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Extraction of Plant Materials: A New Blender Design and the Extraction of Fresh Cassava Roots in Dilute Orthophosphoric Acid
JOURNAL OF FOOD COMPOSITION AND ANALYSIS ARTICLE NO.
10, 358–367 (1997)
FC970548
Extraction of Plant Materials: A New Blender Design and the Extraction of Fresh Cassava Roots in Dilute Orthophosphoric Acid Leon Brimer,*,1 J. D. Kalenga Saka,† and Troels Pedersen‡ *Department of Pharmacology and Pathobiology, Section of Pharmacology and Toxicology, The Royal Veterinary and Agricultural University, 13 Bu¨lowsvej, DK-1870 Frederiksberg, Denmark; †Department of Chemistry, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi, Africa; and ‡IPU Engineering Design, Technical University Denmark, Building 433, DK-2800 Lyngby, Denmark Received April 10, 1997, and in revised form October 1, 1997 A new blender, with two independent motor units and blender heads within the same cabinet, has been developed for wet homogenization of plant materials in weak acid solutions. The blender uses inexpensive disposable cups with lids, which serve as containers for sample collection, processing chambers, and vessels for the storage of the produced homogenate. This construction allows the simultaneous wet processing of two samples and the continuation of work if one of the units break down. Furthermore the design makes the interchange of components between the two units possible as an additional maintenance security.The blender was tested for its efficiency by homogenizing fresh cassava root in dilute phosphoric acid. The efficiency was determined by characterizing the homogenates by sedimentation analysis, sieve analysis of the fluid bed dried pellets, and measuring the total cyanogen content of the supernatants. This consists of the cyanogenic glucosides linamarin and lotaustralin. The homogenate, which combined the (relatively) lowest sedimentation time with the highest total yield of cyanogens, was obtained after 75–80 s of blending.The sediment contained less than 25% matter of 200-mm size. This new blender design gives the same degree of extraction as the traditional Braun/Waring blenders in less time (75–80 s versus 120–195 s) and offers greater ease of emptying and cleaning than the traditional blenders. We believe that the use of our new blender will permit a higher throughput than possible with the more traditional blenders. q 1997 Academic Press Key Words: blender; extraction; homogenization; cassava; cyanogenic glucosides; plant tissues.
INTRODUCTION
Methods, and hence equipment, for the disintegration of samples of biological origin may in general be divided into dry or wet procedures, each used for products with different characteristics. For example, the preparation of flour from cereals involves dry milling of cereals (Kent, 1983), while wet milling is used in the preparation of starch (Whistler et al., 1984). Dry comminutions are usually performed on dry tissues of vegetable origin such as seeds, dried leaves, and roots (List and Schmidt, 1984; Defloor and Delcour, 1993; Schultz, 1989). Wet procedures are classified on whether (i) the fresh tissue holds a substantial amount of water, such as fresh fruits for the production of juices, or (ii) the fresh or dried tissues or microorganisms are mixed with a solvent. Examples of the latter are the production of starch from cereals, cassava roots, and potatoes, where disintegration is obtained using attrition or pin mills (Watson, 1984), rotary saw1
To whom correspondence and reprint requests should be addressed. Fax: (45) 31 35 35 14. 358
0889-1575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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blade rasps or hammermills (Mitch, 1984; Corbishley and Miller, 1984), and the disintegration of microorganisms to extract, e.g., enzymes. Disintegration is a major step in the analysis of raw materials for chemical constituents (MAFF, 1986), or for contaminating microorganisms (Bu¨lte and Reuter, 1984). For chemical analysis, both dry and wet comminutions may be used. Dry materials containing substances which do not evaporate upon comminution, and are not susceptible to oxidation, are usually comminuted and sieved prior to extraction. Fresh or partially dried tissues may also be comminuted directly. However, the inclusion of a solvent is often necessary when some major compounds are volatile, easily oxidizable, or may be hydrolyzed. For example, a number of plant glycosides (e.g., glucosinolates, cyanogenic glycosides, saponins, and cardiac glycosides) may be hydrolyzed by released plant glycosidases (Cheeke, 1989). Also, several organosulfur compounds (e.g., S-alkyl-substituted cystein sulphoxides) in Allium cepa and other Allium spp. (Breu and Dorsch, 1994), and linolenic acid hydroperoxides in cucumber and tomatoes (Baltes, 1992) undergo enzymatic changes, forming well-known aroma and fragrance constituents. Similar chemical changes may occur during drying of fresh material. In conclusion, microorganisms and several fresh plant and animal tissues have to be comminuted in suitable solvents to ensure minimum changes in the content of labile constituents, e.g., by inhibiting the enzymes coreleased. This may also be achieved by disintegration at very low temperatures (Magnusson, 1975) followed by lyophillization or immediate suspension in an extraction medium. The efficiency of the comminution process may be measured by the degree of disintegration or the yield of the required chemical constituent. The former is determined at the cellular level, i.e., the degree of cell disruption, or at the tissue level measuring the resulting particle size distribution. While characterizations of disintegration processes based on their ability to disrupt cells are common when dealing with microorganisms (Magnusson, 1975), comminution of plant materials is often characterized by the particle size obtained. A particle size of 1000 mm will in many cases achieve almost 100% extraction of the desired constituent (MAFF, 1986). Small scale equipment is required to comminute plant materials for analyses. The sharpe knife blender (e.g., Waring and Braun blenders, Omni-Mixers) and the rotorstator homogenizer (e.g., Ultra turrax, Polytron/Megatron, Omni) have proved useful for a wide range of materials. Other systems are generally more restricted in use. For example, the hand operated glass–glass/glass–PTFE Potter–Elvehjem homogenizer (Potter and Elvehjem, 1936) and the motor driven homogenizer (Dingle and Barret, 1972) are suitable for soft animal tissues. Extrusion methods (French press, Lowpressure X-press) (Tagesson et al., 1973) and freeze-pressing (Hughes Press and Xpress) (Edebo, 1960) are used mostly on pastes and suspensions of microorganisms (Edebo, 1983). The Stomacher homogenizer, which by two paddles compresses the contents in a polyethylene bag, is mostly used to homogenize dairy products, or food samples of animal origin, for microbiological analysis (Tuttlebee, 1975). The Waring and Braun blenders have been widely used to homogenize fresh and dried cassava samples (Cooke, 1978). However, these and others blenders have limited throughput, due to among others the complicated procedures for emptying and cleaning the blenders between extractions. A versatile blender which minimizes processing time, labor, and water, for cleaning between samples, is portable (for field studies), and which uses inexpensive disposable cups with lids, which serve as containers for sample collection, processing chambers, and vessels for the storage of the produced homogenate, is needed. The objective of this work was to develop a homogenizer
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with the above attributes and to establish the optimum conditions for extraction of cyanogenic glucosides from fresh cassava root using this blender. EXPERIMENTAL
Apparatus The final design of the new blender appears in Figs. 1 and 2. The overall dimensions (in mm) are 410 1 300 1 425 (height 1 width 1 depth). Motor/gear unit (EW 6114 S, Metabo, Nu¨rtingen, Germany), knife (KNIVHR2810, Item No. 4822 690 40246, Philips, The Netherlands), cups of polypropylene (dimensions (mm): outer height 95, inner diameter at top 85, inner diameter at bottom 60; A 380, Cerbo/Magenta Plast, Copenhagen, Denmark), which could be equipped with a snap on polyethylene lid (Item No. 89DA) were used as sample cups. Gaskets (O-rings ø 70 1 4.5 mm) were of EPDM (ethylene-propylene-diene), i.e., a random copolymer of the ethylenepropylene elastomer class. Chemicals Linamarin (No. L 9131), the standard cyanogen, used in the method of Saka et al. (1998), was purchased from Sigma (St. Louis, U.S.A.). Linamarase (EC 3.2.1.21 from cassava), used in both of the chemical assays, and bispyrazolone (GPR grade), 3methyl-1-phenyl-5-pyrazolone (GPR grade), and potassium cyanide (97%), all used in the method af Cooke (1978), were obtained from BDH Ltd. (Poole, UK). All other chemicals were p.a. quality from Merck (Darmstadt, Germany). Picrate (detection) sheets, for the method of Saka et al. (1998), were prepared from precoated ionexchange sheets (Polygram ionex 24-SB-AC, Cat. No. 806023) from Macherey-Nagel (Du¨ren, Germany), as previously described by Brimer et al. (1983) and Brimer and Rosling (1993). Thus, the ion-exchange sheets were impregnated by consecutive immersion in two solutions: (1) a saturated solution of picric acid in water, followed by air drying, and (2) a 1 M aqueous sodium carbonate solution, followed by air drying. Samples and Sample Preparation Roots from the varieties TMS1430395, MK90/1151, Mbundumali (harvested at University of Malawi, Zomba, Malawi in February 1995), and an unknown cassava variety imported from Brazil, via Amsterdam, were used in the study. Prior to extraction, the roots were cut into cubes with a side length of approximately 1 cm with a sharp knife. The cubes were immediately mixed, and an accurately weighed subsamble was taken randomly and placed in 140 ml 0.1 M of H3PO4 in a cup. Just before blending, the total volume of extractant was made up to the volume needed in the experiment. The mixture was homogenized for the period corresponding to the experiment in question. When using the new blender,the knives and the shaft were washed, replacing the beaker with a new beaker containing 40 ml of the same extractant (blending time 10 s). The washing was mixed with the homogenate. All homogenizations were carried out at room temperature (approx. 22–257C). Homogenates were subjected to one or more of these analyses: (1) sedimentation, (2) sieving, and (3) chemical analysis as follows. Sedimentation The homogenate, diluted to 240 ml with 0.1 M of H3PO4 , was poured into a 250ml measuring cylinder and stoppered. The homogenate was mixed by turning the
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cylinder several times, and the cylinder was left on a table until a clearly defined layer between the sediment and a clear to nearly clear supernatant were obtained. The time needed for this was measured. This was done twice for each homogenate to be analyzed. Analysis of the Particle Size Distribution by Sieving Following the second sedimentation analysis, the supernatant was removed by decanting. The supernatant was used in the chemical analysis (see below). The sediment was washed with distilled water (3 1 150 ml) by shaking and sedimentation. The two first supernatants were discarded. After the third wash the suspension was filtered by suction, and the filter cake was dried at 407C (inlet temp.) in a fluid bed (Uniglatt, Glatt, Binzen, Germany). The drying was run until the outlet temperature reached that of the inlet. The solid was further dried to constant weight at room temperature (20– 227C) over silica-gel. A portion of the dried solid was analyzed for particle size distribution, by sieving, using a Pulverisette Type 03.502 sieve tower (Fritsch), equipped with sieves from Retsch (Haan, Germany). The sieving program comprised amplitude 5 (5 min) followed by amplitude 7 for 1 min. Chemical Analysis The supernatant (see above) was filtered by suction through a glass microfiber filter (GF/A, Whatman, Maidstone, England). A subsample of 0.1 ml was taken, and the total cyanogenic potential (CNp) was determined as described by either Cooke (1978) or Saka et al. (1998). Statistical Analysis All data were subjected to statistical analysis using SPSS for PC, e.g., the facility comparing group means (anova test). Experiments Experiment 1. Blending time versus total cyanogenic potential (CNp) measured: 50 g of cassava root was homogenized in 160 ml of extractant for the times (5, 10, 20, 35, 40, 50, 60, 70, 75, 80, 90, and 120 s). Three different cultivars were used. Experiments were done independently in Copenhagen (Brazil cultivar) and Zomba (cultivars TMS1430395 and Mbundumali). Experiment 2. Effect of cassava chip loading and volume of extractant, on the CNp meaured: homogenizing for 75 s, a number of different combinations of sample size and volume of extractant were tested. The cultivar TMS 1430395 was used. Experiment 3. Comparison to homogenization using a Braun blender (Type 4142) and a blending regime as proposed by Cooke (1978): Using three different cultivars of cassava, samples of 50 g and 160 ml of extractant were homogenized either continously for 75 s (new blender) or for 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (Braun blender). Experiment 4. Particle size distribution as function of time of homogenization: A number of homogenates were produced using the cultivar Brazil (50 g of sample and 160 ml of extractant). The variable was the time of homogenization (5, 10, 20, 40, 80, 120 s). All homogenates were subjected to analysis by sedimentation and sieving (5 s not shown on Fig. 4).
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RESULTS AND DISCUSSION Several basic constructions were analyzed theoretically, before the final design and construction took place. The shaft blender type with two to four sharp to semisharp fast moving knives mounted at the bottom end of a vertical shaft was selected. It was decided to combine this construction with the use of disposable polymer cups (instead of a blender glass) and with an O-ring gasket/lever construction, thus avoiding any screw closure. Using a motor and gear unit providing about 10,000 rpm, and a cylindrical glass of diameter 80 mm, two different sets of knives commercially available were tested by homogenizing 50 g of fresh cassava root in 200 ml of water. The homogenates were visually inspected and the time for clear supernatant formation and complete sedimentation was measured. Preliminary data (not shown) pointed to the knife (KNIVHR2810) as the most effective in this set-up. Also various commercially available motor and gear units were considered as were a number of sample cups. A Metabo unit was chosen for the motor–gear unit, while conical polypropylene cups with a flat bottom, which could be equipped with a snap on polyethylene lid, were used as sample cups. A simple protected shaft connection, between the motor unit and the knife, was constructed, to protect the motor and the operator, and to allow easy rinsing as shown in Fig. 1. Two such motor–knife units were placed in a cabinet as shown in Fig. 2. Levers with safety locks were constructed to press the sample cup against the top at the shaft and hence around the gasket (Fig. 2). The blender was developed primarily for use in the analysis of the content of the toxic cyanogenic glucosides linamarin and lotaustralin in the very important food crop cassava root. Hence a number of tests were made for the characterization of the blender, using roots of different cultivars of cassava. A preliminary experiment to clarify the time needed for total extraction of the cyanogenic glucosides suggested that effective extraction of total cyanogens was obtained after approximately 30 s of homogenization (data not shown); however, data from the sedimentation and sieving experiments led us to select 75 s of constant homogenization as optimal. Using 75 s of constant homogenization, studies were done to determine the influence of cassava loading and volume of acid extractant on the CNp released. Using 160 ml of extractant a preliminary study showed that low (8–40 g) as well as high (more than 60 g) loads of cassava resulted in a low yield (data not shown). A more detailed study showed that identical results were obtained using combinations within the intervals defined by a load of 45–60 g of root and 140– 200 ml of extractant (Table 1). Thus, no sign of correlation between sample weight and volume of extractant, when looking at the resulting CNp, was observed in an analysis of variants with a test for this interaction. For the volume F and P values were (main variable F Å 0.574, P Å 0.644; covariable F Å 0.06, P Å 0.939); for the weight the values were (main variable F Å 1.358, P Å 0.306; covariable F Å 0,294, P Å 0.598). Thus, a method depending on 75 s of homogenization (50 g of sample and 160 ml of extractant) was tested against the homogenization procedure recommended by Cooke (1978) when using a Waring/Braun blender type, i.e., 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (total time 195 s), a procedure also used by Essers et al. (1993), while O’Brien et al. (1991) used a constant homogenization of 120 s. The comparison (using a Braun blender—Type 4142), showed no significant differences in the CNp’s obtained (Table 2). However, since the ordinary Braun/Waring blenders need 120–195 s to produce the same degree of extraction (Cooke, 1978; O’Brien et al., 1991), and since the emptying and cleaning of these are more complicated (time consuming), each unit of the new blender has a significantly greater throughput than an ordinary Braun/Waring blender.
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FIG. 1. Protected shaft connection between motor and knife (1, motor shaft; 2, motor casing; 3, chassis of blender; 4, shielding; 5, shaft; 6, rinsing hole; 7, knife; and 8, check nut). Overall height of shaft, knife, and nut is 80 mm.
FIG. 2. Basic construction details of the blender. On the right and the left are given the front and side views, respectively, showing the interior. For overall dimensions see Experimental.
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BRIMER, SAKA, AND PEDERSEN TABLE 1 Effect of Cassava Loading and Volume of Acid Extractant on Yield of Total Cyanogens for Cultivar TMS 14303951
1 Samples were homogenized for 75 s using the new blender. Each measurement represents one homogenization, the CNp of the supernatant being measured four times (mean { SD; n 0 1), as assayed by Cooke’s method. Means, of columns and rows, respectively, with the same letter, do not differ significantly (P õ 0.05), when analyzed using Newman–Keuls student test. An analysis of variance with test for interaction between solvent volume and sample weight showed no sign of correlation (analysis by weight—multiple R squared 0.284; analysis by volume—multiple R squared 0.136). 2 Grand mean.
Although there is very comprehensive literature on the analysis of cyanogens in cassava roots, no investigations appear to have been published on relations between the extraction efficiency and the particle size of the homogenate. Hence, we decided to analyze the particle size distribution, found in a homogenate, as a function of the time of processing. A preliminary impression of the comminution process was gained TABLE 2 1
Total Cyanogens (CNp) of Fresh Cassava Roots as Affected by the Type of Homogenization Procedure1
1 Homogenization regimes: (a) New blender—75 s of continuous homogenization, (b) Braun blender— 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (total time 195 s). In each case 50 g of sample was homogenized in 160 ml of 0.1 M H3PO4 . 2 Each CNp is the mean ({ SD; n 0 1) of four homogenizations with each supernatant analyzed once. Values between the two homogenizers followed by a different letter are statistically different (P õ 0.05), according to Student’s t test.
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FIG. 3. Sedimentation analysis of cassava root homogenates: sedimentation time as function of the time of homogenization. Fifty grams of cassava root (Brazil cultivar) in 160 ml of 0.1 M H3PO4 . The total volume was made-up to 240 ml (with extractant) prior to analysis.
through a sedimentation analysis (Fig. 3). From this it is further seen that the time for formation of a visually clear supernatant is around 5 min for an 80-s homogenate (complete extraction). This is interesting, since the solid-phase method for determination of total cyanogens in cassava root of Saka et al. (1998) was recently shown to accept the use of such a freely formed supernatant instead of a filtrate. The particle size distribution as a function of the time of processing is shown in Fig. 4. It is noteworthy that the decrease in the weight of particles larger than 2800 mm is rapid for the first 50 s, after which time this fraction is relatively stable. In contrast, the fraction of particles smaller than 710 mm is increasing rapidly in the time interval from 40 to 80 s of homogenization. This pattern may, for example, be due to the existence of a fraction of relatively hard tissue still found on the coarse sieves after the first 40–50 s of homogenization. A further analysis of the results presented in Fig. 4 shows for 40 and 80 s of homogenization, respectively, the following (sieve in micrometers 0 percentage weight at sieve or passing this 40 s/80 s): 2000 0 65.0/ 74.1, 1400 0 53.2/64.8, 1000 0 43.0/55.4, 710 0 32.8/46.4. Thus, a full extraction of linamarin in this case coincides with approximately 70% of the material being of particle size smaller than 2.8 mm and 50% smaller than 2.0 mm. The number of analyses for cyanogens in cassava performed at research and breeding stations per year worldwide is constantly rising, i.e., for purposes of identifying material for breeding programs (Dixon et al., 1994), for check of grown material (Bokanga, 1994), or for investigations concerning the relation of the cyanogenic potential with ecological factors (Bokaga et al., 1994). In the future, it is envisaged that routine analysis will spread to other areas. We believe that the design presented in this report points to construction details that should be considered when looking for extraction equipment that shall handle a large number of samples day after day for
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FIG. 4. Sieve analysis (particle size distribution) of fluid-bed-dried cassava root homogenates (50 g of root—Brazil cultivar—in 160 ml of 0.1 M H3PO4 ). Each point represents the mean from the analysis of three independently produced homogenates.
the routine analysis of constituents such as toxins, pesticide residues or nutrients in cassava roots as well as in other plant tissues. The blender is not commercially available, however, the authors will provide detailed plans to individual readers upon request. Furthermore, the IPU Engineering Design, Technical University, Denmark, may build single copies on request. ACKNOWLEDGMENTS We thank the International Program in the Chemical Sciences (IPICS), Uppsala University, Sweden, and its Director Professor Rune Liminga for funding the development of the blender and a 2-month research fellowship to Dr. Saka at Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Copenhagen, and for a research grant (Project MAL 01). We also thank Professor H. Gjeldstrup and Dr. Ole Jungersen, Department of Pharmaceutics, Royal Danish School of Pharmacy (RDSP), for their assistance in fluid-bed drying of cassava solids and seive analysis, and Dr. Per Mølgaard (RDSP), Department of Medicinal Chemistry, for his help with the statistical analysis.
REFERENCES Baltes, W. (1992). Lebensmittelchemie, 3rd ed. Springer-Verlag, Berlin. Bokanga, M. (1994). Distribution of cyanogenic potential in cassava germplasm. Acta Horticulturae 375, 117–129. Bokanga, M., Ekanayake, I. J., Dixon, A. G. O., and Porto, M. C. M. (1994). Genotype-environment interactions for cyanogenic potential in cassava. Acta Horticulturae 375, 131–151. Breu, W., and Dorsch, E. (1994). Allium cepa L. (onion): Chemistry, analysis and pharmacology. In Economic and Medicinal Plant Research (H. Wagner and N. R. Farnsworth, Eds.), Vol. 6, pp. 115–147. Academic Press, London. Brimer, L., and Rosling, H. A. (1993). Microdiffusion method with solid state detection for determination of cyanogenic glycosides from cassava in human urine. Food Chem. Toxicol. 31, 599–603.
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Brimer, L., Brøgger Christensen, S., Mølgaard, P., and Nartey, F. (1983). Determination of cyanogenic compounds by Thin-layer Chromatography. 1. A Densitometric Method for Quantification of Cyanogenic Glycosides, Employing Enzyme Preparations (b-Glucuronidase) from Helix pomatia and Picrate-Impregnated Ion-Exchange Sheets. J. Agric. Food Chem. 31, 789–793. Bu¨lte, M., and Reuter, G. (1984). Impedance measurement as a rapid method for the determination of the microbial contamination of meat surfaces, testing two different instruments. Int. J. Food Microbiol. 1, 113–125. Cheeke, L. (Ed.) (1989). Toxicants of Plant Origin, Vol. 2 (Plant Glycosides). CRC Press, Boca Raton. Cooke, R. D. (1978). An enzymatic assay for the total cyanide content of cassava (Manihot esculenta Crantz). J. Sci. Food Agric. 29, 345–352. Corbishley, D. A., and Miller, W. (1984). Tapioca, arrowroot, and sago starches: Production. In Starch, Chemistry and Technology (R. L. Whistler, J. N. Bemiller, and E. F. Paschall, Eds.), 2nd ed., pp. 469– 478. Academic Press, Orlando. Defloor, I., and Delcour, J. A. (1993). Impact of milling procedure on breadmaking potential of cassava flour in wheatless breads. Cer. Chem. 70, 616–617. Dingle, J. T., and Barrett, A. J. (1972). Some special methods for the lysosomal system. In Lysosomes in Biology and Pathology (J. T. Dingle and H. B. Fell, Eds.), Vol. 2, pp. 555–566. North Holland, Amsterdam. Dixon, A. G. O., Asiedu, R., and Bokanga, M. (1994). Breeding of cassava for low cyanogenic potential: Problems, progress and prospects. Acta Horticult. 275, 153–161. Edebo, L. (1960). A new press for the disruption of micro-organisms and other cells. J. Biochem. Microbiol. Technol. Eng. 2, 453–479. Edebo, L. (1983). Disintegration of cells by extrusion under pressure. In Enzyme Technology (R. M. Lafferty, Ed.), pp. 93–114. Springer-Verlag, Berlin. Essers, S. A. J. A., Bosneld, M., Grif van der, R. M., and Voragen, A. G. J. (1993). Studies on the quantification of specific cyanogens in cassava products and an introduction of a new chromogen. J. Sci. Food Agric. 63, 287–296. Kent, N. L. (1983). Technology of Cereals, 3rd ed., pp. 86–106. Pergamon Press, Oxford. List, P. H., and Schmidt, P. C. (1984). Technologie Pflanzlicher Arzneizubereitungen, pp. 99–232. Wissenschaftliche Verlagsgesellschaft, Stuttgart. MAFF (Ministry of Agriculture, Fisheries and Food) (1986). The analysis of agricultural materials, 3rd ed., pp. 4–5. Her Majesty’s Stationary Office, London. Magnusson, K. E. (1975). Physical studies on the freeze-pressing of biological material. Linko¨ping University Medical Dissertations No. 28 (Linko¨ping University Linko¨ping). Mitch, E. L. (1984). Potato starch: Production and uses. In Starch, Chemistry and Technology (R. L. Whistler, J. N. Bemiller, and E. F. Paschall, Eds.), 2nd ed., pp. 479–490. Academic Press, Orlando. O’Brien, G. M., Taylor, A. J., and Poulter, N. H. (1991). Improved enzymatic assay for cyanogens in fresh and processed cassava. J. Sci. Food Agric. 56, 277–289. Potter, V. R., and Elvehjem, C. A. (1936). A modified method for the study of tissue oxidations. J. Biol. Chem. 114, 495–504. Saka, J. D. K., Mhone, A. R. K., and Brimer, L. (1998). An improved microdiffusion method with solidphase detection for the determination of total cyanogens in fresh cassava. Comparison to the method of Cooke (1978). J. Sci. Food Agric. 76, in press. Schultz, R. (1989). The hammermill today and tomorrow. Milling Flour Feed 182, 26–31. Tagesson, C., Stendahl, O., Magnusson, K. D., and Edebo, L. (1973). Disintegration of single cells in suspension. Acta Pathol. Microbiol. Scand. Sec. B 81, 464–472. Tuttlebee, J. W. (1975). The stomacher—Its use for homogenization in food microbiology. J. Food Technol. 10, 113–122. Watson, S. A. (1984). Corn and sorghum starches: Production. In Starch, Chemistry and Technology (R. L. Whistler, J. N., Bemiller, and E. F. Paschall, Eds.), 2nd ed., pp. 417–468. Academic Press, Orlando. Whistler, R. L., Bemiller, J. N., and Paschall, E. F. (Eds.) (1984). Starch, Chemistry and Technology, 2. ed. Academic Press, Orlando.
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FC970548
Extraction of Plant Materials: A New Blender Design and the Extraction of Fresh Cassava Roots in Dilute Orthophosphoric Acid Leon Brimer,*,1 J. D. Kalenga Saka,† and Troels Pedersen‡ *Department of Pharmacology and Pathobiology, Section of Pharmacology and Toxicology, The Royal Veterinary and Agricultural University, 13 Bu¨lowsvej, DK-1870 Frederiksberg, Denmark; †Department of Chemistry, Chancellor College, University of Malawi, P.O. Box 280, Zomba, Malawi, Africa; and ‡IPU Engineering Design, Technical University Denmark, Building 433, DK-2800 Lyngby, Denmark Received April 10, 1997, and in revised form October 1, 1997 A new blender, with two independent motor units and blender heads within the same cabinet, has been developed for wet homogenization of plant materials in weak acid solutions. The blender uses inexpensive disposable cups with lids, which serve as containers for sample collection, processing chambers, and vessels for the storage of the produced homogenate. This construction allows the simultaneous wet processing of two samples and the continuation of work if one of the units break down. Furthermore the design makes the interchange of components between the two units possible as an additional maintenance security.The blender was tested for its efficiency by homogenizing fresh cassava root in dilute phosphoric acid. The efficiency was determined by characterizing the homogenates by sedimentation analysis, sieve analysis of the fluid bed dried pellets, and measuring the total cyanogen content of the supernatants. This consists of the cyanogenic glucosides linamarin and lotaustralin. The homogenate, which combined the (relatively) lowest sedimentation time with the highest total yield of cyanogens, was obtained after 75–80 s of blending.The sediment contained less than 25% matter of 200-mm size. This new blender design gives the same degree of extraction as the traditional Braun/Waring blenders in less time (75–80 s versus 120–195 s) and offers greater ease of emptying and cleaning than the traditional blenders. We believe that the use of our new blender will permit a higher throughput than possible with the more traditional blenders. q 1997 Academic Press Key Words: blender; extraction; homogenization; cassava; cyanogenic glucosides; plant tissues.
INTRODUCTION
Methods, and hence equipment, for the disintegration of samples of biological origin may in general be divided into dry or wet procedures, each used for products with different characteristics. For example, the preparation of flour from cereals involves dry milling of cereals (Kent, 1983), while wet milling is used in the preparation of starch (Whistler et al., 1984). Dry comminutions are usually performed on dry tissues of vegetable origin such as seeds, dried leaves, and roots (List and Schmidt, 1984; Defloor and Delcour, 1993; Schultz, 1989). Wet procedures are classified on whether (i) the fresh tissue holds a substantial amount of water, such as fresh fruits for the production of juices, or (ii) the fresh or dried tissues or microorganisms are mixed with a solvent. Examples of the latter are the production of starch from cereals, cassava roots, and potatoes, where disintegration is obtained using attrition or pin mills (Watson, 1984), rotary saw1
To whom correspondence and reprint requests should be addressed. Fax: (45) 31 35 35 14. 358
0889-1575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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blade rasps or hammermills (Mitch, 1984; Corbishley and Miller, 1984), and the disintegration of microorganisms to extract, e.g., enzymes. Disintegration is a major step in the analysis of raw materials for chemical constituents (MAFF, 1986), or for contaminating microorganisms (Bu¨lte and Reuter, 1984). For chemical analysis, both dry and wet comminutions may be used. Dry materials containing substances which do not evaporate upon comminution, and are not susceptible to oxidation, are usually comminuted and sieved prior to extraction. Fresh or partially dried tissues may also be comminuted directly. However, the inclusion of a solvent is often necessary when some major compounds are volatile, easily oxidizable, or may be hydrolyzed. For example, a number of plant glycosides (e.g., glucosinolates, cyanogenic glycosides, saponins, and cardiac glycosides) may be hydrolyzed by released plant glycosidases (Cheeke, 1989). Also, several organosulfur compounds (e.g., S-alkyl-substituted cystein sulphoxides) in Allium cepa and other Allium spp. (Breu and Dorsch, 1994), and linolenic acid hydroperoxides in cucumber and tomatoes (Baltes, 1992) undergo enzymatic changes, forming well-known aroma and fragrance constituents. Similar chemical changes may occur during drying of fresh material. In conclusion, microorganisms and several fresh plant and animal tissues have to be comminuted in suitable solvents to ensure minimum changes in the content of labile constituents, e.g., by inhibiting the enzymes coreleased. This may also be achieved by disintegration at very low temperatures (Magnusson, 1975) followed by lyophillization or immediate suspension in an extraction medium. The efficiency of the comminution process may be measured by the degree of disintegration or the yield of the required chemical constituent. The former is determined at the cellular level, i.e., the degree of cell disruption, or at the tissue level measuring the resulting particle size distribution. While characterizations of disintegration processes based on their ability to disrupt cells are common when dealing with microorganisms (Magnusson, 1975), comminution of plant materials is often characterized by the particle size obtained. A particle size of 1000 mm will in many cases achieve almost 100% extraction of the desired constituent (MAFF, 1986). Small scale equipment is required to comminute plant materials for analyses. The sharpe knife blender (e.g., Waring and Braun blenders, Omni-Mixers) and the rotorstator homogenizer (e.g., Ultra turrax, Polytron/Megatron, Omni) have proved useful for a wide range of materials. Other systems are generally more restricted in use. For example, the hand operated glass–glass/glass–PTFE Potter–Elvehjem homogenizer (Potter and Elvehjem, 1936) and the motor driven homogenizer (Dingle and Barret, 1972) are suitable for soft animal tissues. Extrusion methods (French press, Lowpressure X-press) (Tagesson et al., 1973) and freeze-pressing (Hughes Press and Xpress) (Edebo, 1960) are used mostly on pastes and suspensions of microorganisms (Edebo, 1983). The Stomacher homogenizer, which by two paddles compresses the contents in a polyethylene bag, is mostly used to homogenize dairy products, or food samples of animal origin, for microbiological analysis (Tuttlebee, 1975). The Waring and Braun blenders have been widely used to homogenize fresh and dried cassava samples (Cooke, 1978). However, these and others blenders have limited throughput, due to among others the complicated procedures for emptying and cleaning the blenders between extractions. A versatile blender which minimizes processing time, labor, and water, for cleaning between samples, is portable (for field studies), and which uses inexpensive disposable cups with lids, which serve as containers for sample collection, processing chambers, and vessels for the storage of the produced homogenate, is needed. The objective of this work was to develop a homogenizer
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with the above attributes and to establish the optimum conditions for extraction of cyanogenic glucosides from fresh cassava root using this blender. EXPERIMENTAL
Apparatus The final design of the new blender appears in Figs. 1 and 2. The overall dimensions (in mm) are 410 1 300 1 425 (height 1 width 1 depth). Motor/gear unit (EW 6114 S, Metabo, Nu¨rtingen, Germany), knife (KNIVHR2810, Item No. 4822 690 40246, Philips, The Netherlands), cups of polypropylene (dimensions (mm): outer height 95, inner diameter at top 85, inner diameter at bottom 60; A 380, Cerbo/Magenta Plast, Copenhagen, Denmark), which could be equipped with a snap on polyethylene lid (Item No. 89DA) were used as sample cups. Gaskets (O-rings ø 70 1 4.5 mm) were of EPDM (ethylene-propylene-diene), i.e., a random copolymer of the ethylenepropylene elastomer class. Chemicals Linamarin (No. L 9131), the standard cyanogen, used in the method of Saka et al. (1998), was purchased from Sigma (St. Louis, U.S.A.). Linamarase (EC 3.2.1.21 from cassava), used in both of the chemical assays, and bispyrazolone (GPR grade), 3methyl-1-phenyl-5-pyrazolone (GPR grade), and potassium cyanide (97%), all used in the method af Cooke (1978), were obtained from BDH Ltd. (Poole, UK). All other chemicals were p.a. quality from Merck (Darmstadt, Germany). Picrate (detection) sheets, for the method of Saka et al. (1998), were prepared from precoated ionexchange sheets (Polygram ionex 24-SB-AC, Cat. No. 806023) from Macherey-Nagel (Du¨ren, Germany), as previously described by Brimer et al. (1983) and Brimer and Rosling (1993). Thus, the ion-exchange sheets were impregnated by consecutive immersion in two solutions: (1) a saturated solution of picric acid in water, followed by air drying, and (2) a 1 M aqueous sodium carbonate solution, followed by air drying. Samples and Sample Preparation Roots from the varieties TMS1430395, MK90/1151, Mbundumali (harvested at University of Malawi, Zomba, Malawi in February 1995), and an unknown cassava variety imported from Brazil, via Amsterdam, were used in the study. Prior to extraction, the roots were cut into cubes with a side length of approximately 1 cm with a sharp knife. The cubes were immediately mixed, and an accurately weighed subsamble was taken randomly and placed in 140 ml 0.1 M of H3PO4 in a cup. Just before blending, the total volume of extractant was made up to the volume needed in the experiment. The mixture was homogenized for the period corresponding to the experiment in question. When using the new blender,the knives and the shaft were washed, replacing the beaker with a new beaker containing 40 ml of the same extractant (blending time 10 s). The washing was mixed with the homogenate. All homogenizations were carried out at room temperature (approx. 22–257C). Homogenates were subjected to one or more of these analyses: (1) sedimentation, (2) sieving, and (3) chemical analysis as follows. Sedimentation The homogenate, diluted to 240 ml with 0.1 M of H3PO4 , was poured into a 250ml measuring cylinder and stoppered. The homogenate was mixed by turning the
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cylinder several times, and the cylinder was left on a table until a clearly defined layer between the sediment and a clear to nearly clear supernatant were obtained. The time needed for this was measured. This was done twice for each homogenate to be analyzed. Analysis of the Particle Size Distribution by Sieving Following the second sedimentation analysis, the supernatant was removed by decanting. The supernatant was used in the chemical analysis (see below). The sediment was washed with distilled water (3 1 150 ml) by shaking and sedimentation. The two first supernatants were discarded. After the third wash the suspension was filtered by suction, and the filter cake was dried at 407C (inlet temp.) in a fluid bed (Uniglatt, Glatt, Binzen, Germany). The drying was run until the outlet temperature reached that of the inlet. The solid was further dried to constant weight at room temperature (20– 227C) over silica-gel. A portion of the dried solid was analyzed for particle size distribution, by sieving, using a Pulverisette Type 03.502 sieve tower (Fritsch), equipped with sieves from Retsch (Haan, Germany). The sieving program comprised amplitude 5 (5 min) followed by amplitude 7 for 1 min. Chemical Analysis The supernatant (see above) was filtered by suction through a glass microfiber filter (GF/A, Whatman, Maidstone, England). A subsample of 0.1 ml was taken, and the total cyanogenic potential (CNp) was determined as described by either Cooke (1978) or Saka et al. (1998). Statistical Analysis All data were subjected to statistical analysis using SPSS for PC, e.g., the facility comparing group means (anova test). Experiments Experiment 1. Blending time versus total cyanogenic potential (CNp) measured: 50 g of cassava root was homogenized in 160 ml of extractant for the times (5, 10, 20, 35, 40, 50, 60, 70, 75, 80, 90, and 120 s). Three different cultivars were used. Experiments were done independently in Copenhagen (Brazil cultivar) and Zomba (cultivars TMS1430395 and Mbundumali). Experiment 2. Effect of cassava chip loading and volume of extractant, on the CNp meaured: homogenizing for 75 s, a number of different combinations of sample size and volume of extractant were tested. The cultivar TMS 1430395 was used. Experiment 3. Comparison to homogenization using a Braun blender (Type 4142) and a blending regime as proposed by Cooke (1978): Using three different cultivars of cassava, samples of 50 g and 160 ml of extractant were homogenized either continously for 75 s (new blender) or for 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (Braun blender). Experiment 4. Particle size distribution as function of time of homogenization: A number of homogenates were produced using the cultivar Brazil (50 g of sample and 160 ml of extractant). The variable was the time of homogenization (5, 10, 20, 40, 80, 120 s). All homogenates were subjected to analysis by sedimentation and sieving (5 s not shown on Fig. 4).
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RESULTS AND DISCUSSION Several basic constructions were analyzed theoretically, before the final design and construction took place. The shaft blender type with two to four sharp to semisharp fast moving knives mounted at the bottom end of a vertical shaft was selected. It was decided to combine this construction with the use of disposable polymer cups (instead of a blender glass) and with an O-ring gasket/lever construction, thus avoiding any screw closure. Using a motor and gear unit providing about 10,000 rpm, and a cylindrical glass of diameter 80 mm, two different sets of knives commercially available were tested by homogenizing 50 g of fresh cassava root in 200 ml of water. The homogenates were visually inspected and the time for clear supernatant formation and complete sedimentation was measured. Preliminary data (not shown) pointed to the knife (KNIVHR2810) as the most effective in this set-up. Also various commercially available motor and gear units were considered as were a number of sample cups. A Metabo unit was chosen for the motor–gear unit, while conical polypropylene cups with a flat bottom, which could be equipped with a snap on polyethylene lid, were used as sample cups. A simple protected shaft connection, between the motor unit and the knife, was constructed, to protect the motor and the operator, and to allow easy rinsing as shown in Fig. 1. Two such motor–knife units were placed in a cabinet as shown in Fig. 2. Levers with safety locks were constructed to press the sample cup against the top at the shaft and hence around the gasket (Fig. 2). The blender was developed primarily for use in the analysis of the content of the toxic cyanogenic glucosides linamarin and lotaustralin in the very important food crop cassava root. Hence a number of tests were made for the characterization of the blender, using roots of different cultivars of cassava. A preliminary experiment to clarify the time needed for total extraction of the cyanogenic glucosides suggested that effective extraction of total cyanogens was obtained after approximately 30 s of homogenization (data not shown); however, data from the sedimentation and sieving experiments led us to select 75 s of constant homogenization as optimal. Using 75 s of constant homogenization, studies were done to determine the influence of cassava loading and volume of acid extractant on the CNp released. Using 160 ml of extractant a preliminary study showed that low (8–40 g) as well as high (more than 60 g) loads of cassava resulted in a low yield (data not shown). A more detailed study showed that identical results were obtained using combinations within the intervals defined by a load of 45–60 g of root and 140– 200 ml of extractant (Table 1). Thus, no sign of correlation between sample weight and volume of extractant, when looking at the resulting CNp, was observed in an analysis of variants with a test for this interaction. For the volume F and P values were (main variable F Å 0.574, P Å 0.644; covariable F Å 0.06, P Å 0.939); for the weight the values were (main variable F Å 1.358, P Å 0.306; covariable F Å 0,294, P Å 0.598). Thus, a method depending on 75 s of homogenization (50 g of sample and 160 ml of extractant) was tested against the homogenization procedure recommended by Cooke (1978) when using a Waring/Braun blender type, i.e., 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (total time 195 s), a procedure also used by Essers et al. (1993), while O’Brien et al. (1991) used a constant homogenization of 120 s. The comparison (using a Braun blender—Type 4142), showed no significant differences in the CNp’s obtained (Table 2). However, since the ordinary Braun/Waring blenders need 120–195 s to produce the same degree of extraction (Cooke, 1978; O’Brien et al., 1991), and since the emptying and cleaning of these are more complicated (time consuming), each unit of the new blender has a significantly greater throughput than an ordinary Braun/Waring blender.
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FIG. 1. Protected shaft connection between motor and knife (1, motor shaft; 2, motor casing; 3, chassis of blender; 4, shielding; 5, shaft; 6, rinsing hole; 7, knife; and 8, check nut). Overall height of shaft, knife, and nut is 80 mm.
FIG. 2. Basic construction details of the blender. On the right and the left are given the front and side views, respectively, showing the interior. For overall dimensions see Experimental.
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BRIMER, SAKA, AND PEDERSEN TABLE 1 Effect of Cassava Loading and Volume of Acid Extractant on Yield of Total Cyanogens for Cultivar TMS 14303951
1 Samples were homogenized for 75 s using the new blender. Each measurement represents one homogenization, the CNp of the supernatant being measured four times (mean { SD; n 0 1), as assayed by Cooke’s method. Means, of columns and rows, respectively, with the same letter, do not differ significantly (P õ 0.05), when analyzed using Newman–Keuls student test. An analysis of variance with test for interaction between solvent volume and sample weight showed no sign of correlation (analysis by weight—multiple R squared 0.284; analysis by volume—multiple R squared 0.136). 2 Grand mean.
Although there is very comprehensive literature on the analysis of cyanogens in cassava roots, no investigations appear to have been published on relations between the extraction efficiency and the particle size of the homogenate. Hence, we decided to analyze the particle size distribution, found in a homogenate, as a function of the time of processing. A preliminary impression of the comminution process was gained TABLE 2 1
Total Cyanogens (CNp) of Fresh Cassava Roots as Affected by the Type of Homogenization Procedure1
1 Homogenization regimes: (a) New blender—75 s of continuous homogenization, (b) Braun blender— 15 s at low speed, followed by 2 1 1 min (separated by a 1-min interval) at high speed (total time 195 s). In each case 50 g of sample was homogenized in 160 ml of 0.1 M H3PO4 . 2 Each CNp is the mean ({ SD; n 0 1) of four homogenizations with each supernatant analyzed once. Values between the two homogenizers followed by a different letter are statistically different (P õ 0.05), according to Student’s t test.
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FIG. 3. Sedimentation analysis of cassava root homogenates: sedimentation time as function of the time of homogenization. Fifty grams of cassava root (Brazil cultivar) in 160 ml of 0.1 M H3PO4 . The total volume was made-up to 240 ml (with extractant) prior to analysis.
through a sedimentation analysis (Fig. 3). From this it is further seen that the time for formation of a visually clear supernatant is around 5 min for an 80-s homogenate (complete extraction). This is interesting, since the solid-phase method for determination of total cyanogens in cassava root of Saka et al. (1998) was recently shown to accept the use of such a freely formed supernatant instead of a filtrate. The particle size distribution as a function of the time of processing is shown in Fig. 4. It is noteworthy that the decrease in the weight of particles larger than 2800 mm is rapid for the first 50 s, after which time this fraction is relatively stable. In contrast, the fraction of particles smaller than 710 mm is increasing rapidly in the time interval from 40 to 80 s of homogenization. This pattern may, for example, be due to the existence of a fraction of relatively hard tissue still found on the coarse sieves after the first 40–50 s of homogenization. A further analysis of the results presented in Fig. 4 shows for 40 and 80 s of homogenization, respectively, the following (sieve in micrometers 0 percentage weight at sieve or passing this 40 s/80 s): 2000 0 65.0/ 74.1, 1400 0 53.2/64.8, 1000 0 43.0/55.4, 710 0 32.8/46.4. Thus, a full extraction of linamarin in this case coincides with approximately 70% of the material being of particle size smaller than 2.8 mm and 50% smaller than 2.0 mm. The number of analyses for cyanogens in cassava performed at research and breeding stations per year worldwide is constantly rising, i.e., for purposes of identifying material for breeding programs (Dixon et al., 1994), for check of grown material (Bokanga, 1994), or for investigations concerning the relation of the cyanogenic potential with ecological factors (Bokaga et al., 1994). In the future, it is envisaged that routine analysis will spread to other areas. We believe that the design presented in this report points to construction details that should be considered when looking for extraction equipment that shall handle a large number of samples day after day for
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FIG. 4. Sieve analysis (particle size distribution) of fluid-bed-dried cassava root homogenates (50 g of root—Brazil cultivar—in 160 ml of 0.1 M H3PO4 ). Each point represents the mean from the analysis of three independently produced homogenates.
the routine analysis of constituents such as toxins, pesticide residues or nutrients in cassava roots as well as in other plant tissues. The blender is not commercially available, however, the authors will provide detailed plans to individual readers upon request. Furthermore, the IPU Engineering Design, Technical University, Denmark, may build single copies on request. ACKNOWLEDGMENTS We thank the International Program in the Chemical Sciences (IPICS), Uppsala University, Sweden, and its Director Professor Rune Liminga for funding the development of the blender and a 2-month research fellowship to Dr. Saka at Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Copenhagen, and for a research grant (Project MAL 01). We also thank Professor H. Gjeldstrup and Dr. Ole Jungersen, Department of Pharmaceutics, Royal Danish School of Pharmacy (RDSP), for their assistance in fluid-bed drying of cassava solids and seive analysis, and Dr. Per Mølgaard (RDSP), Department of Medicinal Chemistry, for his help with the statistical analysis.
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