Đề tài Β-D-fructofuranosidase production and application to the manufacture of frutooligosaccharides

Ho Chi Minh City University of Technology Faculty of Chemical Engineering Deparment of Food Technology β-D-FRUCTOFURANOSIDASE PRODUCTION AND APPLICATION TO THE MANUFACTURE OF FRUTOOLIGOSACCHARIDES Supervisor: As.Pro. Le Van Viet Man Students: Le Hoang Du 60600313 Nguyen Minh Long 60601325 Contents Preface 1.Introduction ....................................................................................................... page 1 1.1 β – D – fructofuranosidase ...................................................................... page 1 1.1.1 Catalytic mechanism........................................................................ page 1 1.1.2 Soluble β – D – fructofuranosidase.................................................. page 1 1.1.3 Immobilized β – D – fructofuranosidase ......................................... page 2 1.2 Fructooligosacharides (FOS) ................................................................... page 5 1.2.1 Occurrence ....................................................................................... page 5 1.2.2 Chemical structure ........................................................................... page 7 1.2.3 Enzyme mechanisms........................................................................ page 9 1.2.4 Physicochemical properties ............................................................. page 10 2. β-D-Fructofuranosidase production.................................................................. page 11 2.1 Material .................................................................................................... page 12 2.2 Production line ......................................................................................... page 13 2.2.1 Process discription ........................................................................... page 13 2.2.2 Factors effecting fermentation ......................................................... page 16 2.2.2.1 Time .................................................................................. page 16 2.2.2.2 pH...................................................................................... page 17 2.2.2.3 others factors..................................................................... page 19 3. Fructooligosaccharides production .................................................................. page 21 3.1 Process...................................................................................................... page 21 3.1.1 Enzyme production........................................................................... page 22 3.1.2 Enzyme extraction .......................................................................... page 22 3.1.3 Substrates ........................................................................................ page 22 3.1.4 Cell immobilization ........................................................................ page 23 3.1.5 Enzyme immobilization .................................................................. page 24 3.1.6 Fructooligosaccharides syntheisis.................................................... page 26 3.1.7 Fructooligosaccharide purification ................................................. page 28 3.1.8 Concentration .................................................................................. page 28 3.1.9 Sterilization ..................................................................................... page 28 3.2 Equipment diagram ................................................................................. page 29 3.2.1 Laboratorial scale ............................................................................ page 29 3.2.2 Industrial scale ................................................................................ page 30 4. Application ....................................................................................................... page 30 4.1 β-frucofuranosidase ................................................................................. page 30 4.2 FOS .......................................................................................................... page 31 4.2.1 Apllication........................................................................................ page 31 4.2.2 Market trend .................................................................................... page 31 References LIST OF TABLES Table Heading Page 1 Fructooligosaccharide-synthetic enzymes from plants 6 2 Fructooligosaccharide-producing microorganisms 7 3 Microorganism and medium for the production of FFase 13 4 Optimal pH and temperature for microbial FFase fermentation 19 LIST OF FIGURES Figure Heading Page 1 Dependence of rate of hydrolysis of immobilized (1) and free FFase from yeast on the pH of the medium 2 2 Determination of the pH optima for immobilized (1) and free (2) FFase from yeast 3 3 Thermostabilitis of immobilized (1) and free (2) FFase: A (30 0 C), B (50 0 C), C (55 0 C), D (60 0 C), E (70 0 C) 3 4 Effect of substrate concentration on the reaction velocity of enzyme 4 5 Kinetics of the formation of the products of the enzymatic hydrolysis of sucrose: 1)immobilized enzyme; 2) native enzyme. 4 6 Chemical structure of fructooligosaccharides 8 7 Network of the reaction mechanism for the production of fructooligosaccharides from sucrose catalyzed by fructosyltransferase derived from A. pullans: G, GF, GF 2 , GF 3 and GF 4 means glucose, sucrose, 1-kestose, nystose, and l F fructofuranosyl nystose, respectively (Redrawn from ref, 1955) 10 8 β-D-Fructofuranosidase production line 14 9 FFase production diagram 15 10 FFase production in submerged culture by Saccharomyces cerevisiae. IS-14 (top) and mutant UME-2 (bottom), sucrose concentration 30 g/ L, temperature 30 °C, initial pH 6.0, agitation rate 200 revolutions per minute. Y-error bars indicate standard deviation among three parallel replicates. 16 11 β-Fructofuranosidase (FFase) activity during repeated batch fermentation of sucrose by Aspergillus japonicus 17 immobilized in vegetal fiber 12 Effect of initial pH on the FFase production in submerged culture by the mutant Saccharomyces cerevisiae UME-2. Incubation period 48 h, sucrose concentration 5.0 g/L, temperature 30 °C, agitation rate 200 revolutions per minute. Y-error bars indicate standard deviation among three parallel replicates 18 13 Kinetic behavior of pH during the sucrose fermentation by A. japonicus immo-or not in different lignocellulosic materials. 19 14 Effect of sucrose concentration on the FFase production in submerged culture by the mutant Saccharomyces cerevisiae UME-2. Incubation period 48 h, temperature 30 °C, initial pH 6.0, agitation rate 200 revolutions per minute. Y-error bars indicate standard deviation among three parallel replicates. 14 15 Flow chart of typical process of Sc-FOS production, by free enzyme, immobilized enzyme orimmobilized cells (Pierre F. Monsan, 2008). 21 16 FOS production (B) using A. japonicus immobilized or not in different lignocellulosic materials. 24 17 Time course of the fructo-oligosaccharides production catalysed by soluble Pectinex Ultra SP-L. Experimental conditions: 630 g/l sucrose, 0.3 U/ml, 50mM sodium acetate buffer (pH 5.4) and 60 ◦C. 25 18 Flow chart for producing FOS at laboratorial scale 29 19 FOS production diagram 30 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 β- fructofuranosidase with innovated operations to improve the FOS obtained. 1. Introduction (15%) 1.1 β – D – fructofuranosidase β-D-fructofuranosidase (FFase, EC 3.2.1.26) is a glycoenzyme that hydrolyses β- D-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 D- frucose 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 Glu- 204 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 1.1.3 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 4 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 2 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) 3 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 5 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 1 h, while the native enzyme was inactivated after 15 min. Fig.5. Kinetics of the formation of the products of the enzymatic hydrolysis of sucrose: 1)immobilized enzyme; 2) native enzyme. 4 1.2 Fructooligosacharides (FOS) In response to an increasing demand from the customer for healthier and calorie- controlled 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 β- 2,1 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 1- fructosyltransferase, 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, 5 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 1 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, FO