Luận văn Β-D-fructofuranosidase production and application to the manufacture of frutooligosaccharides

Although many articles on FOSs have been published so far, the extensive data on the physicochemical properties are scarcely available. Gross, 1962 reported chemical properties of ome kestosides such as 1-kestose, 6-kestose, and neokestose. The specific rotation ([α]D20 ) and melting temperature of 1-kestose are 28.5 and 199-200°C respectively. It forms fine white crystals fairly rapidly. The relative sweetness of 1- kestose, nystose, and 1F-fructofuranosyl nystose to 10% sucrose solution are 31, 22, and 16%, respectively. Indeed, FOSs have a nice, clean sweet taste typically 0.3–0.6 times as sweet as sucrose depending on the chain length – sweetness decreases with increasing chain length. FOSs are highly hygroscopic; it is difficult to keep the lyophilized products stable under atmospheric conditions for prolonged periods. The solubility of FOSs is rarely higher (up to 80% in water at room temperature copared to inulin, just 35%, Douwina Bosscher, 2008). The viscosity of an FOS solution is relatively higher than that of sucrose when at the same concentration, and the thermal stability is also higher than that of sucrose (Neosugar User’s guide, Meiji Seika Co., Kawasaki- shi, Japan, 1982). FOSs are highly stable in the normal pH range for food (5.0-10.0) and at refrigerated temperatures over one year. Nevertheless, as a whole, when the pH falls below 4.0 and treatment temperature is high, they can be hydrolyzed. 10 There have been few published studies comparing the physicochemical properties of FOSs from sucrose, there is a strong indication that FOSs resemble sucrose in many properties such as solubility, freezing and boiling points, crystal data, etc. FOSs can also be used to alter the freezing temperature of frozen food to control the intensity of browing due to the Maillard reaction 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-fructofu