Analytical Letters, 41: 1633–1639, 2008 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print/1532-236X online DOI: 10.1080/00032710802122362
MICROWAVE-ASSISTED SAMPLE PREPARATION
Pressurized System and Microwave-Assisted Extraction for Rapid Analysis of Fiber in Animal Feedstuffs G. B. Souza,1 E. A. Menezes,1,2 M. H. Gonzalez,1,2 R. Carapelli,1,2 F. S. Chaves,1,2 W. O. Matos,1,2 and A. R. A. Nogueira1 1
Grupo de Analise Instrumental Aplicada-Embrapa Pecu aria Sudeste, Sa˜o Carlos SP, Brazil 2 Departamento de Quı´mica, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos SP, Brazil
Abstract: A pressurized system under microwave radiation and filter bags to contain the samples was proposed as an alternative to Neutral Detergent Fiber (NDF) determination in animal feed. After optimization by a 23 factorial design, the procedure was applied to nine forage and cereal bran with different amounts of starch (supplied by proficiency testing at various nutrition laboratories). Recoveries from 93 to 110% (0.7 to 7.4 RSD) were obtained. The proposed system allows the NDF simultaneous extraction from 18 samples, increasing the samples throughput from 6 or 40 day 1 to approximately 280 day 1, with low consumption of energy and reagents, producing low amounts of residue. Keywords: Feed analysis, microwave-assisted extraction, neutral detergent fiber, pressurized system
INTRODUCTION Animal feed represents about 70% of the total cost of beef cattle production (NRC 1996). Fiber analysis could provide information to Received 11 October 2007; accepted 2 February 2008. Address correspondence to A.R.A. Nogueira, Grupo de An alise Instrumental Aplicada-Embrapa Pecuaria Sudeste, P. O. Box 339, 13.560-970, Sa˜o Carlos SP, Brazil. Fax: þ 5516 3361 5754; Tel.: þ 5516 3411 5600; E-mail:
[email protected] 1633
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adjust the diet of these animals, and the demand for fiber determination from forages has been documented. Different methods exist for the determination of fiber concentration in feeds. The main challenges facing methods for fiber analysis are reproducibility and relevance, i.e., useful nutrition information (Goering and Van Soest 1970). Besides that, the analysis of fiber in animal feed is one of the most timeconsuming routines in the animal nutrition laboratories (Kitcherside et al. 2000). A gravimetric method to estimate fiber, Neutral Detergent Fiber (NDF), was proposed by Van Soest and Wine (1967). Afterward, the original method was subjected to a range of changes, not only in order to increase the efficiency of the method but also to reduce the costs and the production of toxic residues (Van Soest et al. 1991). The original NDF method was based on refluxing the samples in a detergent solution to make possible the separation of cellular contents: the fraction of the forage soluble in detergent, mainly composed of proteins, ether extract, soluble carbohydrates, pectins and others hydrosoluble constituents, and the fraction of the forage not soluble in detergent, composed of cellulose, hemicellulose, lignin, proteins, and minerals. The NDF determination has been applied in routine laboratories with the purpose of obtaining data about nutritional values of forages used in cattle production systems. However, the reproducibility of the method is affected by operational variation that causes systematic errors. Therefore, a commercial fiber analyzer system that uses filter bags was developed. It allows the mechanization of the process of fiber extraction and a better homogeneity extraction environment, increasing the analytical frequency (Komarek et al. 1993; Ferreira and Mertens 2007). Microwave-Assisted Extraction (MAE) is a process of using microwave energy to heat solvents in contact with a sample in order to partition analytes from the sample matrix into the solvent (Eskilsson 2000). The microwave energy is employed in the sample preparation step to provide reductions of the analysis time, contamination risks, and residue generation. Besides these advantages, it allows the repetition of the same heating program in different microwave oven equipment (Kingston and Haswell 1997). In this work, changes in the NDF extraction by the Van Soest method are proposed, in order to have an efficient and repro ducible method, as well as to reduce costs and time of the fiber analysis. It employees microwave radiation as a source of heating, a commercial pressurized flask system, and filter bags to contain the samples.
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EXPERIMENTAL Samples Two different cultivars of Panicum maximum cv. Tanzania (A and B), one of Pennisetum purpureum, one of Cynodon spp cv. 85, and one of Stylosanthes capitata plus S. macrocephala were the evaluated forage samples with low amount of starch. The evaluated samples with high amount of starch were two different samples of wheat bran (A and B), one of soy bran, and one of rice bran. All samples were supplied by a proficiency study and were previously homogenized and analyzed by 36 laboratories (Souza 2007). These materials had results of NDF determination that had already been statistically evaluated. Factorial Design In order to maximize the efficiency of NDF extraction by using the proposed pressurized system and microwave radiation method, full factorial design for three independent variables was applied (Barros-Neto et al. 1995). The variables were microwave radiation, exposition time, power radiation, and extraction solution volume. The selection of low and high levels for the variable of each constituent was based on literature concerning sample preparation assisted by microwave radiation. A 23 full factorial design was considered for the process of optimization of the experiments. Statgraphics plus software for Windows 3.0 was used to analyze the data from experimental designs. Procedure A mass of 250 mg of samples was directly weighed in previously weighed polypropylene filter bags, 100 g=m2 and 6 5 cm. The filter bags were heat-sealed to encapsulate the samples. The samples with high amount of starch were pretreated under incubation in 120 mL of 8 mol L 1 urea solution (Vetec, Sa˜o Paulo, Brazil) and 4.5 mL of a-amylase (Novozymes=a-amylase termamyl 120 L) heat-stable at 90C in two stages of 15 min for each one. To prevent saturation, the urea and a-amylase solutions were changed at each stage. The samples contained in the filter bags was immersed in 1 L of NDF extraction solution, prepared in agreement with the method proposed by Van Soest and Wine (1967) in a commercial microwave pressure cooker (Tender cooker, Nordic Ware, Minneapolis, MN, USA) and submitted to microwave extraction (Prosd oscimo=Electrolux, Sa˜o Carlos SP,
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Brazil). After the extraction procedure, the filter bags containing the samples were rinsed with deionized water for 5 min, using the same pressurization system and microwave program. The filter bags were then drained and for 3 min immersed in about 150 mL of acetone, then dried for 3 hours in a forced air oven at 105C, and then weighed after the ambient temperature was reached. The NDF concentration from these data was calculated in accordance with the following equation: %NDF ¼
ðP3 ðP1 C1ÞÞ 100 ; P2
where P1 ¼ tare of the filter bag, P2 ¼ sample weight, P3 ¼ sample weight after the extraction process, and C1 ¼ filter bag final weight. RESULTS AND DISCUSSION Factorial Design The experimental parameters time of exposition to the microwave radiation, power, and volume of extraction solution were chosen to apply factorial planning 23 to the procedure of extraction of NDF analysis. For this study, 10 filter bags were used in each experiment. The factorial planning was developed in maximum and minimum levels, and the recovery of fiber was in agreement with the certificate values provided by the proficiency testing. After the data had been processed (Statgraphics plus software), it was observed by the Pareto chart that the variables power and microwave radiation exposition time were the most important factors affecting the NDF extraction with significant effect reliable to 95%. The lack of interactions among the variables and the volume of extraction solution were also verified. Applying higher values of power and extraction time, higher NDF recovery was obtained, whereas the fiber recovery was kept constant after volume of the extraction solution variation. In accordance with the results, the following parameters were chosen: power of 650 W, 20 min microwave radiation exposition time, and 1 L as the extraction solution volume. Operational Optimization After experimental parameters definition, the number of filter bags that could be simultaneously analyzed was evaluated by using a univariated factorial design. The number of filter bags per analysis was varied, and the percentage of fiber recovery was the observed response.
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The numbers of filter bags tested were 10, 12, 15, 18, and 20 units for the same volume of extraction solution, using the parameters established earlier. It was observed that the fiber recovery occurred at a range of 99–109%. Experiments using amounts of filter bags greater than 18 presented problems of rupture and, as a consequence, loss of the samples. These problems can be a result of reduced diameter of the solution container and possible pressure increase, and also, in these conditions, the safety valve of the solution container was activated, making impracticable the procedure due to security problems. Therefore, 18 was the maximum number of filter bags suitable to be simultaneously analyzed. The same extraction solution could be used in three extraction processes without an efficiency decrease. Analytical Application For samples with low amounts of starch, the proposed procedure allowed recoveries from 101 to 110%, with Relative Standard Deviation (RSD) among 0.7 and 6.1, when compared with the results supplied by the proficiency study. This can be observed in samples 1 to 5 of Table 1. Samples with high amounts of starch could be a serious interference to the analysis, owing to the change in the fiber structure, which could form a gelatinous material restrained in the NDF (Southgate 1977). This
Table 1. Concentration of neutral detergent fiber (NDF) in forage and cereal bran samples determined using the MAE proposed procedure and proficiency testing NDF (g kg 1 dry matter) Sample 1. Panicum maximum cv. Tanzania A 2. Stylosanthes Capitata plus S. macrocephala 3. Panicum maximum cv. Tanzania B 4. Pennisetum purpureum 5. Cynodon spp cv. 85 6. Wheat bran A 7. Soy bran 8. Wheat bran B 9. Rice bran
Proposed method
Reference value
756 13 457 28
718 16 414 50
748 10 816 14 803 6 376 4 122 9 372 3 364 17
739 30 788 9 775 17 390 15 116 22 399 47 355 16
Values provided by proficiency testing, including results of 36 different laboratories.
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interference can be avoided by means of a previous incubation with urea and hot-stable a-amylase at 90C (Van Soest et al. 1991). Sample Incubation The necessary time for total hydrolysis of the starch was evaluated. It used 8 mol L 1solution and hot-stable a-amylase at 90C, aiming for a better fiber recovery. Four experiments were carried out, each one with three replicates of three different forage samples. The incubation time was changed as follows: (1) 0 min, (2) 15 min, (3) 30 min, and (4) 45 min. In order to avoid saturation of the starch solution in experiments (3) and (4), the incubation solution was changed every 15 min. After the incubation, the NDF extraction was carried out, and the recovery was evaluated, based on the certificate values provided by the proficiency testing. It was possible to observe that in experiments (1) and (2), the incubation time was not enough to eliminate the interference of the starch, resulting in an overestimated value of fiber recoveries, whereas in experiments (3) and (4), the hydrolysis of the starch was more effective, providing satisfactory results that were confirmed by the recovery percentages. The experiment (3) was the defined procedure for high-starch samples. After 30 min of incubation, recoveries among 93 and 109 %, and the RSD from 0.8 to 7.4 was obtained. The final established procedure, using 18 samples simultaneously, 650 W and 20 min of radiation time, and 30 min of incubation time in samples with high amounts of starch, was applied to six samples of forage and four cereal bran, and the NDF obtained values are shown in the Table 1. When compared with previous NDF procedures that use high-temperature digester blocks and condensed systems and perform a maximum of 6 (in the original Van Soest method) to 40 (with the use of normal digester block) samples day1, the proposed MAE using a commercial pressurized system allow NDF determination of approximately 280 day 1with low consumption of energy and about 80% reagent economy, producing less residue. Even compared with the filter bag mechanized method, the proposed procedure presents similar sample throughput and reduction of approximately 30% of reagent consumption and residue production. REFERENCES Barros-Neto, B., Scarmı´nio, I.S., and Bruns, R.E. 1995. Planejamento E Otimizac¸a˜o De Experimentos, 2nd ed. Campinas: Unicamp, p. 299. Eskilsson, C.S. and Bjo¨rklund, E. 2000. Analytical-scale microwave-assisted extraction. J. Chromatogr., A, 902: 227–250.
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Ferreira, G. and Mertens, D.R. 2007. Measuring detergent fibre and insoluble protein in corn silage using crucibles or filter bags. Anim. Feed Sci. Technol., 133: 335–340. Goering, H.K. and Van Soest, P.J. 1970. Forage fiber analysis (Apparatus, reagents, procedures and some applications). Agric. Handb. Forest Serv., U.S., Washington, 379: 1–20. Kingston, H.M. and Haswell, S.J. (Eds.) 1997. Microwave-Enhanced Chemistry. Washington, DC: American Chemical Society, p. 569. Kitcherside, M.A., Glen, E.F., and Webster, A. J. F. 2000. FiberCap: An improved method for the rapid analysis of fiber in feeding stuff. Anim. Feed Sci. Technol., 86: 125–132. Komarek, A.R., Robertson, J.B., and Van Soest, P.J., 1993. A comparison of methods for determining ADF using the filter bag technique versus conventional filtrationl. J. Dairy Sci., 77 Supplement 1. National Research Council (NRC) 1996. Nutrient Requirements of Beef Cattle. Washington, DC: National Academy of Sciences. Southgate, D.A.T. 1977. The definition and analysis of dietary fibre. Nutr. Rev., 35 (3): 31–37. Souza, G.B. in http://eplna.cppse.embrapa.br/. Accessed 4 October 2007. Van Soest, P.J. and Wine, R.H., 1967. Use of detergents in the analysis of fibrous feeds. IV Determination of plant cell-wall constituents. J. Assoc. Off. Anal. Chem., 50: 50–55. Van Soest, P J., Robertson, J.B., and Lewis, B.A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci., 74: 3583–3597.
