Evaluation of treatments to reduce hardness of agave americana core

ISSN 1330-9862 scientific note (FTB-1544) Evaluation of Treatments to Reduce Hardness of Agave americana Core José A. Ramírez1, Rocio C. Gómez-Ayala1, Cuauhtémoc Jacques-Hernández2and Manuel Vázquez3* 1Department of Food Science and Technology, U. A. M. Reynosa-Aztlán, Universidad Autónoma de Tamaulipas, Apdo. Postal 1015, 88700 Reynosa, Tamaulipas, México 2Centro de Biotecnología Genómica, Instituto Politécnico Nacional, 88700 Reynosa, Tamaulipas, México 3Área de Tecnología de los Alimentos, Departamento Química Analítica, Facultad de Veterinaria, Universidad de Santiago de Compostela – Campus de Lugo, E-27002 Lugo, Spain Received: August 20, 2005 Accepted: November 2, 2005 Summary Agave americana contains inulin as storage carbohydrate. Therefore, agave is interesting to be used for the extraction of inulin by pressing. The yield of the process is low due to the high hardness of the core. The objective of this work was to evaluate pretreatments to reduce the hardness in the process of obtaining inulin by pressing. Treatments with water, sulphuric acid 1 % (by mass) or sodium hydroxide 1 % (by mass) were tested and optimized. The pretreatment of the core of A. americana with sulphuric acid 1 % allowed the reduction of hardness from 30 000 gto 2000 gof breaking force. The mathematical model obtained predicts an optimum processing at 84 °C during 75 min. The treatment with sulphuric acid 1 % also allows white core of A. americana to be obtained, while the other treatments provide yellow core. These results open a good alternative to obtain value added products from this resource. Key words:Agave americana, inulin, hardness, colour Introduction The genus Agave belongs to the Agavaceae family with more than 300 species and occurs natively in arid and tropical regions, particularly in Mexico and Central America. Agave americana L. is a very large plant with rosettes up to 3.5 m wide. The agave can be used to produce two distinct kinds of beverages. The first is dropped as sap from the living plant and consumed fresh, known as aguamiel or fermented, known as pulque. The second kind is the distilled liquors known as mezcal or tequila. The market for tequila is growing due to the recently recognized origin denomination for it by the European Union, which means more tequila will be produced with a substantial improvement of the process and tequila quality. The main Agave used for the production of tequila is Agave tequilana, but analogous products can be obtained with other species of Agave. For instance, A. americana is used to obtain high-fructose fermentable liquor for making mezcal, a distilled alcoholic beverage similar to tequila, by using natural or controlled fermentations. Although agave plants are associated with Mexico, several agave species were introduced in Spain in the 1940s. Actually, in the southeast Spain, a problem exists due to the invasive characteristics of these plants, with high growth rate in sandy soils (1). Finding applications for the agave could promote a reasonable control of their population. 545 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) *Corresponding author; Phone: ++34 982 285 900 ext. 44 139; Fax: ++34 982 285 926; E-mail: [email protected] A new application for this plant is the production of inulin. This is an indigestible oligosaccharide (fructan) that is found in many plants as a storage carbohydrate. Inulin has been defined as a polydisperse carbohydrate material consisting mainly, if not exclusively, of b(2®1) fructosyl-fructose links. It is present in many regularly consumed vegetables, fruits and cereals such as leek, onion, garlic, wheat, chicory, artichoke and banana. Inulin is a dietary fiber that can be used as food additive or ingredient for food products (2). Inulin has good fat mimic properties when combined with water. It can stabilize low-fat mousses and spreadable products, and it shows synergetic gelling effects when combined with gelling agents. Inulin is a prebiotic. Recent nutrition research has shown interesting results with regard to calcium absorption and colon cancer prevention (3). Nowadays, the inulin commercially available on the food ingredient market is either synthesized from sucrose or extracted from chicory roots (Cichorium intybus). A. americana and A. tequilana contain inulin as storage carbohydrate. Therefore, an interesting use of agave is the extraction of inulin by pressing, but the yield of the process is low due to the high hardness of the agave core. After the extraction by pressing, the agave bagasse can be used to obtain agave fiberboards that have moisture and mechanical properties comparable to medium specific gravity fiberboards made by using aspen fiber (4). For that reason, the waste from inulin production process would be eliminated. The objective of this work was to reduce the hardness of the core of Agave americana as a pretreatment in the process of obtaining inulin by pressing. Treatments with water, sulphuric acid or sodium hydroxide were tested and optimized. Materials and Methods Raw material The A. americana used in the experiments was collected in San Carlos county (Tamaulipas, Mexico). The stem was cut to separate the core and the leaves. The core was saved in a single lot in plastic bags, frozen and stored until needed. Treatments of samples The pretreatment using water to reduce hardness of the core for the extraction of inulin from A. americana was selected because it is the usual treatment in the production of tequila. Alternative treatments with acid or alkali solutions were selected on the basis of studies of hydrolysis of other lignocellulose material, such as sorghum straw or wood (5,6). The influence of temperature and processing time during pretreatments on hardness was assessed using the response surface methodology, which has been successfully used to optimize biochemistry and biotechnology processes related to food systems (7). The set of experiments followed a second-order factorial structure (8). Treatments were performed at 80, 100 or 122 °C in media containing 1 g of H2SO4per 100 g of water,1gof NaOH per 100 g of water or only water. The values of acid or alkali concentrations were selected according to the literature for hydrolysis in similar materials (5–9). All experiments were performed using a water/solid ratio (WSR) of5gofwater per1gofagave on dry basis. Samples were collected at treatment times 60, 100 and 120 min and filtrated to separate the core and the liquor. The treated cores were washed with cold sterile water and the hardness was tested as it is shown below. The liquor was used to determine the concentration of carbohydrates released (fructose, glucose and xylose) by HPLC (Agilent Technologies, Palo Alto, CA, USA) and furfural by spectrophotometry (Shimadzu, Kyoto, Japan). The analyses were carried out as shown in the literature (10). Mechanical properties Samples of products of 5 cm×5cm×2cmwereob - tained and equilibrated to room temperature for 30 min in a plastic bag to avoid dehydration before the mechanical properties were measured. The mechanical properties were determined using a TA-XT2i Stable Micro Systems Texturometer (Viena Court, England). The hardness test (Warner Bratzler test) was performed by compressing the samples of the core to 25 % of the initial height using a compression speed of 60 mm/min and an AACC probe of 36 mm. The breaking force expressed in gram-force (g) for each treatment was measured (1 gram-force is 0.0098 N). Samples were placed on the base of the texturometer, taking care that the probe reached the sample at the centre (11). Six samples were analysed for each treatment. Colour attributes Spectral reflectance of A. americana core after the treatments was determined using a HunterLab colorimeter (Model Miniscan, Reston Virginia, USA) calibrated against white tile. CIE L*, a*, and b* values, chroma ([a*2+b*2]1/2), and hue angle (arc tan b*/a*) were calculated based on illuminant C and the 2° standard observer, as referenced in the literature (12). Six samples were analysed for each treatment. Statistical analysis The interrelationship between operational and dependent variables was established through the following equation including linear, interaction and second-order terms: yi=b0i +b 1ix1+b2ix2+b3ix1·x2+b4i x11+b5i x22, where yi (i=1–3) are the dependent, normalized variables and b0i...b5i are regression coefficients calculated from the experimental data by multiple linear regressions. A second-order multiple regression analysis using the least squares regression methodology was performed using Microsoft Excel 7.0 (Microsoft Corporation, Redmond, WA, USA) software. Microsoft PowerPoint 7.0 (Microsoft Corporation, Redmond, WA, USA) was used to plot the experimental data and models. The treatments were performed in triplicate. All texture and colour data presented are mean values of six determinations. 546 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) Results and Discussion Hardness analysis The temperature and time were considered as operational variables (denoted qand t, respectively) and their effects on the selected dependent variable hardness after the treatment (yi) were calculated for each treatment: water y1, acid y2and alkali y3. For computation purposes, the normalized, dimensionless variables x1and x2were defined as: x1=(q–100)/20 and x2=(t–120)/60. Table 1 summarizes the variables involved in the optimization of the pretreatment for the core of A. americana. The operational conditions assayed (in terms of dimensional and dimensionless operational variables) as well as the experimental results determined for yi(i=1 to 3) are shown in Table 2. Empirical mathematical models were obtained by processing these data. Table 3 shows the values of the coefficients from the models and their statistical significance. The range of study for the variable temperature was selected between 80 and 120 °C because 100 °C is the usual temperature commonly recommended for extraction of sugar in the production of tequila or mezcal. The range for the variable time was selected at 60–180 min because the objective is only to decrease the hardness and longer time periods can release sugars to the medium. The experimental values for hardness after the treatment with water (y1) varied over the range 9981–28443 g. The analysis of the main experimental trends and the values of coefficients listed in Table 3 suggest that the effect of temperature and time in the treatment using 547 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) Table 1. Variables used in the study and their nomenclature, units and ranges. The ratio liquid/solid was5gofwater per1gof agave on dry basis (a) Dimensional independent variables Nomenclature Units Variation range Temperature q°C (80–120) Processing time tmin (60–180) (b) Dimensionless, normalized independent variables Nomenclature Definition Variation range Dimensionless temperature x1(q–100)/20 (–1–1) Dimensionless time x2(t–120)/60 (–1–1) (c) Dependent variables Nomenclature Units Hardness after the treatment with water y1g(gram-force) Hardness after the treatment with sulphuric acid y2g(gram-force) Hardness after the treatment with sodium hydroxide y3g(gram-force) Table 2. Operational conditions assayed and experimental results achieved for the pretreatments with water, sulphuric acid or sodium hydroxide Experiment Independent variables Dependent variables Dimensional Dimensionless q/°C t/min x1x2y1/gy2/gy2/g 1 80 60 –1 –1 20254 4085 10114 2 80 120 –1 0 18701 2800 8325 3 80 180 –1 1 22976 7050 7567 4 100 60 0 –1 10703 6958 31096 5 100 120 0 0 9981 5672 20376 6 100 180 0 1 28443 7196 15074 7 120 60 1 –1 23813 20591 14941 8 120 120 1 0 22556 20412 11443 9 120 180 1 1 26340 16947 8303 qis temperature and tis time, x1and x2are the related coded variables, y1is the hardness after the treatment with water, y2is the hardness after the treatment with sulphuric acid and y3is the hardness after the treatment with sodium hydroxide Table 3. Regression coefficients and statistical significance of the empirical models that predict the relationship between temperature and time of the pretreatment and the hardness obtained in the core Coefficients y1 (j=1) y2 (j=2) y3 (j=3) b0j 12480a6046b21425c b1j 963 7335c1466 b2j 2998 –73 –4201a b12j 1201 –1652 –1022 b11j 6897 5372b–12066b b22j 5842 843 1134 acoefficients significant at 90 % confidence level bcoefficients significant at 95 % confidence level ccoefficients significant at 99 % confidence level y1is the hardness after the treatment with water, y2is the hardness after the treatment with sulphuric acid and y3is the hardness after the treatment with sodium hydroxide water do not have a clear effect on the hardness of the core of A. americana. The resulting variation pattern is shown in Fig. 1. Although it appears that a minimum value of hardness exists, the coefficient of determination r2was not significant (0.65). The effect of the treatment with water did not decrease hardness of the core enough to be a good pretreatment for the pressing of the core. The treatment with sulphuric acid1%(y 2) showed a behaviour different from the treatment with water (y1). After the treatment, the hardness varied within the wide range of 2800–20 591 g. The most favourable conditions were defined by low value of temperature and intermediate value of time (experiment 2). The significance analysis of the regression coefficients for y2showed that the hardness was significantly affected by the variable temperature and the second-order term of this variable but was not significantly affected by the variable time. In this case the coefficient of determination r2was significant (0.98). The predictions of the empirical model for the dependence of hardness after the treatment with sulphuric acid1%ontemperature and time are shown in Fig. 1. The optimization of the treatment using the model obtained shows that the best conditions are 84 °C and 75 min, given a core with a predicted hardness of 3154 g. Visual analysis of Fig. 1 allows to conclude that low values of temperature and time produce a core with low hardness (<5000 g). These values of hardness are adequate for the pressing used in the extraction of inulin or the grinding for the hydrolysis of sugars that can be used for fermentation in the production of mezcal or tequila. The last pretreatment, with sodium hydroxide 1 %, allowed hardness in the range of 8303–31 096 gto be obtained. The predictions of the empirical model for the dependence of hardness after this treatment on temperature and time are also shown in Fig. 1. Time and the second-order term of the variable temperature were the terms most influential in the model obtained shown in Table 3. The coefficient of determination r2was significant (0.90), but the values of hardness were not low enough for the posterior processing by pressing. Sugar released It was clear that the best results to reduce the hardness were obtained using sulphuric acid1%atlowtem - perature and time. However, in addition to low hardness, it is also interesting to maintain the sugars in the core for a posterior extraction by pressing or lixiviation (2). Fig. 2 shows the main sugars released to the medium in the pretreatment with water. Fructose, glucose and xylose were always under 3 g/L. The concentration of sugars in the medium increased with temperature and time. The effect of the treatment with sulphuric acid 1 % on the sugars released is shown in Fig. 3. Fructose concentration increased with temperature and time and it was released up to 9.6 g/L. The fructose was a product of the hydrolysis of the inulin from the core. Therefore, a high concentration of fructose in the medium after the treatment is not favourable for reducing hardness if a high yield in the posterior extraction of inulin by pressing is desired. In the conditions for the lowest hardness (around 80 °C and 60 min), the fructose concentration was only 2.6 g/L. The other sugars (glucose and xylose) also showed low concentrations (1.2 and 0.4 g/L). Fig. 4 shows the results for the sugars obtained in the medium after the treatment with sodium hydroxide. The values of fructose concentration were under 1.8 g/L for temperature of 80 or 100 °C. The concentration in- 548 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) Treatment: Water 0 0 0 5000 5000 5000 10000 10000 10000 15000 15000 15000 20000 20000 20000 25000 25000 25000 30000 30000 30000 60 60 60 84 84 84 108 108 108 132 132 132 156 156 156 180 180 180 80 80 80 88 88 88 96 96 96 104 104 104 112 112 112 120 120 120 Time / min Time / min Time / min Temperature/ º C Temperature/ º C Temperature/ º C Hardness/g Hardness/g Hardness/g Treatment: Sulphuric acid 1 % T reatment: Sodium hydroxide 1 % Fig 1. Effect of temperature and time on the hardness parameter for the three pretreatments assayed to reduce hardness in the core of Agave americana creased up to 3.2 g/L when 120 °C was selected. Glucose and xylose concentrations also gave values under 1.4 g/L. Low concentrations obtained for all sugars confirm that the alkali hydrolysis in the conditions of temperature and time studied was moderate and it does not allow a modification of the structure of the system to reduce hardness. Therefore, the acid hydrolysis in these conditions of temperature and time was moderate enough to release sugar, but strong enough to change the structure of the lignocellulose matrix of the core, with the consequent decrease of hardness. These results confirm that the treatment of the core of Agave americana with sulphuric acid1%isanadequate pretreatment to reduce hardness. The analysis of oligomers released from inulin confirmed that the conditions assayed for the three treatments were moderate because the oligomer concentration was always under 1 g/L. The results obtained suggest that a small release of inulin sugars promotes a restructuring of the hemicellulosic matrix causing low hardness of the core. This was observed at low tempera- 549 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) 0 2 4 6 8 10 12 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 0.5 1.0 1.5 2.0 2.5 0 2 4 6 8 0 0 80 ºC 100 ºC 120 ºC 80 ºC 100 ºC 120 ºC 80 ºC 100 ºC 120 ºC 0 50 100 150 20 0 Time / min 0 0 50 100 150 200 Time / min 0 0 50 100 150 200 Time / min 0 g(fructose)/(g/L) ( g l ucose )/( g / L ) g (xylose)/(g/L)g Fig. 3. Effect of the treatment with sulphuric acid1%onthere - leased sugar concentrations from the core of Agave americana 0 0.5 1.5 2.5 g(fructose)/(g/L) 0 0.5 1.0 1.5 ( g l ucose )/( g / L ) g 0 0.5 1.0 1.5 2.0 2.5 (xylose)/(g/L)g 0 1.0 2.0 3.0 0 50 100 150 20 0 Time / min 0 80 ºC 100 ºC 120 ºC 0 2.0 0 80 ºC 100 ºC 120 ºC 80 ºC 100 ºC 120 ºC 0 50 100 150 200 Time / min 0 0 50 100 150 20 0 Time / min 0 Fig. 2. Effect of the treatment with water on the released sugar concentrations from the core of Agave americana tures. Using high temperatures, the initial sugars released can react with other compounds of the core forming new polymers, causing higher hardness and making further release of inulin sugars difficult. Colour parameters Other effects of the treatments of the core of agave could be the changes in the surface colour due to Maillard reactions and caramelization. These reactions should have low intensity because their rates are increased with temperature over 170 °C (13). Table 4 compares the colour attributes for the samples after the treatment with water. The L* values decreased with time from 68.2 to 57.8 at 80 °C, from 65.9 to 57.8 at 100 °C and from 65.8 to 62.1 at 120 °C. This decrease indicates a decrease in the whiteness but the values of L* remain in the range in which the samples can be considered as white. The a* parameter was in the range of 5.4–2.2. There were not significant differences between the samples for this parameter. In the treatment at 80 °C, the b* parameter also decreased from 28.7 to 12.41 with time. This means that the samples lost some yellowness but still showed a colour between white and yellow. Table 4 also shows the colour attributes after the treatment with sulphuric acid 1 %. A high value of L* (72.9) was observed in samples treated at 80 °C for 60 min. The a* parameter was in the range –0.7 and 8.5. This parameter indicates the tendency between green (negative values) and red (positive values). The samples were close to zero, therefore there is not a clear appreciation of these colours in them. The values of b* show that with this treatment a core that was whiter than in the treatment with water was obtained. 550 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006) 0 05 1.0 15 2.0 25 3.0 3 5 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 0. 1. 2. 3 . 0 0 0 0 0 1 1 1 0 0 0 0 0 1 1 0 50 100 150 200 Time / min 0 0 50 100 150 200 Time / min 0 0 50 100 150 200 Tim e/ min 0 80 ºC 100 ºC 120 ºC 80 ºC 100 ºC 120 ºC 80 ºC 100 ºC 120 ºC g(fructose)/(g/L) (glucose)/(g/L)g (xylose)/(g/L)g Fig. 4. Effect of the treatment with sodium hydroxide1%onthe released sugar concentration from the core of Agave americana Table 4. Colour attributes (L*, a* and b*) of cores of Agave americana after the treatments q/°C t/min L* a* b* Treatment with water 80 60 68.2 3.7 28.7 120 63.1 3.8 21.5 180 60.1 2.2 12.4 100 60 65.9 2.2 22.3 120 58.5 3.8 22.6 180 57.8 3.9 14.4 120 60 65.9 5.4 27.4 120 62.5 3.5 23.5 180 62.1 4.5 18.1 Treatment with sulphuric acid 1 % 80 60 72.9 –0.2 10.5 120 72.3 –0.7 11.1 180 68.0 3.6 21.9 100 60 66.1 0.6 18.0 120 62.2 8.5 31.3 180 58.3 3.3 14.6 120 60 71.0 1.0 13.9 120 66.6 8.5 32.1 180 61.0 3.9 12.4 Treatment with sodium hydroxide 1 % 80 60 68.2 0.5 19.2 120 63.8 3.3 19.5 180 63.5 5.0 26.3 100 60 63.5 3.9 23.2 120 53.6 5.3 18.7 180 51.1 3.6 18.8 120 60 65.1 2.6 23.1 120 59.9 3.1 18.5 180 61.0 3.9 12.4 qis temperature and tis time The colour attributes after the treatment with sodium hydroxide1%(Table 4) showed a similar behaviour to the treatment with water. The L* values decreased with time from 68.2 to 63.5 at 80 °C, from 63.5 to 51.0 at 100 °C and from 65.0 to 59.8 at 120 °C. This is indicative of a decrease in whiteness. The a* parameter remains very low and the b* parameter shows a trend to yellowness. After the analysis of the colour attributes, it can be concluded that in the treatment with sulphuric acid 1 % white core of A. americana was obtained, while the other treatments provided yellow core. Conclusions The pretreatment of the core of A. americana with sulphuric acid 1 % allows the reduction of the hardness by a factor of 15, with low release of sugars and remaining white colour of the core. The core after this treatment is suitable to be processed to extract inulin by pressing. This opens a good alternative to obtain value added products from this resource. References 1. E.I. Badano, F.I. Pugnaire, Invasion of Agave species (Agavaceae) in south-east Spain: Invader demographic parameter and impact on native species, Divers. Distrib. 10 (2004) 493–500. 2. C. Blecker, J.P. Chevalier, J.C. Van Herck, C. Fougnies, C. Deroanne, M. Paquot: Inulin: Its physicochemical properties and technological functionality. In: Recent Research Developments in Agricultural and Food Chemistry, Vol. 5, S.G. Pandalai (Ed.), Research Signpost, Trivandrum, India (2001) pp. 125–131. 3. P. Coussement, A. Franck, New food applications for inulin, Agro Food Ind. Hi-Tech, 9 (1998) 26–28. 4. G. Íniguez-Covarrubias, S.E. Lange, R.M. Rowell, Utilization of byproducts from the tequila industry: Part 1: Aga- ve bagasse as a raw material for animal feeding and fiberboard production, Bioresour. Technol. 77 (2001) 25–32. 5. S.J. Téllez-Luis, J.A. Ramírez, M. Vázquez, Modelling of the hydrolysis of sorghum straw at atmospheric pressure, J. Sci. Food Agric. 82 (2002) 505–512. 6. J.C. Parajó, V. Santos, M. Vázquez, Production of carotenoids by Phaffia rhodozyma growing on media made from hemicellulosic hydrolysates of Eucalyptus globulus wood, Biotechnol. Bioeng. 59 (1998) 501–506. 7. M. Vázquez, A.M. Martin, Mathematical model for Phaffia rhodozyma growth using peat hydrolysates as substrate, J. Sci. Food Agric. 76 (1998) 481–487. 8. G.E.P. Box, W.G. Hunter, J.S. Hunter: Statistics for Researchers, Reverté, Barcelona, Spain (1988). 9. R. Aguilar, J.A. Ramírez, G. Garrote, M. Vázquez, Kinetic study of the acid hydrolysis of sugar cane bagasse, J. Food Eng. 55 (2002) 309–318. 10. A. Herrera, S.J. Téllez-Luis, J.J. González-Cabriales, J.A. Ramírez, M. Vázquez, Effect of the hydrochloric acid concentration and time on the hydrolysis of sorghum straw at atmospheric pressure, J. Food Eng. 63 (2004) 103–109. 11. W.Z. Chen, R.C. Hoseney, Development of an objective method for dough stickiness, Food Sci. Technol. 28 (1995) 467– 473. 12. R.M. Uresti, G. Velazquez, M. Vázquez, J.A. Ramírez, J.A. Torres, Restructured products from arrowtooth flounder (Atheresthes stomias) using high pressure treatments, Eur. Food Res. Technol.220 (2005) 113–119. 13. J.M. Jin, Y.J. Zhang, C.R. Yang, Four new steroid constituents from the waste residue of fibre separation from Agave americana leaves, Chem. Pharm. Bull. 52 (2004) 654–658. 551 J.A. RAMÍREZ et al.: Reduction of Hardness of Agave americana Core, Food Technol. Biotechnol. 44 (4) 545–551 (2006)