KEREN HAPUQUE PINHEIRO

Título da Tese: PRODUÇÃO DE CICLODEXTRINAS UTILIZANDO BAGAÇO DE MANDIOCA E DEXTRINA DE MANDIOCA COMO SUBSTRATO: OTIMIZAÇÃO E MODELAGEM MATEMÁTICA

Orientadores: 

 fevereiro de 2014 a março de 2017: Prof. Dr. Flávio Faria de Moraes

A partir de abril de 2017: Profª. Drª. Graciette Matioli

 Data da Defesa: 28/07/2017

RESUMO GERAL

INTRODUCTION. Chemically, the cyclodextrins (CDs) are maltooligosaccharides of cyclic molecular structure in the form of a truncated cone, and have a hydrophobic inner cavity whose dimensions increase with the molar mass of the CD. They are generally produced from starch with the enzyme cyclomaltodextrin glucanotransferase (CGTase) and have the ability to form inclusion complexes with organic and inorganic molecules and, therefore, the CDs have numerous industrial applications, such as: catalysis and inhibition of reactions in the chemical industry; separation of enantiomers in chromatographic applications; preservation of vitamins and additives in the form of inclusion complexes in the food industry; stabilization of fungicides and herbicides in agriculture; and potentiation of the solubility and bioavailability of molecules used in the pharmaceutical industry. Therefore, the production of CDs is the object of a large number of researches, in which the aim is to develop new methodologies for CD production, the use of different substrates and various CGTases, and also the behavior of these enzymes regarding the conversion rate of the substrates, since the rates of the catalyzed reactions are affected by the source of the enzyme and strongly depend on the conditions of the reaction medium. In large-scale CD production, knowledge of kinetic mechanisms and testing of other production alternatives, such as the use of new substrate sources, is of utmost importance for increasing yield, improving catalytic efficiency, and reducing the cost of production of CDs, making it possible to use them in large consumption applications, for which their high costs are still prohibitive. AIMS. This work was accomplished with two objectives. First, the study of CD production with the development of a new mathematical model for the kinetics of the enzymes CGTase Toruzyme® and the CGTase of Bacillus firmus, strain 37. Second, the study of the feasibility of producing CDs (α-, β- and -CD) by the action of the commercial enzyme Toruzyme®, using as substrate an agro-industrial residue called cassava bagasse, currently discarded by the cassava starch plants. MATERIAL AND METHODS. For the kinetic study of the enzymes CGTases, the mathematical model of Souza (2003) and Souza et al. (2013) was expanded, in order to also model the kinetics of α-CD production, in addition to β- and γ-CD already considered in the original model of these authors. The new model considers the reversibility of α-, β- , and γ-CD production reactions, that is, it includes the coupling reaction, and disregards the disproportionation and hydrolysis reactions. It was applied to the experimental data of cyclodextrin production obtained with the commercial CGTase Toruzyme® and CGTase produced locally from the Bacillus firmus, strain 37, at different concentrations of substrate consisting of cassava maltodextrin (20, 30, 50 and 100 g L-1 ). In addition, by adjusting the model to the experimental data, the variations of the kinetic parameters of the production reaction of each of the cyclodextrins were analyzed. These results make possible the future development of a fine regulation of the behavior of the enzymatic reactions and, consequently, the manipulation of the yield of the process. In the production of CDs with cassava bagasse agro-industrial residue, the physicochemical characterization of this potential substrate was first carried out, with moisture content at 105 ºC, protein by the Kjeldahl method, crude fiber by acid and basic digestion, minerals by muffle furnace at 550 ºC, lipids with soxhlet by AOAC methodology (2000), and carbohydrate content by phenol-sulfuric method, water activity in Aqualab model 4TE, and pH in a digital potentiometer. In addition, cassava bagasse was characterized according to the FT-IR and FT-RAMAN analyzes and the thermal profiles of TGA and 10 DSC. Then, the best concentration of cassava bagasse and the best temperature to be used in the production of CDs were determined applying a factorial design 22 with triplicate measurement at the central points. The dependent variables were the temperatures 60 (- 1) ºC, 65 (0) ºC and 70 (+1) ºC and the concentrations of cassava bagasse 2 (-1)%, 3 (0)% and 4 (+1 )%, to a volume of 0.05% (v/v) of the Toruzyme® enzyme. When the best conditions of cassava bagasse temperature and concentration were established for CD production, three experiments were carried out by varying the substrate used (bagasse and cassava maltodextrin) with and without the addition of ethanol. The experiments, both for the mathematical modeling, and the production of CDs from the cassava bagasse were carried out in batches under the enzymatic conditions of each CGTase. The CD analyzes were determined by the High Performance Liquid Chromatography (HPLC) technique. The analysis of the statistical results was obtained through the Statistic 10.0 program, with the main and interaction effects of the independent variables being verified, as they responded to the production of α-, β- and γ-CD in a production period of 12 h, with a confidence limit of 95%. The results of the production of CDs in the best process conditions were submitted to Analysis of Variance (ANOVA) and t-test of comparison of means (p=0.05) in the SAS 9.1.3 program (SAS Institute Inc., Cary, NC). RESULTS AND DISCUSSION. The mathematical model developed in this work, for the kinetic study of the two enzymes CGTase, fitted well to the experimental data collected in a test period of 24 h, showing that the hypotheses considered in the model represent the kinetic behavior of Toruzyme® and the CGTase of Bacillus firmus, strain 37, in the reaction media studied. Comparison of the value of the kinetic parameters generated by fitting the model to the experiments with these enzymes showed variations of kinetic behavior in the coupling reaction and inhibitions by the substrate and product. Toruzyme® presented a higher maximum speed of production of α-CD and β-CD products when compared to the enzyme of Bacillus firmus, strain 37. Under the most favorable conditions tested for each enzyme, Toruzyme®, when compared to the CGTase of Bacillus firmus, strain 37, produces a slightly higher amount of β- and γ-CD and expressive amounts of α-CD. Therefore, if the aim of industrial production is to obtain as much CD as possible, then Toruzyme® should be considered, but if there is a greater interest in the production of β-CD, the CGTase of Bacillus firmus, strain 37 may be more advantageous because it produces less α-CD, and purification of β-CD could be carried out with less effort. It should be pointed out that from the analysis of the results of the modeling it was clear that, in the production of CDs, it is necessary to take into account not only the parameters related to the cyclization step, that is, the direct reactions of formation of CDs, but also those of the kinetics of the reverse reactions, because changing conditions such as enzyme concentration and substrate, change the coupling reactions. This affects the concentration of the final products and, consequently, the inhibition constants of the reactions that produce the CDs, a conclusion that is in agreement with studies carried out by other authors. In the study carried out for the production of CDs using dry cassava bagasse, the chemical analysis of this substrate was enriched by the FTIR/ATR and FT-RAMAN, DSC and TGA analyzes that provide complementary information and confirm the high values of Fibers (16.3%) and starch (on the order of 60%). The bioconversion of this high content of starch to CDs is a way to add value to this substrate, as well as to reduce the cost of producing CDs. Moreover, with the results of the factorial design, it was noticed that the higher temperature and substrate concentration (70ºC and 4%) lead to higher concentrations of CDs. However, considering the interactions between the variables with a confidence limit of 95%, the increase in the concentration of the cassava bagasse substrate was statistically significant (p=0.03), only 11 for the increase of β-CD production, showing positive effects for the production of this CD, in particular. A substrate concentration greater than 4% was not used, because the high fiber and starch contents of the cassava bagasse made the reaction medium highly viscous and thus the production system used (batch with magnetic stirring) was not feasible. When 10% ethanol was added to the CD production medium containing cassava bagasse, an increase of 12.7% was observed in the substrate conversion rate that reached 48.46%. And compared to the conversion of the commercial substrate cassava maltodextrin (without ethanol), this value is 2 times greater. For the tested processes of CD production (cassava bagasse in the presence of ethanol, cassava bagasse in the absence of ethanol and cassava maltodextrin in the absence of ethanol), there was a significant difference between the conversion rates of the substrates and a significant increase in the concentration of α-CD. Conversion rates in other works using conventional substrates, such as several starches and dextrins, among others, are similar to those found in this work with cassava bagasse. This behavior may be related to the high fiber content of this residue, which may help to decrease inhibition of the enzyme by the substrate. In addition, variations in the physical structure of the granules and the properties of their starch, such as the amylose content and liquefaction temperature, can facilitate the production of CDs, increasing their concentrations and the final conversion rate. This indicates that this agro-industrial residue, discarded in cassava starch plants, is a promising substrate for CD production. CONCLUSIONS. Therefore, it is concluded that the more comprehensive study of the kinetic behavior of CGTase, as performed in this work, is essential to increase the yield of the CD production process, since only the parameters of the cyclization reactions are insufficient to model the kinetics, and, in addition, the process efficiency is also related to the inverse reactions, such as the coupling reaction. It is observed experimentally that using cassava maltodextrin and each enzyme tested the production of β and γ-CD is slightly larger with Toruzyme® and much larger for α-CD. The enzyme of Bacillus firmus, strain 37 is indicated for the production of β-CD, since it produces less α-CD and thus facilitates the purification of β-CD. The study carried out with the cassava bagasse for the production of CDs have shown that this agro-industrial waste, discarded in cassava starch plants, can be considered a promising substrate for CD production, because, despite the lower amount of starch in suspension in the reaction medium, when compared to the usual starch substrates, the conversion rates obtained are similar. Thus, in addition to the alternative of using cassava bagasse for the production of CDs, the use of these residues allows mitigating the environmental problems resulting from its elimination. The studies on the use of cassava bagasse as a substrate for CD production and the development of the new kinetic model for the CGTases carried out in this work also allow to increase yield and selectivity in the production of CDs and consequently reduce their costs. Keywords: Cyclodextrins, CGTase, mathematical modeling, cassava bagasse.

Artigos Publicados Vinculados a Tese:

https://link.springer.com/article/10.1007/s00449-017-1789-8

https://onlinelibrary.wiley.com/doi/10.1002/star.201800073