Validación del método para la determinación de pectina en la cáscara de cacao
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Jimenéz Sabogal, Hugo Rodolfo
Delgado Fajardo, Berta Inés
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Abstract This work was developed in Corpoica, its global framework which was implemented at the macro project entitled “PROTOCOLO PARA EL DESARROLLO DE PRODUCTOS CON VALOR AGREGADO PARA MERCADOS NACIONAL E INTERNACIONAL (first approximation)”. The objective of this study was to validate a visible spectrophotometric method for determining the content of pectin in the shell six different clones of Theobroma cacao L (ICS 60, TSH 565, ICS 95, EET 8, CCN 51, SCC 61) of six towns of Tolima, Huila and Santander. The study had five stages: The first one was the selection of the cacao materials, according of the state clones maturity state and appearance. In the second stage clones it was characterized using the methodology proposed by CATIE, finding that the strongest clones come from the town Algeciras. In the third stages was made the pretreatment of biological material, processed at 40°C eliminating volatile components and humidity, with the sections of cocoa dry fruit was continued to milling and screening, reducing the size of the particle to 0,5 mm for storage and codification. After was done the pectine extraction according to the methodology propose by Mollea y col. and Emaga y col. The quantification of pectin based in the methodology proposed by Blumenkrantz and Asboe-Hansen, and Ibarz and col. Finally, in the last stage of the validation, were applied the criteria according to the the Colombian technical standard NTC-ISO/IEC 17025 of 2005, which was considered the guided (appendix K) of the AOAC for the validation of botanical methods and dietary supplements for quantifying majority components. The parameters evaluated were linearity, detection limit, quantification limit, selectivity, precision and accuracy. The linearity was measured using the calibration curve with an intercept of 0, 00382, a slope of 0,00942 , a coefficient of correlation of 1,00, a determination coefficient of 1,00; also to estimate the linearity using statistical analysis the t- Student test with n-2 liberty degrees obtaining a value of 199,98 and was compared with a value t tabulated for a confidence level of 95% equal 2,776 checking the linearity of the system. The confidence limits of the slope were 0.00942±0, 0000156 and 0,00382±0,000607 in the intercept. The detection limit obtained for response of twelve blanks was 5,62 mg/L. The quantification limit was of 6,96 mg/L. The selectivity was established according the processing of specters of absorption of reagent blanks, the acid α-D-galacturonic and two clones per town, obtaining a maximum of absorption of 523 nm, for the standard and the clones. Nonetheless, for rigorous analysis, was realized the correction proposed by Kintner and Van Buren (1982) modified for measure the content of α-D-GalA in the pectine. For that a calibration curve of glucose (the only carbohydrate revealed by TLC), and based in this curve, it was constructed the correction factor obtained by the ratio of the absorbance of the blank and the absorbance generated by the concentration of Glc. The precision of the analytic method was evaluated on the system and the method of repetitive and reproducible conditions, obtaining a CV≤ 5%. The accuracy of the method was determined using the clon EET 8 from Chaparral for five copies in repeatable and reproducible conditions, obtaining a CV≤ 5. The accuracy was evaluated in the system and method. The accuracy of the system was determinated for three different concentrations of the α-D-GalA standard and was obtained a relative error less than 5% and a CV≤ 5%. For the accuracy of the method was chosen the clon CCN 51 from Villarrica for being enriched on three concentration levels, obtaining a recovery rate of 99%. Also the accuracy was estimated using t-Student, with n-1 liberty degrees for a confidence level of 95%,, concluding that test concluding that there is no significant difference between the mean obtained recovery and 100%. In the dtermination of the study matrix, first it was determinate the stability of 5FFA/MHDF complex, for knowing the reading time where the less error margin. After it was quantified in triplicate, each of the four replicates field. Finally it was realized the analysis of variance to estimate if the clones present significative differences and then, the Tukey's test was performed to estimate outliers among clones of each municipality.