Preface Oligosaccharides, especially fructooligosaccharides (FOS) are relatively new functional food ingredients that have great potential to improve the quality of many foods In addition to providing[.]
Preface Oligosaccharides, especially fructooligosaccharides (FOS) are relatively new functional food ingredients that have great potential to improve the quality of many foods In addition to providing useful modifications to food favors and physiochemical characteristics, many of these sugar possess properties that ar beneficial to the heath of consumers These include non-cariogenicity, a low calorific value and the ability to stimulate the growth of beneficial bacteria in the colon Both the production and the applications of food-grade oligosaccharides are increasing rapidly Major uses are in beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy desserts Research continues into the development of new oligosaccharides with a range of physiological properties and applications in the food industry FOS has been attracted attention of many researchers with its prebiotical property recently In industrial scale, immobilized fungal β-fructofuranosidase or immobilized cells are used for the manufacture of FOS To improve the yield of FOS, so many studies have been done For this reason, this report will represent some new methods for the production of β-fructofuranosidase and the combination of immobilized fungal βfructofuranosidase with innovated operations to improve the FOS obtained Introduction (15%) 1.1 β – D – fructofuranosidase β-D-fructofuranosidase (FFase, EC 3.2.1.26) is a glycoenzyme that hydrolyses βD-fructofuranoside such as sucrose, raffinose, stachyose, ( α-D-Fructofuranosides and β -D-fructopyranosides are not hydrolysed ), also named invertase FFase catalyses the hydrolysis of sucrose into fructose and glucose In addition to releasing D-glucose and Dfrucose from sucrose, some microbial β-D-fructofuranosidase may catalyse the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to sucrose by different glycosidic bonds depend on the enzyme source (Sangeetha et al., 2005) This enzyme has been used in food industry to produce inverted sugar and mostly used for the preparation of jams, candies and soft-centered chocolates (Aranda C, 2006) FFase has been found in many different plants and microorganisms FFase from different sources differs in optinum pH of activity (which may be neutral, acid or alkaline) (Winter H, 2000), optinum temperature of activity, 1.1.1 Catalytic mechanism There have been many researches on the amino acid residues that present at the active site of FFase However, amino acid involved at the active site of enzyme from different source may various According to the study of Reddy and Maley (1996), the active site of FFase from Saccharomyces cerevisiae consists of imidazole, carboxylic and thiol groups Reddy and Maley also indicated that carboxylic groups from Asp-23 and Glu-204 play an important role in the catalytic process Nevertheless, amino acid that participate in the catalytic process of FFase from Arabidopsis thaliane’cell wall (Arabidopsis thaliane is a small flowering plant native to Europe, Asia, and northwestern Africa) are Asp-23 and Glu-203 (M Verhaest, 2006) The catalytic sucrose process of FFase is divided into three steeps: - First, FFase links with sucrose to form enzyme-substrate complex at the Glu204 by hydrogen bond - Second, fructosyl residue on sucrose molecule combines with Asp-23 of FFase by valent bond to break the glycosidic bond between glucose and fructose After that, α-glucose receives proton from Glu-204 and releases from enzyme active site - Finally, fructose residue combines with free water in media and separate from Asp-203 1.1.2 Soluble β – D – fructofuranosidase Commercial FFase is often powdered in shape and slight yellow in colour Soluble FFase may be produced from many sources but it mainly produced from Saccharomyces cerevisiae, As.niger, As.japonicus Soluble enzymes have a high activity but sensity to temperature, pH, During use, the activity of soluble FFase decreases due to the change in pH, temperature, conformational changes as a result of friction, amostic pressure imposed by the environs of their use Furthermore, since it is soluble, its cover from a mixture of subtrate and product for use is not economically practical Thus, the advance of immobilized enzyme technology has led to increasing efforts to replace conventional enzymatic process with the preparation as immobilization 1.1.3 Immobilized β – D – fructofuranosidase The immobilization of invertase broadens the field of its application, since it prevents the crystallization of sugar in food products and the assimilation of alcohol in fortified wines (D N Klimovskii, 1967) and provides the possibility of regulating the composition of the volatile components of wine, brandy, and aqueous liqueurs (S Kh Abdurazakova, 1978) Characteristics of enzymes important for their practical use are their dependences on the pH, the temperature and substrate concentration - The effect of pH In general, immobilized enzyme are more stable with the effect of pH than free enzyme As we can see from Fig.1 below that FFase was immobilized to polyamide was more stable than free FFase (D T Mirzarakhmetova, 1998) The activity of free FFase reached the maximum level at pH around and fell down quickly after that On the other hand, immobilized FFase had pH optimum in the 4.5-5.0 region and a narrower symmetrical profile According to D T Mirzarakhmetova, the shift of the pH optimum into the neutral region is probably due to a change in the local concentration of hydrogen ions in the microenviroment of the enzyme through the introduction of amino groups during the modification of the support The observed narrowing of the pH profile of the immobilized FFase may be a consequence of the selective binding of the more neutral forms of the enzyme with the modified support in the immobilization process Fig.1 Dependence of rate of hydrolysis of immobilized (1) and free FFase from yeast on the pH of the medium - The effect of temperature The same as the effect of temperature, immobilized enzyme are more stable with the effect of temperature than free enzyme The optimal pH of FFase from Fig.2 Determination of the pH optima for immobilized (1) and free (2) FFase from yeast The thermostability cureves are shown in Fig.3 As can be seen, the free enzyme was inactivated completely at 60-700C for 0.5-1h In contrary, immobilized enzyme was not inactivated at 700C, event after 3h Fig.3 Thermostabilitis of immobilized (1) and free (2) FFase: A (300C), B (500C), C (550C), D (600C), E (700C) - The effect of substrate and product concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum After this point, increases in substrate concentration will not increase the velocity (Worthington, Biochemical corporation, 1972) This is represented graphically in Fig.4 Fig.4 Effect of substrate concentration on the reaction velocity of enzyme Acid invertases from plants are also inhibited by their reaction products, with Glc acting as a non-competitive inhibitor and Fru as a competitive inhibitor Figure shows the dependence of the concentration of reaction products on the time for the immobilized and native enzymes The activity of the immobilized enzyme was stable for h, while the native enzyme was inactivated after 15 Fig.5 Kinetics of the formation of the products of the enzymatic hydrolysis of sucrose: 1)immobilized enzyme; 2) native enzyme 1.2 Fructooligosacharides (FOS) In response to an increasing demand from the customer for healthier and caloriecontrolled foods, a number of so-called alternative sweeteners such as palatinose and various oligosaccharides including isomaltooligosaccharides, soybean oligosaccharides and especially, fructooligosaccharides have emerged since the 1980s They are important primarily because of their functional properties rather than sweetness All of the new products introduced so far, microbial fructooligosaccharides (FOS) from sucrose have attracted special attention and are attributed to the expansion of the sugar market by several factors First, mass production is not complicated Second, the sweet taste is very similar to that of sucrose, a traditional sweetener Various fructans of higher molecular weight have been produced by the action of transfructosylation activity from many plants and microorganisms Depending on the enzyme sources, they have difference linkages; for instance, fructosyltransferase from fungi such as Aureobasidium pullulans and Aspergillus niger produce only the lF-type FOS while Claviceps purpurea enzymes and asparagus enzymes produce both lF- and 6G type oligofructosides It is an accepted opinion that fructooligosaccharides is a common name only for fructose oligomers that are mainly composed of 1-kestose (GF), nystose (GF), and lF-fructofuranosyl nystose (GF) in which fructosyl units (F) are bound at the β2,1 position of sucrose (GF), respectively, which should be distinguished from other kinds of fructose oligomers (Hidaka H Eida, 1986 and Hayash, 1989) The production yield of FOS using enzymes originated from plants is low and mass production of enzyme is quite limited by seasonal conditions; therefore, industrial production depends chiefly on fungal enzymes from either Aureobasidium sp (Yun, J W Jung, 1992) or A niger (Hidaka H Eida, 1986) In 1984, Meiji Seika Co in Japan first succeeded in the commercial production of FOSS (commercial name is Neosugar) by A niger enzyme 1.2.1 Occurrence Plants The fructooligosaccharides are found in several kinds of plants, such as onion, wheat, asparagus root, (Shiomi N, 1976) Allen and Bacon, 1956 found transfructosylation activity derived from the leaves of suger beet and were led to the conclusion that in the presence of sucrose, the products of transfer are mainly 1-kestose ( lFfructosylsucrose) with some neokestose (6G-β-fructosylsucrose) An enzyme which transfers the terminal fructosyl residue from the trisaccharide to sucrose to reform a donor molecule was discovered in the Jerusalem artichoke (Edelman, 1966) Onion and asparagus are also important sources of fructosyltransferase (Edelman, 1980) Shiomi et extensively studied the fructosyltransferase extracted from asparagus roots They isolated eleven components of FOS Asparagus oligosaccharides are produced by cooperative enzymatic reactions with at least three kinds of fructosyltransferase: sucrose 1fructosyltransferase, 6”- fructosyltransferase, and lF-fructosyltransferase They further purified and characterized the individual fructosyltransferases It was found that the general properties resembled those of the Jerusalem artichoke, but its substrate specificity differed Satyanarayana, 1976 described the biosynthesis of oligosaccharides and fructans from agave He isolated various oligosaccharides, (DP 3-15), synthesized them in vitro, and proposed a reaction mechanism Unlike most enzymes, this agave enzyme is capable of synthesizing inulotriose from inulobiose The naturally occurring oligosaccharides in agave consists of l-kestose, neokestose, 6-kestose, and their derivatives These oligosaccharides arise not only by transfructosylation reactions but by the stepwise hydrolysis of the higher oligosaccharides and fructans catalyzed by the inherent hydrolytic activity of the enzyme Table show the fructooligosaccharide-synthetic enzymes from plants that were discover by some workers in the past Table 1: Fructooligosaccharide-synthetic enzymes from plants(1) Microoganisms On the other hand, industrial fructooligosaccharides are mainly produced from sucrose by fungal enzyme During the cultivation of several fungi in the sucrose medium, the synthesis of FOSs was observed When the the concentration of sucrose supply in the medium was inadequate, FOSs were ultilized as energy source (Arcamone, 1970) Pazur, 1952 studied transfructosidation of an enzyme of A.oryzae He found two β-2,1 linked triand tetrasaccharides named provisonly 1-inulobiosyl-Dglucose and 1-inulotriosyl-Dglucose, respectively (they seem to be 1-kestose and nystose, according to Jong Won Yun, 1996) The action of C.purpurea enzyme on sucrose also gives rise to a number of oligofructosides including 1-kestose and neokestose (Dickerson, 1972) Fusarium oxysporum is another important enzyme source functioning transfructosylation activity, which has been studied by many workers Maruyama and Onodera, 1969 isolated two kinds of enzyme showing transfructosylation activity That is everything sciencetists did prior to 1980s Enzymes with the potential for achiving a high yield of FOS production were found in the late 1980s and early 1990s Hidaka et al, 1988 studied A niger enzymes; they fully characterized this enzyme and virtually developed it into the industrial production of FOS syrup By using A niger enzyme, the maximum FOS conversion reached 55 - 60% (w/w) based on total sugars Van Balken et, 1991 reported another fructosyltransferase showing high activity from Aspergillus phoenicis; they produced FOSS at a 60% yield Furthermore, Takeda et al, 1994 reported a new fungal strain, Scopulariopsis brevicaulis This strain has the ability of selective production of 1kestose, a major component of FOS Table shows microorganisms that were discovered as the fructooligosaccharide-producing sources Table 2: Fructooligosaccharide-producing microorganisms Moreover, using imobillized cell and enzyme for the production of FOS has been developing Production of FOS from sucrose catalyzed by β-D-fructofuranosidase was achieved by Chien and Lee (2001) with the use of mycelia of Aspergillus japonicus immobilized in gluten One gram of mycelia-immobilized particials having a cell content of 20% (w/w) was incubated with 100ml sucrose solution with an initial content of 400g.L-1 After a reaction period of 5h, the FOS yield was 61% of the total sugars When Aspergillus oryzae was used, the cultural conditions and reaction parameters have been standardized to get FOS yield of 58% (Sangeetha, Ramesh and Prapulla, 2002) Besides fungi, bacterial strain have been reported to produce FOS A transfructosylating enzyme, which produces FOS from sucrose, have been isolated from Bacillus macerans FG-6 which, unlike other FTases, produced selectively GF5 and GF6 fructooligosaccharides The final yield of FOS was reported to be 33% when 50% sucrose was used as substrate (Park and Oh, 2001) Lactobacillus reutri strain 121 has been reported to produce 10g.L-1 (95% kestose and 5% nystose) in the supernatants when grown on sucrose containing medium (Van Hijum, Van Geel-Schutten, 2002) More recently, high content FOS is produced by removing the liberated glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS This aspect will be represented later 1.2.2 Chemical structure FOS are easily understood as inulin-type oligosaccharides of D-fructose attached by β-(2 ->1) linkages that carry a D-glucosyl residue at the end of the chain They constitute a series of homologous oligosaccharides derived from sucrose usually represented by formula GFn as depicted in Figure Fig Chemical structure of fructooligosaccharides A research group of Meiji Seika Co, the first commercial producer of FOS, introduced the chemical structure of FOS produced from A niger fructosyltransferase Until now, FOSs are widely known that oligosaccharides contain 1-kestose, nystose and 1f - fructofuranosyl nystose However, this definition is not completely true FOSs are not only contain these sugar but also others sugar with higher polymerization Aspergillus sydowi produce six different FOSs showing a high degree of polymerization (DP 3-13) (Muramasu, 1988) Structure analysis is important in the study of FOSsbecause, as the mentioned above, the degree of polymerization and linkages of FOSs vary with the enzyme sources 1.2.3 Enzyme mechanisms The reaction mechanism of the fructosyltransferase to form FOSs depends on the source of the enzyme In plants and some microorganisms, a series of enzymes act together whereas a single enzyme works in most other microorganisms For instance, fructosan metabolism in Jerusalem artichoke is established by the combination of two enzyme: sucrose:sucrose l-fructosyltransferase (SST) and β (2->1) fructan:β(2->1) fructan l-fructosyltransferase (FFT) In the first instance, SST converts sucrose into glucose and an oligosaccharide but unable to promote polymerization above the trisaccharide level; further higher polymers are consecutively synthesized by FFT The overall reaction mechanism was expressed as follows: where GF is a sucrosyl group and n is the number of extrasucrosyl fructose residues Agave enzyme catalyzed a stepwise transfructosylation reaction to give rise to higher FOS formation where synthesis of FOSs from sucrose takes place as follows: GF + fructosyltransferase -> F-fructosyltransferase +G F-fructosyltransferase + GF -> GF2 + frucotsyltransferase Here, it is notable that glucose, not fructose, acts as the acceptor of the fructose molecule from sucrose GF2, GF3, and GF4, cannot act as donors of the fructosyl moiety for the synthesis of higher oligosaccharides but act as acceptors of fructose from sucrose only for the synthesis of higher oligosaccharides This mechanism is identical with that of chicory enzyme reported by Singh and Bhatia, 1971 Gupta and Bhatia, 1980 proposed a model for the fructosyltransferase in F oqsporum They suggested that fructose is transferred from the donor site to the fructosylated nucleotide bridge and this, in turn, transfers the fructose moiety to the sucrose at the acceptor site to form GF2, GF4 was the highest glucofructosan, suggesting that the acceptor site is perhaps just big enough to accommodate up to GF4 This seems a similar result with the cases of fructosyltransferase from A niger (Hirayama, 1989 and Hidaka, 1988) and A pullulan (Yun, J W, 1992 and Hayashi, 1991) in that GF4, is the biggest molecule of FOS in both cases The enzyme reaction mechanism (Figure 7) can be expressed as follows: GFn + GFn -> GFn-1 + GFn+1 ... α -D- Fructofuranosides and β -D- fructopyranosides are not hydrolysed ), also named invertase FFase catalyses the hydrolysis of sucrose into fructose and glucose In addition to releasing D- glucose and. .. Immobilized β – D – fructofuranosidase The immobilization of invertase broadens the field of its application, since it prevents the crystallization of sugar in food products and the assimilation of. .. cooled to about 30 ºC The production line of FFase production is shown below: 13 Nutrients Sterilize Cool Inoculum Inoculate Ferment Purify and concentrate β- D- Fructofuranosidase Fig 8: β- D- Fructofuranosidase