STUDY OF THE INULIN CONCENTRATION BY PHYSICAL METHODS

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1 Drying 2004 Proceedings of the 14th International Drying Symposium (IDS 2004) São Paulo, Brazil, August 2004, vol. B, pp STUDY OF THE INULIN CONCENTRATION BY PHYSICAL METHODS Juliana Tófano C. Leite 1, Paula Martinelli 1, Fernanda Elisabeth. X. Murr 1 and Kil Jin Park 2 1. School of Food Engineering Campinas State University (UNICAMP), P.O.Box 6121, Campinas, SP, Brazil, juliana@agr.unicamp.br, fexmurr@fea.unicamp.br 2. School of Agricultural Engineering Campinas State University (UNICAMP), P.O.Box 6011, Campinas, SP, Brazil, kil@agr.unicamp.br Keywords: inulin, physical separation, experimental design ABSTRACT This research work reports the development and optimization of a process to obtain an inulin concentrated solution from chicory roots which will be employed latter to feed a spray drier to produce a final powder product. The initial inulin solution was obtained from milled chicory roots through a diffusion process in hot water. The liquid extract obtained was filtered and concentrated by evaporation. The concentrated extract was then submitted to a precipitation process by temperature lowering and subsequent phases splitting by centrifugation. Variables involving inulin precipitation yield (w/w) were identified and studied as: precipitation temperature (from -24 to 16ºC), centrifugation velocity (from 6,000 to 10,000 rpm) and centrifugation time (from 15 to 45 minutes). A central composed experimental design with five variation levels was set and the response surface methodology was applied for data analysis. Statistical analysis showed that inulin precipitation yield was affected by the precipitation temperature. INTRODUCTION Inulin is a reserve carbohydrate consisting in a chain of fructose molecules with a terminal glucose molecule. It may be classified as a fructoligosaccharide (FOS), which can be found in more than 30,000 vegetable products. Amongst these vegetables, chicory roots are outlined for inulin production in industrial scale due to its stability in producing GFn long chains and regular growing, even in moderate climates (Van Loo et al., 1995; Silva, 1996). The application of inulin in the food industry, at first, was restricted to the drink production similar to coffee, due its bitter taste. However, it was recently 868

2 discovered that inulin could act as a sugar or fat substitute, with the advantage of exhibiting a very low caloric value. From then on, inulin has been used as an ingredient in foods with reduced or no sugar and fat at all, such as chocolates, ice creams and yogurts, among others. Its use in products with low calories and reduced fat levels is already diffused in Europe, in the United States and in Canada. Besides acting as a sugar and fat substitute, inulin also presents some functional properties. It acts in the human organs in a similar way as dietary fibers, contributing to the gastrointestinal system overall conditions improvement (Roberfroid, Gibson, Delzene, 1993). Due to above mentioned properties, food and pharmaceutical industries have found applications for inulin in functional food production, in nutritional composites and medicines. Inulin is mostly often commercialized as a powder product. This type of product presents advantages due to its easier manipulation, transportation, storage and consumption. The most often used method for this form of the inulin obtainment is the liquid extract drying through a spray drying process, which requires a significant amount of energy. Concentration by liquid extract evaporation before drying is the procedure generally adopted to reduce energy expenses. In accordance to comments presented by Silva (1996), inulin is water soluble and its solubility varies according to temperature. At 10 o C, the solubility is 6%, compared with 35% at 90ºC. Due to its low solubility at low temperatures, the inulin liquid extract, suffers a splitting of phases when cooled or frozen. The most concentrated phase precipitates as a pastelike substance (precipitated), with a higher inulin concentration (Leite, 2001; Park et al., 2000). In accordance to these authors, the cooling of the inulin liquid extract followed by the splitting of phases through a physical method using only the precipitated phase on the drying by atomization, would be an alternative for the concentration of inulin liquid extract. The objective of this work was to optimize a process to obtain an inulin concentrated solution to feed the spray dryer to produce inulin powder. The initial inulin solution was obtained from milled chicory roots through a diffusion process in hot water. Then, the liquid extract was filtered and concentrated by evaporation up to 16ºBrix. The concentrated extract was then submitted to a lowering temperature process and a subsequent phases splitting by centrifugal operation. For the optimization of the precipitation process of the inulin the yield of inulin precipitation (w/w) was studied in terms of three variables: precipitation temperature (-24, -16, -4, 8 and 16ºC), centrifugation velocity (6,000; 6,810; 8,000; 9,190 and 10,000rpm) and centrifugation time (15, 21, 30, 39 and 45 minutes). A central composed experimental design with five levels was used and a response surface methodology was applied for the analysis of the data. Obtainment and preparation of raw material METODOLOGY Chicory seedlings have been carried out in the experimental field of Faculty of Agricultural Engineering at UNICAMP (FEAGRI/UNICAMP) located in Campinas, state of São Paulo. There have been used seeds of Cichorium intybus species var Orchies provided by CPQBA/Unicamp. The sowing was done in the beginning of March, in styrofoam trays, using vermiculite as substrate. Irrigation was made by a diffuser, at intervals of 12 hours, in the beginning and at the end of the day. The seedlings were transplanted to the field a month after the sowing. The planting was made in seedbeds, spaced 50 cm between lines and 25 cm between plants. Sprinkle irrigation system was adopted keeping four sprinklers equally spaced, located at the center of the planting area. Chicory harvest was made during the spring time, at the end of October, before flowering had occurred. That was the period of highest inulin concentration in the roots (Figueira, 2001). After flowering, inulin begins to be broken into fructose to be consumed by the plant, leading to a concentration decrease. 869

3 Chicory roots were washed with pressurized water jet in order to remove soil and other remaining impurities. In sequence, deteriorated parts were removed and the roots were sliced to begin inulin extraction process. Obtainment of the concentrated inulin liquid extract. The extraction of inulin was made through diffusion in hot water. Roots were firstly triturated in a multiprocessor of Stephani brand. Extraction was carried out in an ICMA vaporized boiler, with an agitation device. The extraction process was made by adding hot water to the triturated roots, in a 1:2 proportion (roots:water). The mixture was then kept at the average temperature of 80 ± 2 C for 1 hour, with constant agitation. Extraction was made in four stages, according to the boiler capacity. Liquid inulin extracts obtained at the end of each stage were filtered in a cloth to remove large root pieces, afterwards, filtered in coffee strainer to remove smaller particles. The filtered extracts were submitted to a concentration process by evaporation by means of a Flender Himmel vacuum evaporator. To avoid foaming it has been added a silicone based antifoamer to the filtered extract, in a concentration of 300 ppm. The concentration process by evaporation has been carried out at an average temperature of 70 ± 3 C, until the extracts presented a soluble solids concentration of approximately 16 Brix. The process took place under a vacuum of 15 ± 2 inches of Hg, associated to a water flux in the pump of 250 L/h. The concentrated liquid extracts obtained at each stage were mixed and homogenized at 80 C for an hour with constant agitation. Inulin precipitation process Inulin precipitation was made by a reduction of temperature followed by a centrifugation to separate the phases. At the first stage, there had been taken samples of approximately 600g of concentrated extract and put in plastic recipients. These recipients were stored in five different environments with temperatures varying between 16 and 24 C, according to the levels defined in the experimental design. To obtain the precipitated inulin, centrifugation process was performed after temperature reduction. The simply cooled samples were taken off from the refrigerators and placed directly into the centrifuge. Frozen samples were primarily defrosted at room temperature and next centrifuged. The centrifugation process was performed at a temperature of 5 C in a Sorvall RC 26 Plus centrifuge, using a superlite GSA Sorvall rotor, model SLA1500, with 250 ml tubes being the velocity and centrifugation time varying according to the experimental design levels. For the centrifugation process, 540g of sample have been weighted, equally divided in three tubes with 180g each. The analysis has been made in duplicate. Precipitation mass yield was calculated as the ratio between the total precipitated mass of inulin and the concentrated extract mass put inside the centrifuge tubes. Average mass yield was calculated as the average between the trials repetitions. To optimize the inulin precipitation process, it was adopted a 2 3 factorial experimental design of 6 trials plus 3 central points, totalizing 17 trials. Three independent variables were evaluated: cooling/freezing temperature of the concentrated extract or precipitation temperature (Tp), centrifugation velocity (v) and centrifugation time (t). The precipitation mass yield (PY) response was analyzed based in the results obtained by Leite (2001). The analysis of the independent variable effects on the precipitation mass yield was assayed by a mathematic model of second order, using the software Statistica 5.0, according to the Equation (1) 870

4 2 2 2 PY = bo + b1tp + b2v + b3t + b11tp + b22v + b33t + b12tp v + b13tp t + b23vt (1) The validity of the obtained mathematic model was verified through the calculation of analysis of variance and relative deviations between the provided values by the mathematic model and the experimental values of precipitation mass yield. In Table 1, the real values corresponding to codified levels of the experimental design are presented. The 17 trials corresponding to the experimental design can be seen in Table 3, where the precipitation mass yield results are presented as well. Table 1 Real values of experimental design levels Variables Codified Levels Precipitation Temperature (ºC) Centrifugation velocity (rpm) 6,000 6,810 8,000 9,190 10,000 Centrifugation time (min) Extraction process results RESULTS AND DISCUSSION There had been harvested 369 plants, resulting in kg of root, 76.15% of which were healthy, with white surfaces. In 88 roots, corresponding to 23.85% of the total harvested plants, a darken of some external superficial parts and also of some internal area was observed, beyond an extremely unpleasant smell. In some plants, a deterioration of 100% of the root has been observed with a consequently total loss of plants at the experimental field. In other plants, the affected region was smaller, making possible the use of non-affected areas. In Table 2, water and root masses used in each one of the four extraction stages are presented, as well as the soluble solid concentration and mass of the resulting filtered extract. Table 2 Data from the stages of extraction process of inulin from chicory roots Extraction process Data Process nº Total Root mass (kg) Water mass (kg) Filtered extract mass (kg) Soluble solids ( Brix) Concentration by evaporation of the filtered liquid extracts had an average yield of 34.05%, resulting in kg of concentrated liquid extract, with 16 Brix of soluble solids concentration. Inulin precipitation process The precipitation mass yields obtained from different trials varied from 40.8 to 23.1%, as shown in Table 3. The best result was obtained from the trial 9, which precipitation temperature was of 24 C, 871

5 centrifugation velocity of 8,000 rpm and centrifugation time of 30 min. In contrast, the worst result corresponded to the temperature of 16 C, to the same conditions of velocity and time of centrifugation. These results show a tendency of an increasing inulin precipitation mass yield when precipitation temperature decreases, independently of centrifugation velocity and time. Table 3 Average mass yield corresponding to the inulin precipitation essays of a 2 3 complete factorial experimental design, with 6 axial points and 3 central points. Variables PY (w/w) Trial Std Variation Number Tp V T Average(%) Deviation(%) Coeff. (%) To verify the effect of each variable on the precipitation mass yield, an optimization of the precipitation process of inulin was performed by using the software Statistica 5.0 and the data presented in Table 3. The adjustment coefficients of the experimental data to the mathematic model represented by the Equation (1) are presented in Table 4. Table 4 Regression coefficients of the modified mathematic model -95% +95% Regression Standard Effect t (7) P Coefficients Error Conf. Conf. Limit Limit b o * b 1 * b b b b b b b b Statistically significant coefficients at 95% of confidence 872

6 The results presented in Table 4 show that, at a confidence level of 95%, only coefficients b 0 and b 1 were statistically significant. This means that, from the studied variables, only the precipitation temperature had a statistically significant effect on the precipitation mass yield, in the considered interval. The negative value of coefficient b 1 shows a tendency of an increasing precipitation yield when the temperature is reduced, independently of the centrifugation velocity or time. Based on these results, the codified mathematic model that represents the precipitation process can be reduced to a linear model of only two parameters with a correlation coefficient of R 2 = 0.906, as shows the Equation (2). PY ( w / w) = T p (2) To verify the validity of the model represented by the Equation (2), the analysis of variance correspondent to the precipitation mass yield was performed. The results can be seen at Table 5. The absolute average deviation of the precipitation mass yield obtained from Equation (2) in relation to the experimental values was of 4.63%. In the majority of the trial, the deviations were under 10% and the maximum deviation observed was of 12.08%, in the trial 12. Table 5 Analysis of variance correspondent of the codified mathematic model reduced from the inulin precipitation mass yield *, evaluated with a 95% of confidence. Source of variation Sum of squares Degrees of freedom Mean Squares Regression Residual Lack of fit Pure error Total * Regression coefficient R 2 = Ftest F 0.95;1; F 0.95;13; By observing the values presented in the analysis of variance, it can be noted that, to a confidence of 95%, the F value calculated between the regression and the residues is almost 40 times larger than the value of F from table and that the value of F calculated between the lack of adjustment and the pure deviation is around 24 times less of the value of F from table. These results, allied to the low values observed for the relative deviations and to the high value of the regression coefficient (R 2 = 0.906), indicate that the proposed model to describe the response surface of the inulin precipitation mass yield in function of the precipitation temperature is adequate. The mathematics model obtained to the inulin precipitation mass yield (Equation 2) was used to construct a response surface as a function of the precipitation temperature, which can be seen in Figure

7 above (a) Response Surface above t (min) Tp (ºC) (b) Contour line Figure 1 Response surface and contour lines obtained from the reduced codified model correspondent to precipitation mass yield From Figure 1, it can be noted that that the precipitation temperature has a negative effect on the mass yield, in the studied values range. As it can be seen from Figure 1b, the temperature of 24 C was the one that presented better precipitation mass yield, independently of the used centrifugation velocity and time. This result is in agreement with the observed by Leite (2001), indicating that the reduction in the storing temperature of the inulin concentrated extract results in a better precipitation mass yield. According to the tendency of the curves observed in Figure 1, the reduction of the precipitation temperature to values inferiors to 24 C would lead to a mass yield even better. However, due to the unavailability of equipments that can reach such temperatures, it was not possible to evaluate the process in this region of inferior temperatures. CONCLUSIONS The obtained results lead to the conclusion that the best condition to the obtainment of inulin, inside the range of studied values, would be storing the extract at the temperature of 24 C. Meanwhile, the centrifugation process could be taken at the velocity of 6,000 rpm and at a time of 15 min, as these variables do not present statistically significant effects on the precipitation mass yield. ACKNOWLEDGMENT We acknowledge to the State of São Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq) to the financial support. NOTATION B Regression Coefficient T Temperature ºC T Centrifugation time min PY Precipitation yield of inulin (w/w) V Centrifugation velocity rpm 874

8 GFn Chemical structure of inulin formed by n fructose molecules *(F) and a terminal glucose molecule (G) Subscripts O Average effect P Precipitation 1 Linear effect of the precipitation temperature 2 Linear effect of the centrifugation velocity 3 Linear effect of the centrifugation time 11 Quadratic effect of the precipitation temperature 22 Quadratic effect of the centrifugation velocity 33 Quadratic effect of the centrifugation time 12 Interaction effect between Tp and v 13 Interaction effect between Tp and t 23 Interaction effect between v and t LITERATURE Figueira, G. M. (2001). Agritechnological development of Cichorium intybus L species. Ph. D Thesis of Agricultural Engineering College, State University of Campinas, Campinas. 68p. Leite, J. T. C. (2001). Obtainment of inulin extract from chicory (Cichorium intybus) by lowering temperature and spray drying. M. Sc. Thesis of Agricultural Engineering College, State University of Campinas, Campinas. 112p. Park, K. J.; Park, T. H. K. B.; Park, K. J. B; Nogueira, R. I.; Leite, J. T. C. (2000), Process of inulin concentrate obtainment by lowering temperature and physical separation. BR Patent number PI August 17, Roberfroid, M.; Gibson, G. R.; Delzene, N. (1993), The biochemistry of oligofructose, a nondigestible fiber: an approach to calculate its caloric value. Nutrition Reviews. Vol. 51, no. 5, pp Silva, R. F. (1996), Use of inulin as a natural texture modifier. Cereal Foods World. Vol. 41, no. 10, pp Van Loo, J.; Coussment, P.; Leenheer, L.; Hoebregs, H.; Smits, G. (1995), On the presence of inulin and oligofructose as a natural ingredients in the western diet. Critical Reviews in Food Science and Nutrition. Vol. 35, no. 6, pp

Many plants use starch polysaccharides as a form of energy storage Two non starch polysaccharides used in much the same way are

Many plants use starch polysaccharides as a form of energy storage Two non starch polysaccharides used in much the same way are Inulin Konjac glucomannan Inulin is composed of beta D fructofuranosyl units