Đồ án Β-D-fructofuranosidase production and application to the manufacture of frutooligosaccharides

n in heat-processed food. They also provide high moisture retaining activity preventing excessive drying (Mussato and Mancilha, 2007). The caloric value of value of purified fructooligosaccharides (%Sc-FOS > 95%) has been estimated to be 1.5–2.0 kCal/g. This is approximately 40–50% the caloric value of digestible carbohydrates such as sucrose. 2. β-D-Fructofuranosidase production Yeast FFase have been widely studied in Saccharomyces cerevisiae (Taussig and Carlson, 1983; Reddy and Maley, 1990,1996), Schwanniomyces occidentailis (Miguel Alvoro-Benito, 2007), Aspergillus niger (Ashok Kuman Balasub ramaniem, 2001), Aspergillus japonicus (S.I Mussatto, 2009), Aspergillus aculeatus (Iraj Ghazi, 2005),…Among of them, S.cerevisiae is considered as the organism of choice for FFase production because of its hight sucrose fermentability (Rouwen horst et at., 1991). A mutant with improved FFase production and the provision of appropriate fermentation conditions are required for better yield of enzyme (Gomez et al., 2000; ShaWq et al., 2004). Furthermore, immobilized cells are also a good choice for the production of high yield of FFase due to they promote an increase in fermentor cell density that consequently contribute to increased productivity. The production level of FFase depends to a great extent on the microorganism, basal substrate and microbial production process. Moreover, the fermentation operation mode also influences the efficency of the process. Submerged fermentation has been preferred over the solid-state for FFase production as it is inviromentally friendly, requires less manpower and give higher yields (Koo et at., 1988). For this reason, most researches these days concentrate on submerged fermentation for FFase production. Moreover, Compared with the traditional batch operation, repeated batch, fed-batch, or continuous operating modes often improve the efficiency of the fermentation process (Liu Y, Liu D, 2004). Repeated batch cultivation is a well-known method for enhancing the productivity of microbial cultures because it skips the turnaround time and the lag phase, thus increasing the process productivity (Radmann EM, 2007; Huang W-C, 2008). In addition, cell immobilization is particularly feasible for repeated batch fermentation because the process is characterized by its easy operation, convenient separation of cells from the broth, and high density of cells (Liu Y, Liu D, 2004). Furthermore, fermentation with immobilized cells is a convenient manner to reduce the fermentation time during repeated batch fermentation due to the elimination of the time needed for cell growth (Yang X, 2005). S.I.Mussatto, 2009 also studied a system by using A.japonicus immobilized in vegetable fibe as a feasible operation strategy to increase the process yield. With these recent achievement, this report will represent the production of FFase by using submerged fermentation with some new methods to increase the yield of product. 11 2.1 Material Material for the production of FFas are microorganism and nutrient. Saccharomyces cerevisiae: is oftens isolated from different soil samples and fruits such as plum, peach, banana, mango,… S.cerevisiae best grows in yeast peptone sugar agar (YPSA) medium containing (g/l): yeast extract 3.0, peptone 5.0, sucrose 20.0 and sugar 20.0 at pH 6.0 and room temperature (Ikram ul – Haq, 2006). In order to increase the yield of obtained, some workers mutated S.cerevisiae by different methods such as UV irradiation or chemical mutagenesis,… (Ginka et at, 2004; Ikram ul – Haq, 2006). Subsequently, mutant S.cerevisiae is cultured in YPSA medium, harvested during the exponential phase growth (about 1.6.106 cells/ml), wash with distilled water and plated on suitable medium before fermentation. Medium for the production of FFase by S.cerevisiae was improved by many authors. In general, medium have to contain sucrose or raffinose which known as the best carbon sourse to get the highest yield of FFase. For instance, S.cerevisiae which mutated by UV irradiation was inoculated in sterilized medium containing (mg/ml): yeast extract 3.0, peptone 5.0, raffinose 20.0, agar 20.0 and 2-deoxy-D-glucose 0.02-0.10 (Ikram ul – Haq, 2006). Aspergillus japonicus is also considered as a potential source for FFase production. There have been so many research on the production of FFase by As.japonicus such as Wen Chang chen, 1997; Ching-shan chien, 2001; S.I Mussatto, 2009;… As.japonicus can produce both intra- and extracellular FFase. As.japonicus is maintained on potato dextro agar (PDA) medium at 40C and spores are maintained by mixing with glycerine solution in ultrafreeze at -800C. Spores are produced by growing the strain on PDA medium at 300C for 7-8 days. The best culture for the fermentation containing (mg/ml): sucrose 20.0, yeast extract 2.75, NaNO3 0.2, K2HPO4 0.5, MgSO4.7H2O 0.05 and KCl 0.05 (S.I. Mussatto, 2009). Before use, the medium is sterilized at 1210C for 20min. Spore suspension used in the fermentation contains around 1.8.107 spore/ml. Besides S.cerevisiae and As.japonicus, some other microorganism such as Schwaniomyces occidentialis (Miguel Alvaro Benito, 2007); Bifidobacterium lactis (Carolina Janer, 2004); Aspergillus aculaeatus (Iraj Ghazi, 2005);… were disicribed as a good souce for FFas production. Table 3 shows the microorganism and the medium for the production of FFase which have been researched recently. 12 Table 3: Microorganism and medium for the production of FFase Microorganism Medium Author Sch.occidentalis YEPD (1%, w/v, yeast extract, 2%, w/v, peptone, 2%, w/v, glucose) or Lactose Medium (0.3%, w/v, yeast extract from Difco, 0.35%, w/v, bactopeptone, 0.5%, w/v, KH2PO4, 0.1%, w/v, MgSO4·7H2O, 0.1%, w/v, (NH4)SO2, 2%, w/v, lactose). Miguel A´ lvaro- Benito, 2007 S.cerevisiae (mg/ml) yeast extract 3.0, peptone 5.0, raYnose 20.0, agar 20.0 and 2-deoxy-D-glucose 0.02–0.10 Ikram ul-Haq, 2007 (% w/v) sucrose 20.0, yeast extract 2.75, NaNO3 0.2, K2HPO4 0.5, MgSO4. 7H2O 0.05, and KCl 0.05 S. I. Mussatto, 2009 As. japonicus 20% sucrose, 2% yeast extract (Difco), 2% NaNO3, 0.05% MgSO4-7H20, and 0.5% K2HPO4. Wen-Chang Chen, 2001 As. niger (g/l): (NH4)2SO4-45; KH2PO4-23; FeSO4-0.1; MgSO4 · 7H2O-7; sucrose-50; urea-11 and yeast extract-5, initial pH 5. Ashok Kumar Balasubramaniem, 2001 2.2 Production line 2.2.1 Process discription Microorganism preparation was described above. After inoculating, the fermentation experiment is carried out in a fermentor. In laboratory scale operation, microorganism is cultivated in flasks. The flasks are cultivated in a rotary shaking inoculator at 30°C for 48 h. The agitation rate is often kept at 200 revolutions per minute. On the other hand, in large scale operation, the fermentation process is taken place in a dadecated fermentor with contains drive motor, heaters, pumps, gas control, vessel, intrumenstation and sensors. These base components combine to perform some important functions such as: maintain a specific temperature, provide adequate mixing and aeration, allow monitoring and/or control of dissolved oxygen, allow feeding of nutrient solutions and reagents,… The production medium is sterilized by heating it to 121ºC at a pressure of 1.2 Kgf/cm2 and maintaining those conditions for 30 minutes. Heat is supplied by circulating steam through the fermenter jacket. Air is filtered by passing it through polypropylene filter. Cold water is then circulated through the fermenter’s jacket and the broth is cooled to about 30 ºC. The production line of FFase production is shown below: 13 β-D-Fructofuranosidase Sterilize Cool Inoculate Ferment Purify and concentrate Nutrients Inoculum Fig 8: β-D-Fructofuranosidase production line The process is monitored continuously through periodic measurement of the following parameters: temperature, pH, activity,… When the peak activity is reached, the batch (crude enzyme) is harverted. The crude enzyme is purified by different methods such as: ultrafiltration, gel filtration, ion-exchange chromatography,…Ultrafiltration is used to separate the biomass from the culture fluid, which is later used as a source of fructosyltransferase for the production of FOS. For commercial FFase, purified FFase is dried by spray drier or freeze drier to obtain powder product. To determine the highest yield of FFas in the process, the activity is examed during the operation. Depending on the authors, FFase activity is defined by different ways. For instance, one FFase activity unit is defined as the amount of enzyme which released 1.0 mg of sucrose in 5 min at 350C and pH 5.5 (Ikram ul-Haq, 2007). In most experiments, FFase activity is measured by exame the amount of glucose released in the 14 whole time of the reaction. The mount of glucose is measured by determining color intensity by a UV/Vis spectrophoto meter after glucose reacts with DNS reagent. microfilter Gel filter waste Fig 9: FFase production diagram 15 2.2.2 Factors effecting fermentation 2.2.2.1 Time In batch fermentation, enzyme submerged culture production begin after a lag phase of approximately 8-12h and reached a maximum at the onset of stationary phase. Afterwards, enzyme productivity declined sharply possibly due to the decrease in nutrient avaiblability in the medium or carbon catabolite repression, and the expression of FFase in yeast is repressed by monosaccharides such as glucose and fructose (Herwig.et.at., 2001). Therefore, the growth stage of a culture is a critical factor for the optimal enzyme production. IKram ul.Hag, 2008 studied mutant S.cerevisiae to improved the production of FFase by submerged fermentation. Time course profiles for FFase production by wildstyle S.cerevisiae IS-14 and mutan S.cerevisiae UMF are shown in fig 10. As the result, maximum FFase production by mutant S.cerevisiae (34.72±2.6U/ml with 17.05±1.2 g/L sugar consumption and 7.85±1.8 g/L dry cell mass) was observed 48 h after the onset of incubation. Therefore the rate of volumetric productivity was improved approximately 31-fold over the parental strain. Longer incubation times did not increase FFase production possibly due to the 16 Fig 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. decrease in available nitrogen, the age of the cells, inhibitors produced by yeast itself and protease production. Other workers have reported maximum FFase production by S. cerevisiae incubated for 48 (Barlikova et al., 1991; Gomez et al., 2000). Not only S.cerevisiae but also a mould, aspergillus japonicus can produce FFase with hight activity. The same as the S.cerevisiae, the maximum enzyme productivity was obtained at 48h after inocubation (Wen-chang chen, 1997; S.I.Mussatto, 2009). There are few reports on the medium improvement of the production of FFase. But incrase level by medium improvement is not high as the increasae level by immobilized cells. Recently, S.I.Mussatto, 2009 studied a system using As.japonicus immobilized in vegetal fiber as a feasible operation strategy to increase the yield of the process. The maximum jield obtained also at 48h. Fig 11 showns the FFase activity during the repeated batch fermentation of sucrese by As.japonicus immobilized in vegetal fiber. As can se, in the subsequence seven cycle, enzyme production remain almost satble at 40.6U/ml and this value decreased (22%) only at the end of the eighth cycle. This is an interesting result because demonstrates an important increase in the productivity of the process to obtain a higer yield of FFase. Fig 11. β-Fructofuranosidase (FFase) activity during repeated batch fermentation of sucrose by Aspergillus japonicus immobilized in vegetal fiber 2.2.2.2 pH The production of FFase is largely dependent on the initial pH of medium. The effect of initial pH on enzyme production by S.cerevisiae UME-3 is shown in Fig12. Maximum production of FFase was obtained when the pH of the medium was 6.5 (IKram ul-Hag, 2006). Similar result was abtained by Silveira et at (1996) who also observed maximum FFasae production by yeast at pH 6.5. For As.japonicus, the FFase production and growth was maximum at pH 5.5, being restrained at acidic conditions or pH greater than 5.5. According to S.I.Mussatto, 2009 who research the FFase production by 17 As.japonicus immobilized on lignocellulosic material, the pH of media was set at 7.0 before inoculation and was not controlled during the experiment, being gradually decrease during the cultivation (Fig 13). Fig12. 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. As can see in Fig13, the final pH of the fermented media was just around 5.5. This fac could explain the hight activity (48.81U/ml) obtained at the fermentation’s end. Similar result was obtained by R.C.FErnadez, 2007; L.L.Hocine, 2000 who also investigated the FFas production by using cells immobilized on corn cobs and the final pH obtained was near to 6.0 , value close to the one reported as optima for the FFase activity by others fungus strain. Some others research on the production of FFase by A.niger also showed that the highest level of FFase obtain at pH around 5.5. 18 Fig13. Kinetic behavior of pH during the sucrose fermentation by A. japonicus immo-or not in different lignocellulosic materials. 2.2.2.3 Others factors: not the same as pH, the temperature of the medium is controlled during the cultivation to ensure that the obtained yield is maximum. In general, the optimal pH for yeast and fungi is from 28 to 300 and bacterium is around 370C. Table 4 shows the optimal pH and temperature that have been reported recently Table 4: optimal pH and temperature for microbial FFase fermentation Microorganism Temperature (0C) pH Authors S.cerevisiae 30 6.5 Ikram ul-Haq, 2006 28 5.0 (initial pH) S.I. Mussatto, 2009 As.japonicus 28 5.5 (initial pH) Wen chang chen, 1997 30 5.0 (initial pH) A.K.Balasubramaniem,2001As.niger 28 6.5 (initial pH) Quang D. Nguyen, 2004 Bifidobacterium lactis 37 6.4 (initial pH) Carolina Janer, 2004 Scopulariopis brevicaulis 30 6.0 (initial pH) Yoshiaki hatakey ama, 1996 Beside time, pH and temperature, subtrate concentration is one of the most important factors that affects the enzyme production. In FFase production the effect of sucrose concentration must be observed. Depending on the microorganic strain, suitable amount of sucrose should be used. According to Ikram ul – Haq 2006, maximum FFase activity produced by S.cerevisiae obtained at a sucrose concentration of 5.0g/l. Sucrose concentrations higher than 5.0g/l caused an increase in sugar consumption and cells 19 biomass but there was no net increase in FFase productivity (Fig 14). The reason may be the generation of invertase sugar in the medium at a leval that results in glucose-induced repression of FFase. At the concentration of sucrose less than 5.0g/l, enzyme production was significantly less. As sucrose is the carbon source in the medium, lower concentrations might limit growth of yeast, resulting in a lower yield of FFase (Arifi et at., 2003). In FFase production by As.jaonicus IIT-90076, the optimal enzyme production occurred at 25% sucrose concentration (Wen chang chen, 1996). According to Wen chang chen, optimal cells mass resulted from 10% sucrose. The results suggest that a sucrose concentration below 10% means that large portion of carbon source is metabolized for supporting cell growth but when sucrose concentration is higher, substrate inhibitor of cell growth occure; however, more inducer may induce mor enzyme expected. Therefore, one of the most important factors in the production of FFase from A.japonicus is not final cell mass but sucrose concentration (Wen chang chen, 1996). Fig 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. 20 3. Fructooligosaccharides production 3.1 Process Recent developments in industrial enzymology have made possible the largescale production of Sc-FOS by enzymatic synthesis. It appears that industrial production of Sc- FOS by enzymatic synthesis can be divided into two types of processes. The first one is the batch system using soluble or immobilized enzyme and the second one the continuous process using immobilized enzyme or whole cells. FOS syrup 50-60% Sucrose syrup Extract enzyme Immobilize enzyme Immobilize cell Batch systhesize Continious reactor Systhesize FOS Purify Concentrate Sterilize Final FOS>=95% Industrial processes for the synthesis of fructooligosaccharides were studied using enzymes with high transfructosylating activity. The best enzymes described were from fungi such as Aspergillus niger, Aspergillus japonicus and Aureobasidium pullulans. The industrial processes for fructooligosaccharide synthesis are schematized in Figure 15. The steps are described in detail below. Enzyme production (cell culture) 21 Fig 15: Flow chart of typical process of Sc-FOS production, by free enzyme, immobilized enzyme orimmobilized cells (Pierre F. Monsan, 2008). 3.1.1 Enzyme production For some fungal strains, production of fructosyl transferase can be by aerobic submerged culture, by fluid-bed culture, culture broth or semi-solid culture medium. Fermentation parameters (temperature, pH, aeration, agitation) should be established for each microorganism. Generally, sucrose is the best carbon source for both cell growth and enzyme activity production. The optimum temperature for growth is often around 300C and the pH above 5.5. The enzymes thus obtained are cell bound and require complex operations for separating the enzyme from themycelium. At the end of the culture period, cells are easily harvested by a basket or continuous centrifuge, and the enzyme extracted. Cells can also be directly immobilized for Sc-FOS production. Non cell-bound enzyme can be obtained using semi-solid Aspergillus niger 489 culture medium, with the addition of cereal bran. The enzyme thus obtained was easily separated from the cells without any extraction step (Park and Pastore, 1998). The example of Aspergillus oryzae grown in liquid medium with sucrose and yeast extract was also described (Rao et al., 2005) for soluble extracellular enzyme production – a process that is easy to carry out and that is of low cost. Using an optimized medium, Aspergillus oryzae produces round pellets which were stable during the fermentation period (Sangeetha et al., 2005a) or industrial scale fermentation, these pellets consisting of compact masses of hyphae are advantageous, since the filamentous form of fungi may wrap around the impeller and damage the agitator blade. Also pellet formation makes downstream processing easier in industrial fermentation. The enzyme production was also described in details above (at II part). 3.1.2 Enzyme extraction At the end of the log-phase period, cells are collected by centrifugation and washed twice with deionized water, physiological saline solution or buffer. For mycelium-bound enzyme, washed cells are resuspended in water or buffer before enzyme extraction. Different methods can be used for enzyme extraction: ƒ Ultrasonication ƒ Lysozyme ƒ Cell grinding An additional centrifugation or filtration step is then needed to produce clear supernatant containing free enzyme. In some cases (Yun and Song, 1999), the enzyme solution thus obtained required an additional concentration step by ultrafiltration or dialysis (molecular weight cutoff 10 KDa). The resulting enzyme is used crude without further purification. 3.1.3 Substrates Sucrose is the natural substrate of fructosyl transferase. The effect of the sucrose concentration was investigated to minimize the hydrolysis reaction. The maximum yield of Sc-FOS is currently obtained with an initial sucrose concentration of 55 Brix. Moreover, there is a consensus that a high concentration of commercial food grade sucrose syrup (60–70 Brix) should be used for both the batch and continuous process of 22 fructooligosaccharide production. At such high concentrations, sucrose syrups have the benefit of a low water activity reducing the risk of contamination during the enzymatic synthesis step and reducing the final evaporation costs (Pierre F. Monsan, 2008). Commercial food grade Sc-FOS is being produced from pure food grade sucrose. In order to use Sc-FOS as animal feed additive, it would be necessary to reduce production costs. With batch production based on free enzyme or cells, the use of molasses was described to produce 166 g/l fructooligosaccharides from 360 g/l molasses sugar (Shin et al., 2004). Raw sugar can also be used. 3.1.4 Cell immobilization When enzymes are cell-bound, a method in which microorganisms are directly immobilized on a carrier can be used for Sc-FOS synthesis. This method does not require separation of enzyme from cells and can prevent the reduction of enzyme activity during the extraction step. Meiji Seika Co., which was the first to produce Sc-FOSs, used initially a continuous process involving immobilized Aspergillus niger cells entrapped in calcium alginate beads. Yun and Song (1999) described Aureobasidium pullulans cell immobilization with calcium alginate. Cells were suspended with 3% sodium alginate solution and extruded through syringe needles to form small beads dropping into calcium chloride solution. Cheil Foods and Chemicals Co. also developed a continuous process using immobilized cells of Aureobasidium pullulans entrapped in calcium alginate beads. Two one-cubic meter packed bed reactors have been in commercial operation since 1990. With concentrated sucrose as substrate, the stability of the immobilized cells is about 3 months at 500C (Yun, 1996). β-fructofuranosidase from Aspergillus japonicus mycelium was also immobilized by entrapment in calcium alginate gel (Cheng et al., 1996; Rubens Cruz, 1997). A packed bed reactor was employed for the production of fructooligosaccharides at 420C. The reaction was continued for 35 days and only 17% of enzyme activity was lost during this period. S.I.Mussatto, 2009 described Aspergillus japonicus cell immobilization with corn cobs with was appreciated as the best support material since it gave the highest yield of FOS compared with wheat straw, coffee husks, cork oak, and loofa sponge (Fig 16). 23 3.1.5 Enzyme Immobilization Fig 16. FOS production (B) using A. japonicus immobilized or not in different lignocellulosic materials. A column reactor filled with immobilized cells, is hard to operate at high flow rate due to a diffusional limitation of substrate and products within the calcium alginate gel matrix. The immobilized enzyme column is essentially superior to the immobilized cell column from the practical point of view: ƒ The immobilization process is quite simple. ƒ The unit volumetric immobilized activity is higher. ƒ There are fewer diffusional limitations. For these reasons, a lot of studies on fructosyl transferase immobilization have been done. Several of adsorbents, ion exchangers and carriers for covalent enzyme linkage have been studied for the immobilization of fructosyl transferase. The following examples are illustrative of some of the relevant studies for industrial immobilization of fungal fructosyl transferase. Yun and Song (1999) described Aureobasidium pullulans fructosyl transferase immobilization process on a highly porous anion exchange resin (Diaion HPA25, Samyang Co., Korea). The support consisted of styrene-divinylbenzene polymer with quaternary alkylamine as functional group. Immobilization was very simple: after intracellular enzyme extraction with lysozyme, soluble enzyme was passed through a column containing the porous ion exchange resin (10 h at room temperature). After immobilization, only washing was done. In order to simplify the immobilization procedure, no activation of the support was carried out before or after immobilization. Kono et al. (1994) patented for Meiji Seika Co. a fructose transferring enzyme immobilization on a granular carrier with or without crosslinking agent. Fourteen carriers were tested and in accordance with the results obtained, anion exchange resin which carrying primary to quaternary amines as functional groups were defined to be essential for immobilization. Immobilization resulted in 63–100% relative activity. 24 More recently, another industrial immobilization process was described for Aureobasidium pullulans fructosyl-transferase (Vankova et al., 2008). As in the previous case, an anion-exchanger suitable for food applications (Dowex Marathon MSA resin, Dow Chemical, USA) which is a styrene-divinylbenzene matrix with quaternary amine as a functional group was used. For enzyme extraction, the cells were disrupted in a bead mill and the debris removed in a centrifuge. In order to increase the efficiency of the immobilization process, the low molecular weight solutes present in the enzyme solution were removed by ultra-filtration. For immobilization, resin particles were simply mixed gently with free purified enzyme, for 12 h at 120C and washed to eliminate unbound compounds. Immobilization resulted in 65% relative activity. Pectinex ultra SP-L (a commercial preparation from Novozymes) containing fructosyl transferase activity, was covalently immobilized onto Eupergit C (Tanriseven and Asla, 2005). Immobilization resulted in 96% relative activity and immobilized enzyme retained its activity for 20 days without any decrease. This enzyme was also covalently immobilized on an industrial polymer polymethacrylate- based (Sepabeads EC) activated with epoxy groups. The influence of pore volume and average pore size on biocatalyst performance was studied. Several parameters that affect immobilization, such as buffer concentration, pH and amount (mg) of protein added per gram of support were analyzed. Authors found that Pectinex Ultra SP-L can be efficiently immobilized on these supports without adding any external salt or buffer (Ghazi et al., 2005). Fig 17 shows the the dependence of FOS and sucrose concentration on time course (Ghazi et al., 2005). Fig 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 3.1.6 Fructooligosaccharides syntheisis Two enzymatic reaction systems were described for industrial fructooligosaccharide (Sc-FOS) production: ƒ A batch system with soluble fructosyl transferase or cells containing fructosyl transferase activity. ƒ A continuous system using immobilized cells entrapped in calcium alginate gel or immobilized enzyme on insoluble carrier. Batch Production For batch production of fructooligosaccharides, enzymes are suspended in high sucrose concentration syrup (55–70 Brix) and incubated at 55–600C for 20–25 h with gentle agitation. From a practical standpoint, the batch production with free enzyme needs an additional process to destroy and remove the residual enzymes or cells after conversion. This process results in high costs for the production of a unit amount of Sc- FOS, therefore, a continuous process employing immobilized enzyme or cells is a better alternative. The use of Aspergillus oryzae mycelium pellets was also described (Sangeetha et al., 2005a) for batch production of fructooligosaccharides. The pellets, incubated with 60% sucrose solution at 550C, gave a Sc-FOS yield of 53%. This Sc-FOS yield was maintained up to the sixth cycle. However, it was not possible to recycle the pellets beyond the sixth cycle due to their disintegration. Compared to conventional batch production, the system was described as advantageous and economical because it does not require supplementation of any additional nutrients or the use of fresh inocula. In the process of Sc-FOS production with fructosyl transferase, the main problem is the generation of by-products, principally glucose and residual sucrose. At the end of the production step, the maximum Sc-FOS content is known to be 55–60% per unit dry substance. Some authors have focused on searching for an economical method for the production of higher Sc-FOS contents. This issue will be represented below. Continious production From the standpoint of production cost, a continuous process using an immobilized enzyme or an immobilized microorganism is preferred to a batch process with native enzyme. For continuous production of fructooligosaccharides, particles of immobilized biocatalyst were packed into fixed bed reactors. Industrial reactor volumes are conventionally between 1 and 2 m3. To avoid channeling problems in the reactor, column operation is facilitated by the upward flow of substrate, in this way, compression of the immobilized enzyme is minimized (Yun and Song, 1999). The reactor temperature is kept constant by circulating hot water through a jacket. In case of fructosyl transferase from Aureobasidium pullulans, for cell immobilization fixed bed reactors, the initial flow rate at 500C is about 0.15–0.3 BVH and 1.5 to 2 BVH for immobilized enzyme fixed bed reactors (Yun and Song, 1999). Authors concluded that immobilized enzyme column is essentially superior to the immobilized cell column. A method for the continuous production of FOS was studied by Chien et al. (2001) immobilizing the mycelia on gluten particles and packing it into a column reactor. 26 FOS yield of 173 g h-1 L-1 of reaction volume was achieved at a flow rate of 0.8 ml min- 1. The mass fraction of FOS increased from 0.2 to 0.54 (w/w) as the flow rate decreased from 1 to 0.1 mL min-1, corresponding to an increase in the residence time from 0.35 to 3.5 h. The immobilized preparation was reported to be stable in long term operation since gluten was found to be adequate as the base material to immobilize mycelia-associated enzymes. However, the half-life of the enzyme was found to be 34 days. For immobilized fructosyl transferase from Aureobasidium pullulans CCY 27-1– 94, the initial flow rate described (Vankova et al., 2008) was about 0.8 BVH for a constant temperature of 500C. Production of high content FOS High content FOS was produced by removing the liberated glucose and untreated sucrose from the reaction mixture resulting in up to 98%. As mention above, industrial production of carried out with fungal transfructosylase have been found to give a maximum theorical yield of 50-60% based on the initial sucrose concentration. The FOS yield dose not increase beyond this value because glucose liberated during the enzymatic reaction acts as a competitive inhibitor (Yun, 1996). To inhance the FOS conversion by removing the liberated glucose, the use of mixed enzyme systems has been recommended by many authors. ƒ Studies were carried out on mixed enzyme systems using a commercial enzyme, with glucose oxidase and catalase, and mycelia of A. japonicus CCRC 93007 and A.niger ATCC 20611 with b-fructofuranosidase activity to produce high yields of FOS. The reaction was performed in an aerated stirred tank reactor maintained at pH 5.5 by a slurry of CaCO3. Glucose, an inhibitor of β-fructofuranosidase, produced was converted by glucose oxidase to gluconic acid, which was then precipitated by slurry of CaCO3 to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dryweight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate (Jung et al, 1993). The same result also described by Sheu, Lio, Chen, Lin,& Duan, 2001. ƒ Another process with simultaneous removal of glucose was described with free purified enzyme (Nizikawa et al., 2001). A membrane reactor system was developed using a nanofiltration membrane through which glucose permeated but sucrose and fructooligosaccharides did not. Percentage fructooligosaccharide reaction product was thus increased to above 90%, a yield comparable to that of the product obtained following chromatographic separation. A forced flow membrane reactor system for transfructosylation was investigated by Nishizawa, Nakajima, and Nabetani (2000) using several ceramic membranes having different pore sizes. β-Fructofuranosidase from A. niger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane coupling reagent. Transfructosylation took place while sucrose solution was forced through the ceramic membrane by cross flow filtration and the yield of FOS was reported to be 560 times higher than that is reported in a batch system. The half-life of the immobilized enzyme on the membrane was estimated to be 35 days by a long-term operation. 27 3.1.7 Fructooligosaccharide Purification In 1984, the separation of Sc-FOS from residual sucrose and monosaccharides was performed by Meiji Seika Co by simulated moving bed chromatography (SMB). But as would be expected, the recovery yield of this process was far too low to be applicable to the production scale (Yun, 1996). Over the last two decades there has been remarkable progress toward the understanding of the phenomenon of chromatographic separation. This has expanded the field of application of this technology and led to major improvements in process control and column design. More recently, Vankova et al. (2008) described an optimized simulated moving bed separation for fructooligosaccharide purification. A cation exchange adsorbent tailored for saccharide separation (Amberlite™ 1320 Ca, Rhom and Hass Company, France) which is based on a poly(styrene-codivinylbenzene) matrix with a functional group – (SO3)2Ca2+, was used. The Sc-FOS solution obtained contains less than 5% monosaccharides and disaccharides. The separation yield of Sc-FOSs in recent SMB processes is 95%. This type of separation allows high yield at low capital investment and running costs. β-fructofuranosidase was purified from A. niger ATCC 20611 with 76% recovery by Nizhizawa et al., 2001) after calcium acetate precipitation, anion exchange chromatography and gel filtration chromatography. The potential of cross flow nanofiltration has been assessed in the purification of oligosaccharide from mixtures containing contaminant mono and disaccharides (Nobre et al., 2006). The work showed the concentration of the high molecular weight sugar fraction from a Sc-FOS mixture obtained by fermentation. Fractionation of sugar was studied using 0.5 KDa membranes. The retention yields obtained for the oligosaccharides were 27 and 8% for mono and disaccharides with a volume concentration ratio of 3.6 as compared to the initial conditions. The fraction of Sc-FOS was concentrated during the separation process from 52 to 72%. To be applied for fractionation of oligosaccharides on a large scale, this process requires further optimization. 3.1.8 Concentration For concentration, evaporation of the reaction products is performed by a traditional process for sucrose concentration. For the final dry product, spray drying of purified syrup can also be carried out. 3.1.9 Sterilization In the sterilization procedure, heat, ultraviolet radiation or sterilizing filtration can be used. To avoid coloring the reaction products, high-temperature sterilization is not recommended. 28 3.2 Equipment diagram 3.1.2 Laboratorial scale Fig18. Flow chart for producing FOS at laboratorial scale 29 3.2.2 Industrial scale FFase immobilized on ceramic gel filtration ion-exchanged system spray dryer hot air sucrose concentration FOS powder ion-exchanged columnliberated glucose, untreated sucrose concentration . Fig 19: FOS production diagram 4. Application 4.1 β-frucofuranosidase As mention above, β-frucofuranosidase has both sucrose hydrolase and transfructosylation activity. β-frucofuranosidase with high transfructosylation activity is mainly used as an enzyme source for the production of FOS while the other one is used to produce inverted syrup (HFS). Recently, immobilized have been described by some authors for the high fructose syrup’s production. Presently, the HFS bulk production is based on the continuous isomerization of glucose by immobilized glucose isomerase in a packed-bed reactor but HFS is also produced by alternative process. This process is the continuous hydrolysis of sucrose by invertase conducted in a continuous stirred-tank reactor such as a membrane reactor. The increase of the concentration of fructose in the mixture can be achieved by passing the HFS through a chromatographic column (Godfrey & West, 1996). Ester Junko Tomotani, 2006 described the performance of immobilized invertase in a continuous process using a membrane bioreactor to produce HFS. As the result, after sucrose was hydrolysed, the inverted sugar was submitted to a chromatographic separation through a column packed with DOWEX-50W 8x100 resulting in a 70% high-fructose syrup (HFS). 30 4.2 FOS 4.2.1 Apllication Oligofructose and both inulin ingredients that deliver a number of important nutritional benefits as well as contribute functional properties that enhance shelf life and taste profile of various food products like nutrition bars (Izzo & Niness, 2001). Development of synbiotics (synbiotics is the combination of pro- and prebiotics to obtain synergistic system) combining FOS with targeted probiotic strains are increasingly described. These synbiotics promote the growth and/or activate the metabolism of various health promoting bacteria. Fructooligosaccharides are used in dairy products for sugar reduction and prebiotic effects. They are also associated with probiotic bacteria in yoghurts and fermented drinks. In dairy products, FOS improves the mouth feel by increasing unctuousness, which is appreciated in low-fat reduction creams, sorbets, chocolate, mousse, milk, jam, etc. Examples of the use of FOS in food products include the following: ƒ .Light jam products: FOS can be used as the sole sweetening agent and gives 34% calorie reduction compared with sucrose standard. Organoleptic characteristics of the products are claimed to be very similar, with the test sample having a lower sweetness and a softer texture. ƒ Ice cream: FOS can be used with inulin to replace all the sugar and reduce the fat content and give excellent mouthfeel characteristics. Since the freezing point depression is less with oligofructose than with sugar, the texture can be harder. ƒ Confectionery applications: Hard candies, gums, and marshmallows can be made while achieving significantly reduced energy values (Murphy, 2001). Other applications in food industries include desserts such as, fruits preparations, cereals, bakery products, biscuits, sweet, gums, soup,salad dressing. Fructooligosaccharides also find uses in processed meat, canned fish, sauces and pet food with health and nutrition claims. 4.2.2 Market trend Prebiotics concern a very small part of the functional food market but this is growing rapidly. Prebiotics are associated with breakfast cereals, baked goods, cereal bars, baby food as well as some dairy products. The main commercial food oligosaccharides (DP 2–10), used as prebiotic agents, are essentially obtained by enzymatic technology. They include non-digestible oligosaccharides such as inulin, fructooligosaccharides, galacto-oligosaccharides, lactulose, isomalto-oligosaccharides, soybean oligosaccharides, xylo-oligosaccharides. In Japan the demand for prebiotic oligosaccharides was 69,000 tons/year. Among these oligosaccharides the demand for starch oligosaccharides was the largest. The fructooligosaccharide market was only 6.5% of the global demand of prebiotics (Nakakuki, 2002). FOS products were first approved as food for specified health uses in 1993 and are sold as functional ingredients in Japan. In Europe, total sales of functional food was estimated in 2005 at around eight billion euros while the prebiotics sector was valued at 87 million euros, with expectations to reach 180 million euros in 2010 (Source: Frost and Sullivan). Currently, the European market is believed to be dominated by fructans and galacto-oligosaccharides. Resistant starch products are at the development stage. The production of prebiotic fibers in Europe 31 is estimated of about 30,000 tons, including inulin, oligofructose, FOS, GOS and lactulose. FOS are available worldwide, mainly in East Asia, North America and Europe as prebiotics and/or dietary fiber like oligosaccharides. Market growth of oligosaccharides is about 15% per year. 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