-] n where n=2-4 inulin F fructooligosaccharides 1 2 O 1 2 F O 1 el o Maltooligosaccharides Glucooligosaccharides Palatinose (isomaltulose) oligosaccharides Xylooligosaccharides Gentiooligosaccharides Soybean oligosaccharides* Cyclodextrins F ng 2 O 1 2 ati on F endoinulinase O 1 transglycosylation of sucrose by dextransucrase/alternansucrose synthesis of platinose by palatinose synthetase. Ltd. malto-. Unlike other oligosaccharides. USA Beneo-Orafti. 48 (4) 435–450 (2010) 441 of novel fructooligosaccharides can also be carried out by substrate and enzyme engineering (97).. Data obtained by surveying the major manufacturers of food-grade oligosaccharides Table 6.20.. they are manufactured enzymatically by two different processes Inulin *data obtained by surveying major manufacturers of food-grade fructooligosaccharides . soybean oligosaccharides are extracted directly from the soybean whey and do not require enzymatic manufacturing process. Enzymatic synthesis of food-grade oligosaccharides Class of oligosaccharides Galactooligosaccharides Fructooligosaccharides Isomaltooligosaccharides Production process transgalactosylation of lactose by b-galactosidase transfructosylation of sucrose by b-fructofuranosidase or hydrolysis of inulin by endoinulinase debranching of starch by isoamylase or pullulanase. and then hydrolysis by specific oligosaccharide forming a-amylase hydrolysis of starch into maltose by a-amylase and b-amylase.95.

The maximum yield of fructooligosaccharides using soluble and immobilized endoinulinase was 72 and 83 %. A. The enzymatic synthesis route to FOSs using FTase from Aspergillus niger was first developed by Meiji Seika Kaisha Ltd. France to produce FOSs marketed as Actilight®. Continuous production of FOSs from chicory juice was also carried out using the polystyrene-immobilized endoinulinase from Pseudomonas sp. having both hydrolytic and transfructosylating activity were used for the production of FOSs and high yield was obtained (111). The composition of products from sucrose by transfructosylation is given in Fig. Then. Endoinulinases act randomly and cleave b-2. Endoinulinases purified from different microorganisms have been used for the production of FOSs (43. 48 (4) 435–450 (2010) Transfructosylation of sucrose by b-fructofuranosidases FOSs can be manufactured from sucrose by glycosyl transfer reactions (17. SINGH: Fructooligosaccharides from Inulin as Prebiotics. 4.2-glycosidic bonds and terminated with a-D-glucose residue. Penicillium citrinum (107). not all the b-2.44. where n=2–9.442 R. In this case.110). the FOS mixture produced from inulin hydrolysis contains longer (DP2-9) chains compared to that (DP2-4) obtained from sucrose by transfructosylation process (95). rugulosum. On the contrary. P. The enzyme reactor was successfully run for 28 days at 55 °C achieving a yield of 82 % without any significant loss of enzyme activity. sucrose plays the dual role of fructose donor and fructose acceptor (95). A. The hydrolytic conditions and the yield of FOSs from inulin by various endoinulinases are listed in Table 8 (43. A. 4. Bacterial strains producing FTase are rare and only a few reports on Bacillus macerans (102) and Lactobacillus reuteri (103) are available in literature. Fusarium oxysporum (17) and Aureobasidium pullulans (108) have been reported to produce FOSs from . FOSs produced by this process contain 2–4 fructosyl units linked by b-1. where n=1–9 and a-D-Glu-(1®2)-[b-D-Fru-(2®1)-]n. several fungal strains. The whole cells of Aspergillus sp.19. Chemical structure of fructooligosaccharides produced from sucrose by b-fructofuranosidase action of b-fructofuranose on 1-ketose produces nystose. The G O G O G O F O F O F O sucrose. Fructosyl tranferases (FTases) are the enzymes for the microbial production of FOSs. FTases have a great potential to manufacture prebiotics and also offer some process enhancements using thermophilic enzymes (114).. FOSs can be purified by chromatographic or membrane processes to remove glucose and a small amount of fructose formed as a by-product as well as unreacted sucrose (95). Inulin was completely hydrolyzed to FOSs with DP ranging from 2 to 7. under optimal conditions. High concentration of starting material is required for efficient transfructosylation (99–101). (116).51). Aspergillus ficuum (51). Furthermore. Batch production of FOSs from inulin has been compared using soluble and immobilized endoinulinases from Pseudomonas sp. japonicus (105). phoenicis (17). SINGH and R.50. (44). Hydrolysis of inulin by endoinulinases The second approach used for the production of FOSs is the controlled hydrolysis of inulin by endoinulinases. In all the cases. The use of mixed enzyme system of fructosyltransferase and glucose oxidase for the production of high content of FOSs has been investigated and highly concentrated (90–98 %) FOSs were obtained (109. Japan to launch the commercial product Meioligo®. FOSs produced from sucrose have a lower DP range (2–4) than inulin-derived FOSs and are frequently described as short-chain FOSs (22).0–7.20). 46–48. are known to produce extracellular or intracellular FTases. The developed system showed a long operational stability and half life of the immobilized enzyme was estimated to be 35 days. Aspergillus niger (104). frequentens. A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module to produce high-content FOSs in a countinous system initiated by a batch process has also been reported (113). Most of the microorganisms used for the production of FOSs are bacteria except for one fungus. USA to make FOSs under the trade name NutraFlora®. and also with GTC Nutrition. respectively. Biotechnol. oryzae (106). After the completion of the reaction. Both pure inulin and naturally occurring inulin-rich substrates have been used for the production of FOSs. The composition of the product was considerably affected by inulin concentration and enzyme form.51). this company also established a joint venture with Beghin-Meiji Industries.50.S.1-linked fructosyl chains end with a terminal glucose moiety. Food Technol.1 linkages of inulin to yield FOSs containing a mixture of b-D-Fru (2®1)-[b-D-Fru-(2®1)-]n.46–48. FOSs produced from inulin closely resemble the mixture of FOSs obtained by transfructosylation of sucrose by FTases (17). Temperature range of 37–55 °C and pH optima of 6. Therefore. niger on ceramic membranes of varied pore sizes has been developed for transfructosylation of sucrose (112). and on nystose.0 have been used for the hydrolysis of inulin. especially from Aspergillus sp. The first reaction of b-fructofuranosidase on two sucrose molecules leads to 1-ketose and glucose.P. no marked difference has been reported in operational stability between F GF2 1-ketose F O F O F GF3 nystose F O F GF4 1F-fructofuranosyl nystose Fig. it produces 1F-fructofuranosyl nystose. P. Furthermore. FOS yield ranged from 60 to 86 % under optimal hydrolysis conditions. high content of FOSs can be produced by these techniques. A forced flow membrane reactor system by immobilizing b-fructofuranosidase from A.44. However. Recombinant FTases also have interesting biocatalytic properties (115).

Food Technol. Their best known nutritional effect is the stimulation of growth of bifidobacteria in the intestine.R. Their nutritional and health benefits have been well proven (17.0 NS 6.5. a b Enzyme dosage U/g 10 15 20 NS 840 NS a w(inulin) % 5b 5 5 3b 5b 5 Inulin source Jerusalem artichoke tubers dahlia tubers NS chicory roots chicory roots dahlia tubers pH 6.0 Temp. Because of the . 2008 a prebiotic was defined as 'a fermented ingredient that results in specific changes.95. FOSs as potent prebiotics Prebiotic FOSs are gaining increasing recognition as agents to modulate the colonic microbiota in humans and animals.00 Ref. The inulin extracted from chicory contains some FOSs in addition to polysaccharides.20. sucrase. because they are nondigestible food ingredients that selectively stimulate growth and/or activity of a number of potentially-stimulating bacteria.0 6. Consequently. Raftiline® HP contains mainly inulin (995 g/kg) and is devoid of fructans with lower DP.90 86.44 46 47 48 50 units/gram of substrate inulin-rich natural substrates were used NS – not specified the two reactors fed with pure inulin solution and chicory juice as a substrate. FOSs have a wide range of food applications. large number of health promoting functions. 48 (4) 435–450 (2010) 443 Table 8. In batch system.S. etc. maltase. USA makes the inulin and inulin-derived products from chicory roots under the trade names Raftiline® and Raftilose®. Endoinulinase gene (inu1) of Pseudomonas sp.). The most convincing studies were done with the use of an ileostomy model which provides a valuable alternative to the study of digestive physiology in man (126). these fructans are resistant to hydrolysis by human digestive enzymes (a-glucosidase. Due to the b-configuration of the anomeric C2 in their fructose monomers. Its DP ranges between 10 and 60 (average DP of 25). Continuous production of FOSs was also carried out at 50 °C using a bioreactor packed with recombinant cells immobilized on alginate and the system was successfully operated for 15 days without significant loss of initial activity. The small loss during the passage through the small intestine may be due to fermentation by the microbiota colonizing the ileum. in the composition and/or activity of the gastrointestinal microbiota. 79 % yield of FOSs was obtained under optimal conditions of 55 °C.19.118). thus conferring benefit(s) upon host's health' (95). coli and used for the production of FOSs in a batch and continuous system (117). It contains mainly FOSs (950 g/kg) and is a mixture of b(2®1) fructans with a DP ranging from 3 to 7 (average DP of 4).00 74. the attempts have been made to redefine prebiotics on the basis of new outcomes on prebiotic interventions. Both inulin and oligofructose have been termed as 'prebiotics' (125).00 60. Biotechnol. Functionalities and Applications of FOSs FOSs have received considerable attention due to their functional properties. coli containing inu1 gene from Pseudomonas were also used as a biocatalyst at 50 °C for FOS production from inulin in a batch and continuous system and a high yield of 78 % was obtained (118). Edible parts of many plants containing inulin and oligofructose are used in daily diet of many of the world's populations (35). The companies manufacturing FOSs from inulin and their trade names are listed in Table 7.2 NS 7. There are a few reports on the use of recombinant endoinulinases for the production of FOSs from inulin (117. pH=7. 51 43.P. which is why they are classified as nondigestible fructans (123). This new definition reaffirms that prebiotics act by inducing specific changes but allows changes in wording such that the term could be used for extra-intestinal applications. substrate concentration 10 % and enzyme dosage of 20 U/g of substrate. isomaltase. SINGH and R. SINGH: Fructooligosaccharides from Inulin as Prebiotics. Another explanation was acid and/or enzymatic hydrolysis of the low-molecular-mass fructans which are more sensitive to stomach and/or small intestinal hydrolysis than the high-molecular-mass components.60 71. Hydrolysis conditions and yield of fructooligosaccharides (FOSs) from inulin by various endoinulinases Enzyme source Aspergillus ficuum Pseudomonas sp. Xanthomonas campestris Xanthomonas oryzae Xanthomonas sp. Raftilose® is an inulin-derived product produced by partial enzymatic hydrolysis of chicory inulin. The whole cells of recombinant E. However. Continuous production of FOSs under optimal conditions was achieved with a productivity of 150 g/(L·h) for 17 days with significant loss of enzyme activity of immobilized biocatalyst. was successfully cloned in E. Inulin and inulin-derived products are marketed under different trade names. Streptomyces sp. Orafti Active Food Ingredients. These so-called prebiotics were first defined in 1995 as 'nondigestible food ingredient(s) that beneficially affect host's health by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon' (124).119–123). At a meeting of International Scientific Association of Probiotics and Prebiotics (ISAPP) in London in November. In the ileostomy effluent 86–88 % of inulin and oligofructose were recovered. °C 45 55 40 37 45 45 Time h 72 48 24 24 4 60 FOS yield % 70.37 75. Similar inulin-hydrolyzed products on the market are Fibrulose® and Inulin FOS®. which supports the idea that these fructans are indigestible in the small intestine of man.

17.95). There has been a lot of interest in the potential of prebiotics to increase mineral absorption from the gut.127). thereby leaving the long-chain prebiotics for more distal colonic activity (128). Food Technol. FOSs are known to prevent colonization of human gut by pathogenic microorganisms. implantation and growth of newly added probiotic strains (119).167). it has been shown that FOSs have triacylglycerol. The bifidogenic effect of inulin and its long-chain FOSs in humans are well established (123). making long-chain fructans as prototype prebiotic (125.144). Mg.123. In a number of studies. One of the important nutritional attributes of inulin and FOSs is their action as dietary fibre. mainly calcium and magnesium (95). FOSs from inulin are long-chain fructans (DP<9) as compared to the fructans (DP<4) from sucrose (95). Inulin and FOSs are often used in combination with 'probiotics'. It has been established that fructan-containing diets increase the number of a bacterial population (Clostridium coccoides–Eubacterium rectale cluster) other than bifidobacteria or lactobacilli. three have shown no effects on blood levels of cholesterol or TAG. The combinations of pre. Out of nine studies reviewed on the response of blood lipids to inulin and FOSs (151). Although the mechanism of the effect of fructans on calcium is not known with any certainty. which influence the lipid metabolism in humans (151). cellulose/FOS feeding enhanced the apparent absorption and apparent retention of Ca. are also able to exert systemic effect by modifying the hepatic metabolism of lipids in many animal models (150). Other functionalities of FOSs Apart from their bifidogenic effect. It has been concluded that the scores were dependent on both the probiotic bacterial strains and the type of prebiotic utilized. many companies are manufacturing them from inulin by enzymatic synthesis. The bifidogenic effect of inulin and oligofructose and its consequences for gut health have been reviewed recently (123). both of which are considered to be beneficial to the host (125. In a comparative study on the effects of FOSs on lipid metabolism in man and in animal models. It has been reported in an animal study on obese Zucker fa/fa rats that dietary enrichment with FOSs can reduce both the fat mass development and the occurrence of hepatic steatosis (152). Furthermore. Bifidobacteria and lactobacilli are known to produce a range of antimicrobial agents from short-chain fatty acids to peptides (161– 163). FOSs resist digestion and absorption in the stomach and small intestine of humans (145). It has been established that short-chain FOSs are fermented in the proximal colon. Biotechnol.19–21.(TAG) and cholesterol-lowering effects in rodents (153). Although inulin and FOSs are present in significant amount in several edible fruits and vegetables. Fructans promote the growth of existing microflora in the colon and also act to improve the survival. Short-chain fructans have been shown to lower serum total and LDL-cholesterol in non-insulin dependent diabetic patients. It is well established that FOSs.131–133). A number of in vitro and in vivo studies have confirmed that inulin long-chain FOSs are fermented into lactic and short-chain carboxylic acids (123. Inulin®-S (DP=2–60). Zn and Fe in rats (160).119. particularly in constipated patients (146) and/or lead to softer stools (147. while four have shown modest reductions in total and LDL cholesterol. It has been reported that Fn type of fructans had a similar prebiotic effect in humans as GFn type of fructans (130). Raftilose® P95 (DP=2–7). which are added to the host's diet to promote health. 48 (4) 435–450 (2010) The bifidogenic effect of inulin and oligofructose has been well established (95. FOSs also have many other health-promoting functions (15. The influence of oligofructose and long-chain inulin on the gut microbial ecology was investigated in vivo in rats (135). but not in healthy subjects (154). SINGH and R. The results in man were more conflicting. Functional activities of some commercial prebiotics like NutraFlora® P-95 (DP=2–4). These bacteria are also known for creating conditions unfavourable for the growth of potentially pathogenic organisms such as enterobacteria and certain clostridia (143. SINGH: Fructooligosaccharides from Inulin as Prebiotics. They may also have a protective effect on the accumulation of TAG in the liver and the development of steatosis in animals. Listeria monocytogenes and Salmonella typhimurium (165). several hypotheses have been put forward (159). FOSs have been found effective in inhibiting colonization by Clostridium difficile (164). three have shown significant reductions in TAG. Colonic fermentation of FOSs results in the synthesis of short-chain fatty acids.P. They influence intestinal function by increasing stool frequency.S.130. Fructans have also been evaluated for their ability to act synergistically with probiotics to inhibit pathogens (166. In an animal study. The role of FOSs in the control of diabetes is also the subject of study by many workers (17).444 R. a prebiotic activity score was calculated for different strains of lactobacilli and bifidobacteria. That is why there has been much interest in the potential of using prebiotics to reduce the risk of acute infections.148).and probiotics have synergistic effect referred to as 'synbiotics'. It has been reported that the daily consumption of 20 g of FOSs decreases basal hepatic glucose production in healthy sub- . The highest score was obtained for Lactobacillus paracasei on inulin. Their enzymatic synthesis from inulin produces a mixture of fructans containing Fn and GFn types of oligofructose. besides their effect on the gastrointestinal tract.128. The increase in stool output could be ascribed to a significant increase in faecal bacterial mass and to a significant increase in water content (149). Fermentation of FOSs to short-chain fatty acids reduces pH in the colon and this facilitates the absorption of mineral ions from the intestine.127–129). Raftiline® HP (DP>23) and GOS® (DP=2–4) were assessed in in vitro studies (134). The synbiotic health concept using FOSs is being used in many dairy products in European countries (119). Inulin and FOSs are known to increase daily stool output (125). From the biomass data. Animal studies provide strong evidence that FOSs inhibit secretion of TAG-rich very low density lipoprotein (VLDL) particles via inhibition of de novo fatty acid synthesis (152). both in vitro and in vivo.136–142). it has been well demonstrated that inulin and FOS selectively stimulate the growth of bifidobacteria or lactobacilli. Most of the animal studies show that fructan consumption increases calcium absorption (155–158). in vivo studies in humans have demonstrated that this fermentation leads to the selective stimulation of growth of bifidobacteria.

Confectionery items like hard candies. Microbiol. Proghurt (Ja! Natürlich Naturprodukte. The inulin type fructans may be helpful for non-insulin-dependent diabetes patients (169). 81 (1999) 35–52. Switzerland). Biochem. Since the freezing point depression of such products is lower with fructans than with sugar. Chi. P. Vifit (Sudmilch/Stassano. A significant reduction of cholesterolemia in diabetic subjects receiving a diet supplemented with fructans has been reported (170). A. Gill. 5. J. Z. Calabrò. SINGH and R. Austria). 4. Food Technol. Fysiq (Mona. 126 (1972) 569–573. Pandey. V.95). UK). they are often used in combination with high intensity sweeteners to replace sugars and provide a well-balanced sweetener profile and mask the bitter aftertaste of aspartame or acesulfame K (173). D. Applications of FOSs in food industry FOSs have many interesting nutritional and functional properties which enhance shelf-life and taste profile of various food products (172). FOSs with inulin can be used in ice creams to replace all the sugar and reduce the fat content. Currently. 82 (2009) 211–220.S. so consequences of their long-term consumption should be investigated (95). That is why FOSs are used as a sugar substitute mainly in dairy and bakery products (21). Puri. the Netherlands). 17 (2007) 733–738. The development of functional foods is a unique opportunity to contribute to the improvement of the quality of consumer's health and well-being. Ricca. R.19. V. Singh. G. may be one of the aspects of future research on prebiotics. it is also expected that these foods should be convenient and affordable.S. Rev. Fyos (Nutricia. 7. The effect of inulin-type fructans on the reduction of colon cancer in experimental animals and humans has been extensively reviewed (171). Xue. etc. With this knowledge and enzyme engineering.S. P. Liu. S. 3. both are strongly hydrolyzed into fructose. Belgium). Furthermore.P. 6. Biotechnol. The effect of chronic ingestion of FOS on plasma lipid.R. Since then they have been widely used in confectionery and dairy industry. and infant milk formulations (20). The current applications of FOSs include desserts such as jellies and ice creams. Biotechnol.D. Soccol. Biochem. Crit. Appl. The use of inulin or inulin-derived fructans is not possible in most of the soft drinks and fruit jams.20. Production of inulinases: Recent advances. and marshmallows can be made while achieving significantly reduced energy values (174). . 44 (2006) 151–162. SINGH: Fructooligosaccharides from Inulin as Prebiotics. and it also gives excellent mouthfeel characteristics (17).R. Japan in 1984. Biotechnol. Vijayaraghavan.C. Fontana. are fat. bakery products including biscuits. Switzerland). 48 (4) 435–450 (2010) 445 jects without any effect on insulin-stimulated glucose metabolism (168). Biotechnol. Microbiol. Future Perspectives Today's consumers hold high standards for the foods they consume. FOSs are the ingredients of the future that meet the needs of the food industry today and are on the leading edge of the emerging trend towards functional foods. highly efficient purification systems and/or new substrates should be explored. and it was found that FOSs did not modify fasting plasma glucose and insulin concentration or basal hepatic glucose production. Trends in inulinase production – A review. A. Biotechnol. Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high fructose syrup. N. Z. L. Recent developments in microbial inulinases: Its production. Of course. Germany. The use of oligosaccharides in infant food formulations has been extensively reviewed (175). FOSs can be used as the sole sweetening agent in light jam products. More emphasis should also be given to elaborative characterization of FOSs using sophisticated analytical techniques (17). Singh.K. their safety and beneficial effects on health. Sowdamini. hepatic glucose production and inulin resistance was evaluated in type 2 diabetic subjects. Some of such commercial products are Aktifit (Emmi.P. Dhaliwal. 29 (2009) 67–77. Selvakumar. Zhang. due to their recognized bifidogenic action. Probioplus (Migros. glucose concentration. R. Crit. J. and are also interested in such foods that provide added health benefits. Symbalance (Tonilait. M. Food Technol.and/or calorie-reduced. Soccol. b-fructofuranosides from roots of dandelion (Taraxacum officinale Weber).. Novel production techniques for FOSs using native/recombinant enzymes. 27 (2007) 129–145. The substantial market of FOSs as food ingredients supports a wide scope of isolation of novel FTase. Novel FOSs can also be produced by substrate and enzyme engineering (97). FOSs were first introduced into the market as foodstuffs by Meiji Seika Co. Biotechnol. K. Chi. a sound understanding of the structure-function effects of the FOS prebiotics or the mechanisms behind their specific metabolism are not known (95). Yamini. Which microbiological outcomes a prebiotic should target. Inulinase-expressing microorganisms and applications of inulinases. represent one of the most widely produced oligosaccharide groups and those most used in food industry (17. They are ingredients of the future that meet the needs of the food industry today. P. Rutherford. In such acidic foods with long shelf-life. and are on the leading edge of the emerging trend towards functional foods. being low-calorie sweetener. Rev. Appl. FOSs are also included in probiotic yoghurt and dairy drinks to produce 'synbiotic' products. Curcio. Belgium. T. G. Lorio. The studies with inulin and FOSs have shown reduction of chemically induced aberrant crypts and prevention of colon cancer. N. properties and industrial applications. Ambika. 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