Orientadora: Profa. Dra. Graciette Matioli

Data da defesa: 27/03/2015


Microbial synthesis of polysaccharides has emerged as a relevant source of new biopolymers for industrial application. Among them stands out curdlan, hydrocolloid with unique gelling characteristics. Curdlan is a neutral linear exopolysaccharide that is composed exclusively of more than 12000 subunits of glucose linked by β-(1,3)-glycosidic bonds, with an average molecular weight of 74000 Da. It is commercially produced by the bacterium Agrobacterium sp biovar 1. Its water suspension can form distinct gels by temperature setting: a weak gel thermo-reversible obtained by heating at 60 °C and subsequent cooling, and a strong gel thermo-irreversible obtained at temperatures above 80 °C. Gels with different strengths are formed by temperature variation, heating time and curdlan concentration. Furthermore, the gels may be formed in a wide pH range in the presence of sugars, starches, salts, fats and oils. The particular properties of the curdlan satisfy the needs of the food industry, where it is used as a gelling agent and stabilizer, as well as to improve viscosity, texture and stability of the foods. In most foods it is used in the form of athermo-irreversible gel, being stable during distillation processes, severe frying and cycles of freezing and thawing. This biopolymer also presents a high potential for application in the pharmaceutical industry, due to its potent biological activities, besides being considered dietary fiber with possible prebiotic activity. Its versatility in combination with its health benefits is certainly a valuable tool for the development of new products. Studies have been conducted to optimize the production of curdlan and attend the great demand in their applications. The immobilization of microorganisms is an alternative to be explored, and immobilization matrix which is one focus of interest is the loofa sponge. It is abundantly available, inexpensive, easy to apply, highly stable during repeated use, porous, resistant to sterilization, and pH and temperature variations. The application of successive cycles of immobilized cells allows recovery and reuse adapted cells, resulting in a production with high yield and productivity. In this context, the study of the interaction of microorganisms with the immobilization matrix is necessary, and the technique Fourier transform infrared - attenuated total reflectance (FTIRATR) spectrometry can be used for this purpose, because it allows evaluation of the physico-chemical structure of materials, by detecting the absorption bands of chemical groups molecular and the occurrence of modifications. To the knowledge of the authors of this study, the current literature contains no studies of the immobilization of bacterial cells for the purpose of curdlan production. Therefore, the aim of this study was to evaluate the curdlan production by Agrobacterium sp. IFO 13140 immobilized on loofa sponge, alginate, and a combination of loofa sponge with alginate. Subsequently, the aim was to use the immobilization matrix which showed the best performance to optimize the immobilization and the curdlan production, evaluate the production in operating consecutive cycles, and analyze the interaction of the cells with the matrix and its effect on the matrix by the ATR-FTIR technique. The control was represented by the application of free cells for the curdlan production. Firstly, we evaluated the immobilization of the microorganisms in different matrices: loofa sponge, alginate, and combination of loofa sponge with alginate. The curdlan produced was quantified and the immobilization was analyzed by the scanning electron microscopy technique. There was no significant difference in curdlan production and the cells immobilization was detected on all evaluated matrices. The loofa sponge was chosen because of its 10 practical application and economy and because it provides a high stability through its continued use. Immobilization of the microorganism on loofa sponge was optimized by evaluating the combination of the variables time of incubation, initial biomass and shaking rate. The curdlan production was evaluated under different shaking rate of the production medium, under the best conditions for cell immobilization. Then, a kinetic study of the curdlan production was conducted and glucose and nitrogen consumption were evaluated, as was the production of curdlan and biomass over 14 days. Cycles were performed over 10 days and, at the end of each cycle, the loofa sponges containing immobilized cells were removed from the medium and transferred to a new production medium. The biomass present in the production medium was evaluated at the end of each cycle. The biomass immobilized in the matrix was determined after the first production cycle of curdlan. Microorganism cells were immobilized and stored at 4 °C, lyophilized and stored at -18 °C. The storage stability was determined during 300 days. The FTIR-ATR technique was used to characterize the interaction of the Agrobacterium sp. IFO 13140 with the loofa sponge fibers and its influence on the structure of this matrix. Three sponges with and without the presence of the microorganism were analyzed at the end of the first, third and fifth curdlan production cycle. Samples of loofa sponge, pre-inoculum medium, production medium, and loofa sponge with pre-inoculum medium were also analyzed. The curdlan produced was quantified, characterized by FTIR and FTIR-ATR, and its purity was determined. The results were compared with the results obtained for commercial curdlan. The curdlan was applied in yogurt produced with mixed culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus. The acidity and the pH of the product were evaluated during 28 days of storage at 4 °C. The results were compared with the results obtained for the control yogurt without the presence of curdlan. Yogurt with and without the addition of curdlan was prepared and stored for 1 day at 4 °C. Subsequently sensory analysis was performed with 120 untrained panelists. A 9-point hedonic scale was used to evaluate the acceptability of the texture, taste, color, and acidity of the products. Purchase intent was also analyzed. Scanning electron microscopy showed a large number of Agrobacterium sp. IFO 13140 cells that adhered to the surface of the loofa sponge fibers. The loofa sponge is composed of fibers which present a vascular system arranged in a multidirectional way, and its interconnections form an open structure. Thus, the microorganism was able to keep your metabolism and, consequent, the curdlan production. The best conditions for immobilization were 50 mg of cell, 200 rpm, and 72 h of incubation. The immobilized cells produced more curdlan compared with free cells, 19.45 g.L-1 and 14.50 g.L-1 , respectively. Changes in the microenvironment caused by the matrix provided beneficial and convenient immobilization and production of curdlan. For each amount of cell used for immobilization and for each incubation time evaluated, the curdlan production was higher with the increase in the shaking rate. This could be a sign of mass transfer, combined with the increased oxygenation. The regression model used to analyze the results adjusted to the experimental data adequately, with a statistically significant R2 ADJ of 0.82. According to the equation obtained for the coded variables, synergistic interactions were observed among the factors evaluated, demonstrating that the isolated analysis of each variable alone is not appropriate. The process of Agrobacterium sp. IFO 13140 immobilization significantly increased the production in 17.27% of curdlan. The immobilizationof microorganism for 72 h showed better results compared with those for 48 h and resulted in an increase of 7.92% in the curdlan production. The maintenance of immobilized cells in the shaking rate at 200 rpm promoted 11 an increase of 3.65% in the curdlan production. The 150 rpm shaking rate was more favorable for curdlan production by immobilized cells compared with the shaking rates of 110 and 200 rpm, and by free cells. Control of the shaking rate is an important factor for curdlan production because shaking may not provide adequate oxygen supply for an increase in biomass, whereas too much shaking can lead to a stressful environment for a microbial cell. About kinetic study of curdlan production, curdlan production by immobilized cells started 34 hours earlier than that observed in free cells. Glucose exhaustion and maximum curdlan production occurred at approximately 10 days in the assay. The operating stability was evaluated, and until the fourth cycle, immobilized cells retained 87.40% of the production of the first cycle (20.62 g.L-1). The highest volumetric productivity was achieved by immobilized cells on the first cycle, 0.09 g.L-1 .h-1 , while for free cells it was 0.06 g.L-1 .h-1 . The specific rate of production obtained in the immobilization system was 0.37 g curdlan.g-1cells.h-1 , and in the cell free system was 0.03 g curdlan.g-1células.h-1 . The highest production of curdlan by immobilized cells is due to better biocatalytic activity provided by the system. The immobilized cells showed high operational stability. Cell viability and metabolic activity were verified after 300 days of storage at 4 °C and -18 °C. The presence of the microorganism adhered to the loofa sponge was evidenced by the change in the spectrum obtained by FTIR-ATR for the loofa sponge with immobilized cells compared with the spectrum obtained in the absence of cells. This change was represented by the transmittance band at 1080 cm-1 , during all the production cycle, which suggests the presence of curdlan on loofa sponge. This evidences the possibility of the curdlan to be produced also for cell attachment to immobilization matrix. The results showed no changes in the fibers of loofa sponge over five production cycles. The curdlan produced by Agrobacterium sp. IFO 13140 showed the same chemical structure of commercial curdlan, once there was no difference in the spectrum obtained for both molecules. The purity was 87.41 and 91.28%, respectively. The use of curdlan in yogurt resulted in a more stable functional product with lower post-acidification and syneresis compared with the control yogurt. Moreover, the product was not statistically different from the control yogurt in sensory characteristics, which would allow marketing it without impacting its consumer acceptance. The results of this study demonstrate success in immobilizing cells of Agrobacterium sp. IFO 13140 for curdlan production. An innovative process, potentially suitable for industrial scale-up. Furthermore, the use of curdlan in yogurt allowed the development of a more stable product, with high acceptability. Key words: Agrobacterium sp., curdlan, loofa sponge, immobilization, FTIRATR, yogurt.


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