Recovery of Linamarin by Adsorption of Cassava Extract onto

The unavailability of linamarin and its use in various applications such as determining the cyanide content in cassava flour and chips and measuring g...
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SEPARATIONS Recovery of Linamarin by Adsorption of Cassava Extract onto Activated Carbon Sunny E. Iyuke* and Christopher A. Idibie School of Chemical and Metallurgical Engineering, UniVersity of the Witwatersrand, P/Bag 3, Wits, 2050 Johannesburg, South Africa

The unavailability of linamarin and its use in various applications such as determining the cyanide content in cassava flour and chips and measuring glucose in biomedical applications and hydrogen cyanide in the environment using linanmarin/linamarase/SnO2 electrode biosensors have necessitated its recovery from cassava tissues. A 0.2 µm membrane was used at a filtration rate of 1.44 mL/min for the initial isolation of linamarin, followed by extract adsorption onto activated carbon. U-shaped isotherms were observed for the adsorption of bulk cassava jelly-like solid (BJS) extract onto activated carbon. The phenomenon can be described by the selective adsorption of the solvent (B) while the solute (A) is left concentrated, resulting in an initial increase in A until equilibrium is reached and A starts to drop. This process in which the concentration of the solute in the presence of the solvent results from competitive adsorption (solute-solvent) is referred to as surface excess adsorption. Integration of the results from both 1H NMR and IR analyses indicates that the product was linamarin. The decomposition temperature of the purified product was found to be in the range of 138140 °C, which corresponds to values reported in the literature. The Freundlich empirical adsorption equation was used to simulate surface excess adsorption where values of the volume of solute per mass adsorbed (V) at 25 and 65 °C were found to be 0.434 and 3.950 mL/g, respectively. In comparison, the values calculated using the Freundlich equation fitted the experimental data quite well. Introduction Linamarin, 2-(D-glucopyronosyloxy-2-methylpropanenitrile), is a cyanogenic glucoside found in plants such as cassava (Manihot esculenta Crantz). Cassava has been reported to contain an estimate of 225-1830 mg/kg of the nitriloside linamarin.1 It is also believed that the cassava plant uses linamarin in its defense mechanism when it hydrolyzes with the enzyme linamarase (β-glucosidase, also contained in the plant) to release deadly cyanohydric acid (known as prussic acid) against predators such as herbivores.2 The hydrolysis of linamarin involves the formation of an intermediate, acetonecyanohydrin, which breaks down spontaneously or by hydroxynitrilylase action to form acetone and hydrogen cyanide3,4 as shown in Figure 1. Cassava serves as food for more than 500 million people worldwide,3 and its constituent linamarin has been known since 1891.5 It has been implicated as an aggravating factor in iodine deficiency disorder as a result of malnutrition. Its role in neurological disease and some tropical variants of diabetes mellitus has also been reported.6 As a cyanoglucoside, linamarin is chemically related to amygdalin (laetrile), although the two compounds have different molecular weights and structures. As compounds of cyanogenesis (especially laetrile), both materials have been vehemently rejected for therapeutic applications in cancer treatment across the world.7-9 This controversial natural product, linamarin, has been shown to have applications as a standard in determining cyanide content in cassava-related food products such as gari, cassava flour, and chips, and in linan* To whom correspondence should be addressed. E-mail: [email protected]. Tel.: +27 (0)11 717 7594. Fax: +27 (0)11 717 7591.

marin/linamarase/SnO2 electrode biosensors4 used for measuring glucose in biomedical applications or hydrogen cyanide in the environment. Cassava plant tissues contain many macromolecules and minerals including cyanide and tannin, as summarized in Table 1. Recently, a ban was imposed on the off-the-shelf sale of linamarin by the World Health Organization (WHO) to prevent its indiscriminate use. Because its production is synthetic, it is therefore necessary (as s natural plant) to devise an isolation procedure for its recovery from the cassava plant for valuable uses such as those mentioned above. However, its isolation from cassava plant is very difficult. Such difficulty is not encountered in the recovery of narirutin by adsorption from water extract of citrus unshiu peels.11 The difficulty is associated with the numerous minerals, proximate materials, and antinutrient substances that are contained in the plant, as presented in Table 1. In an attempt to improvise a convenient and simple linamarin isolation protocol, this article discusses a membrane filtration technique followed by a batch adsorption process using activated carbon to isolate and recover linamarin by adsorption from the multicomponent12 cassava extract. The recovery protocol is presented in Figure 2 and discussed in the later sections. The isolated and recovered linamarin could be used to functionalize carbon nanotube (CNT) biosensors, which will be reported in future articles. Experimental Section Materials and Equipment. Analytical-grade (98-99.5% purity) sodium carbonate, picric acid, 0.1 M phosphate buffered solution (PBS) (pH 6.0) and 0.1 M acetate buffered solution (pH 5.5) were acquired from Sigma-Aldrich (Milwaukee, WI).

10.1021/ie061512a CCC: $37.00 © 2007 American Chemical Society Published on Web 06/14/2007

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Figure 1. Linamarin hydrolysis with linamarase.

Figure 2. Schematics of the recovery of linamarin, which involves its isolation, purification, and analysis: (A) extraction of linamarase, (B) linamarin isolation followed by purification using adsorption on activated carbon, (C) characterization of linamarin, its hydrolysis with linamarase, and calibration. Table 1. Proximate, Mineral, and Antinutrient Contents in Unfermented Cassava Products10 proximate

mineral

% of dry matter

antinutrient

mg/100 g of dry matter

dry matter

component

flour

gari

component

flour

gari

component

flour

gari

protein crude fat ash crude fiber

4.4 2.6 2.1 3.8

3.6 3.6 1.9 3.7

Zn Mg Fe Ca Na K

13.1 43.4 26.0 61.6 43.8 49.8

5.8 27.7 2.3 16.7 51.4 55.6

cyanide (mg/kg) tannin (%)

21.3 0.2

14.6 0.2

Methanol and ammonium sulfate were obtained from Merck Chemicals (Modderfontein, South Africa). Commercial granular activated carbon was obtained from Associated Chemical Enterprises (Johannesburg, South Africa) and had the following physical properties: particle size, 8-30 mesh; bulk density, 483 kg/m3; moisture content, 2.4 wt %; specific surface area, 746 m2/g; and average pore size, 26 Å. A MiniFlex Ultrafiltration (UF) system built around a tubular module was purchased from Schleicher and Schuell, Dassel, Germany. It contained polyethersulfone membranes (0.2-0.45 µm) of polypropylene screens and silicone adhesives, with a

nominal molecular weight cutoff of 0.2 µm and 50 kD inclusive and a membrane surface area of 2.4 mm2. Other parts of the UF system included a constant-pressure variable-speed peristaltic pump, pressure gauges to measure the retention inlet and outlet pressures, connecting tubes for feed flow, and two 140 mL graduated containers for feed and permeate. Procedures Used in the Recovery of Linamarin from Cassava Tissues. The procedures included the following stages: Preparation of Picric Paper. Whatman No. 1 filter papers were dipped into an alkaline picrate sodium solution (0.5% w/v in 2.5% w/v sodium carbonate), which immediately turned the

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paper from white to yellow. After 30 min, the filter papers were air-dried. The picric papers were stored in a dark medium until use. These papers were used in the spectrophotometric analysis of concentration of linamarin throughout the study. This analysis included taking 4 mL samples in a vial and adding 11 mL of linamarase. Picric paper, cut to size, was suspended above the samples, and the vials were immediately stoppered. The vials were held at 30 °C overnight. A change in the color of the picric paper to orange and brown was observed. This is in accordance with previous studies conducted by Cooke.13 The papers were removed and immersed in 50 mL of distilled water. After 30 min, the elutes were collected, and the absorbance was read at 510 nm against a blank solution. Extraction of the Enzyme, Linamarase. Figure 2 presents a schematic outline of the major processes involved in the recovery of linamarase and the analysis of linamarin from cassava tissues. The enzyme, linamarase, was extracted as described previously13,14 with modifications. Fresh cassava peels (locally produced) were crushed and homogenized in 0.1 M acetate buffer (pH 5.5). The solution was centrifuged at 10 000 rpm for 30 min, and the supernatant was decanted into a saturated solution of ammonium sulfate (60% w/v) and kept at 4 °C for 16 h. This solution was then centrifuged at 10 000 rpm for 1 h. The precipitate thus obtained was dissolved in 0.1 M phosphate buffered solution (PBS) at pH 6.0 and dialyzed using a membrane with a 0.45 µm pore size. Further purification was achieved using a 0.2 µm membrane that permeated the cyanogenic component, where the purified enzyme was collected as retentate over the 50 kD membrane cutoff. The enzyme solution was stored at 4 °C. RecoVery of Linamarin. Cassava roots (1 kg) were diced, crushed, and homogenized in boiling methanol (99.5%). The bulk solution was allowed to settle for 1 h, after which the relatively clear supernatant was decanted from the sediment. This solution was filtered using a 0.2 µm membrane in the UF unit. Membranes with different pore sizes (0.45 µm, 0.2 µm, and 50 kD) were used for the preliminary experiments with 1 kg of bulk cassava roots. The concentrations of linamarin obtained were 0.04 g/mL at 1.52 mL/min, 0.07 g/mL at 1.44 mL/min, and 0.08 g/mL at 0.62 mL/min for the 0.45 µm, 0.2 µm, and 50 kD membranes, respectively. Because the concentration of linamarin (0.07 g/mL) obtained from the 0.2 µm membrane was relatively high at the filtration rate of 1.44 mL/ min, the 0.2 µm membrane was used throughout the study. The isolated linamarin mixture was then evaporated at 45 °C. A dark brown jelly-like solid (BJS) was obtained. This preparation was repeated for 1.5 and 2.5 kg of cassava roots. Then, 60 g of the crude cassava extract, referred to as BJS, was dissolved in 250 mL of distilled water, which was then placed in contact with 80 g of activated carbon. All batch adsorption experiments were performed by placing known amounts (60, 80, and 100 g) of activated carbon into contact with 150 mL of BJS aqueous solution of known concentrations and stirring at 190 rpm at room temperature (25 °C) for varying periods of time until the solution turned colorless after filtration. After each contact time and filtration, 4 mL of filtrate was collected and added to 11 mL of linamarase for spectrophotometric analysis. The procedure was also repeated at different temperatures to study the effect of temperature on the adsorption. It was also repeated with longer contact times of 62, 72, and 84 min with 60, 80, and 100 g, respectively, of BJS. A total of 1.7 g of white granules was recovered from 60 g of BJS. Subsequent isolations from crude extracts of 80 and 100 g yielded 2.0 and 2.5 g of linamarin, respectively.

Figure 3. Determined standard parameters: optimum quantity of enzyme activity (error bars represent standard deviation of n ) 3 experiments) and linamarin calibration curve with a standard deviation of 0.2.

Nuclear Magnetic Resonance and Infrared Analysis. The recovered linamarin is characterized using 1H NMR and IR spectroscopies. Routine 1H NMR spectra were recorded on Brucker 400 spectrometers. D2O solvent was used to dissolve 5 mg of purified product, and tetramethylsaline (TMS) was used as the internal standard. Typical results are shown in Figure 6 below. 1H NMR (300 MHz, D2O): δ (ppm) 1.44 (d, 3H, J ) 5.9 Hz, CH3), 1.33 (d, 3H, J ) 6.9 Hz, CH3), 2.40 (s, 2H, CH2OH), 3.06 (t, 1H, J ) 8.12 Hz, 8.12, HCH2OH), 5.41 (d, 4H, J ) 3.58 Hz, 4CHOH) (where J is the coupling constant and s, d, and t indicate singlet, doublet, and triplet peaks, respectively). Infrared spectra were recorded using a Bruker Vector 22 model instrument. For each spectrum, 5 mg of purified product was dissolved in 1 mL of deuterated water (D2O) to form a film. The film was introduced onto a sodium chloride plate, and the infrared spectrum was recorded in the range of 14543419 cm-1. Results and Discussion Enzyme Activity. To determine the specific activity (units/ mass of protein) of linamarase, a solution containing 0.42 units/ mL of the enzyme was prepared using 0.08 g of the protein. This gave a specific activity of linamarase of 52 × 10-6 mol/ (µg min) [where 1 enzyme unit (EU) ) 1 µmol/min]. Subsequently, varying amounts (2-13 mL) of linamarase solution were mixed with 2 g of linamarin, where 2 g of linamarin was determined as the optimum quantity of linamarin from the range of 1.5-2.5 g of linamarin as reported elsewhere.15 The hydrolysis of linamarin by linamarase was found to be proportional to the amount of the enzyme used. Figure 3 presents the absorbance readings, which represent HCN equivalents released4,13,14 from linamarin (Lmr), against the quantity of enzyme used. As can be seen from the figure, the absorbance increases with increasing volume of enzyme until equilibrium is reached at 11 mL of enzyme solution. The curve obtained (Figure 3) is characteristic of enzyme-catalyzed reactions, which can normally be analyzed using the Michaelis-Menten kinetics (which does not fall within the scope on this article). Therefore, 11 mL of the enzyme solution was used in all subsequent sample analyses. The calibration curve obtained with different standard solutions of linamarin to account for the complete range of concentrations needed in the entire set of experiments is also presented in Figure 3. It is a plot of HCN released, represented as spectrophotometric absorbance, versus standard concentration (0.3, 0.6, 0.9, 1.2, 1.5, 1.8, and 2.1 g/mL) of linamarin, which gave a linear relationship. Therefore, with the measured spec-

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Figure 4. Varying concentrations of linamarin isolated from different weights of cassava root with time, including the flux dynamics of BJS obtained from the membrane.

trophotometric absorbances of subsequent samples, the concentrations of linamarin could be read from the calibration curve. Linamarin Isolation from Cassava Extract. Figure 4 presents varying concentrations of linamarin isolated from different weights (1, 1.5, and 2.5 kg) of cassava roots with time, including the flux dynamics of BJS obtained from the membrane after the permeate had been evaporated. The concentration of linamarin from bulk cassava extract (BJS) was high at the initial time of operation and started to decrease after 250 min until equilibrium was reached (forming a plateau). All of the curves increased with time for all quantities of cassava roots used and followed the same pattern. The fluxes decreased with time, which is normally a direct result of a decrease in driving force and an increase in the hydraulic resistance of the membrane due to membrane surface fouling, which, in turn, can be caused by the excessive accumulation of particulates at the membrane surface or in the pores. This can also be due to chemical polarization caused by the accumulation of retained solutes or particles on the membrane or in the boundary layer adjacent to the membrane surface. Normally, the concentration is highest at the membrane surface and decreases exponentially toward the solution. In the case of higher concentrations of BJS, the solubility limit might be reached at the membrane surface. The precipitated layer could act as a secondary membrane referred to as the “gel layer”. Gel layer formation could be responsible for the decrease in flux. The gel layer might also have a higher retention than the membrane itself, which increases the actual retention of the membrane as the filtration process proceeds. The concentration polarization is reduced by decreasing the quantity of BJS in solution from 2.5 to 1 kg as reflected in the flux data presented in Figure 4. The points of intersection between the data on flux and those on the concentration of linamarin with time decreased from 32.9 to 26.6 and 20.5 mg/mL at 270 min for 1, 1.5, and 2.5 kg of cassava roots, respectively. The flux also decreased from 5.08 to 4.11 and 3.16 at 270 min for 1, 1.5 and 2.5 kg of cassava roots, respectively. This decline in the flux and the concentration of BJS is a result of the high concentration of cassava extracts, which reduced the membrane flux, which is similar to results reported earlier.16,17 The gradual decline in membrane performance observed during the process can also be attributed to membrane fouling and concentration polarization.18,19,20 Thus, 1 kg of cassava root was used in subsequent experiments because the filtration process can be run for a longer period of time and more filtrate can be obtained than with the other quantities (1.5 and 2.5 kg of cassava roots). Linamarin Recovery from BJS by Adsorption on Activated Carbon. As shown above, the dark brown jelly-like solid

(BJS) obtained from the evaporation of the permeate from the 0.2 µm membrane was shown to contain linamarin. To recover the pure linamarin from this mixture, the BJS was dissolved in distilled water to obtain a standard solution, which, in turn, was subjected to a stirred batch adsorption process with activated carbon as discussed above. Figure 5 represents adsorption kinetics in terms of the change in concentration of linamarin on different weights (60, 80, and 100 g) of activated carbon (Figure 5a) and for different initial concentrations of BJS (Figure 5b). As shown in Figure 5a, with 60 g of activated carbon, there was an increase in the change in concentration of linamarin up to 20 min of adsorption, when equilibrium was attained. Different scenarios where seen when 80 and 100 g of activated carbon were used. There was an initial increase in the change in concentration of linamarin, until equilibrium at 20 min for 80 g and 17 min for 100 g of activated carbon. The equilibrium stage was followed by a monotonic drop in the change in concentration of linamarin to zero. It is interesting to note that the decrease in the concentration of linamarin was more rapid when 100 g rather than 80 g of activated carbon was used in the adsorption process. As a result, 80 g of activated carbon was used in future experiments. As can be seen in Figure 5b, similar adsorption kinetics as for 80 and 100 g of activated carbon were observed with varying weights of BJS. This unique adsorption behavior of BJS on activated carbon observed in an effort to recover linamarin from complex and concentrated BJS solutions can be taken as similar to a phenomenon described earlier by Treybal21 as adsorption from concentrated solutions.22,23 Figure 5 therefore presents the apparent adsorption of the constituents of BJS, which consisted of linamarin, A, as the solute and the solvent water, B, containing the impurities, which are the proximate materials, minerals, and antinutrients (Table 1). The constituents of B are not individually analyzed in this article. The phenomenon can be described as the preferential adsorption of B, whereby A is initially concentrated, as indicated in the initial increase of A, until equilibrium (maximum point) is reached and A starts to be adsorbed, which is often referred to as surface excess.24 Surface excess is a measure of the extent to which the bulk B is depleted of one component, A, because the surface layer of the activated carbon used is correspondingly enriched in the sorbate. It is the competitive character of adsorption processes occurring at liquid-solid interfaces.25 Characterization of the Recovered Linamarin. The 1H NMR spectrum (Figure 6) of the purified product has been discussed previously. It suffices to note that the doublet obtained from the 4H of the CHOH corresponds to the anomeric proton of linamarin26 as a β-glucoside. The remaining 4H band of the HOCH is inseparable from the OH band of the D2O (deuterated water used as the solvent), which appears as the largest absorption band in the spectrum, indicating a total of 17 H atoms. This is always the case for protons of NH and OH whenever D2O is used as a solvent.27 The infrared spectrum of the purified product is shown in Figure 7. All of the functional groups that constitute linamarin as a compound were identified at their different wavelengths (cm-1): 3419 (O-H), 2930 (CCH3), 2721 (C-H), 1649 (-CN), 1576 (-CHO), 1454 (-CH2). Integration of the results from both 1H NMR and IR analyses indicated that the condensed formula of linamarin was C6H11O6C(CH3)2CN or C10H17NO6, giving a molecular weight of 247.24 g/mol. The decomposition temperature of the purified product was found to be 138-140 °C, which corresponds to

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Figure 5. Preliminary determination of optimum parameters: (a) different weights of activated carbon on 80 g of BJS, (b) different weights of BJS on 80 g of activated carbon. (Error bars represent the standard deviation for three experiments.)

Figure 6.

1H

NMR spectrum of purified linamarin.

values reported in the literature.28 The structure of linamarin thus determined corresponds to that presented in Figure 1. Apparent Adsorption Isotherm of Linamarin from Solution onto Activated Carbon. If the aqueous solution of the BJS is assumed to be a binary mixture consisting of the solute linamarin as component A of mole fraction x1 and component B, which is the solvent water and the impurities with mole fractions x2, x3, x4, etc., there are three degrees of freedom upon adsorption of the binary liquid mixture. However, the system is reduced to two independent variables, because the liquid and solid phases are both condensed and thus the effect of pressure becomes negligible. These are the temperature, T, and x1, the mole fraction of linamarin in the bulk liquid phase at equilibrium. The models used for the measurement of the surface excess, n1e, as a function of x1 along an isotherm for a binary solution are given as29

0

n1e ) n0(x10 - x1)

(1)

n1e ) n1x2 - n2x1

(2)

where x1 is the mole fraction of component 1, linamarin, in the bulk liquid solution before it is placed into contact with the activated carbon; n0 is the total number of moles of bulk liquid solution per unit mass of adsorbent (mol/kg); and n1 and n2 are the specific amounts (mol/kg) of components 1 and 2 adsorbed. It is extremely difficult to measure n1 and n2 experimentally and simultaneously because of uncertainty in the location of the interface between the bulk liquid and the adsorbed phase. Because n2, x2, x3, ..., xN were not measured in the study, eq 1 was used to analyze the apparent adsorption isotherms. Figure 8a presents the surface excess as a function of x1 at different temperatures. A pattern of data characteristics similar to that

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Figure 7. Infrared spectrum of purified linamarin.

Figure 8. Effect of temperature on (a) surface excess and (b) parameters in the Freundlich equation (experimental data, points; best-fit linear regression, smooth lines).

shown in Figure 8a was seen earlier in Figure 5. Both are general cases of U-shaped isotherms, as have also been reported in the literature.10,24,29,30 As shown in Figure 8a, the maximum surface excess was almost the same at all temperatures, but the areas under the curves decreased with increasing temperature. The latter observation could mean that the adsorption of linamarin as displayed at the right-hand side of the curves increases with decreasing temperature, which is normal in the adsorption of a solute onto activated carbon. Adopting Freundlich Equation. Treybal21 stated that, over a small concentration range, adsorption isotherms can be described by the empirical Freundlich equation as

c* ) k[V(c0 - c*)]n

(3)

where V(c0 - c*) is the apparent adsorption per unit mass of adsorbent c0 is the initial solute concentration c* is the final equilibrium solute concentration (mass of solute/volume liquid) k and n is the constants V is the volume of solute per mass adsorbed.

Table 2. Parameters in the Freundlich Equation temp (oC)

n

ln k

k

linear regression coefficient (R2)

standard error

25 35 45 55 65

1.514 0.662 0.671 0.666 0.638

0.993 -0.661 -0.681 -0.614 -0.616

2.699 0.516 0.506 0.541 0.540

0.992 0.993 0.993 0.992 0.736

0.0176 0.0177 0.0194 0.0190 0.0141

Taking the logarithm of both sides of eq 3 gives

ln c* ) ln k + n ln[V(c0 - c*)]

(4)

Thus, a plot of ln c* versus ln[V(c0 - c*)] (Figure 8b) using eq 4 gave excellent straight lines with slopes n and intercepts ln k at different temperatures, as presented in Table 2. A linear regression between the experiments and the model gave regression coefficients (R2) of 0.992, 0.993, 0.993, 0.992, and 0.736 at the adsorption temperatures of 25, 35, 45, 55, and 65 °C, respectively, with standard errors ranging from 0.0176 to 0.0141 (Table 2). It is obvious from Figure 8b that there is less degree of regression between the experiments and the model

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data reported, the Freundlich empirical equation was used to simulate the data. In comparison, the values calculated using the Freundlich equation fitted the experimental data quite well. Acknowledgment

Figure 9. Comparison of experimental and calculated surface excess at 25 and 65 °C with standard errors of 0.640 and 0.754, respectively (experimental data, points; simulation, smooth lines).

at a temperature of 65 °C than at the other temperatures. However, the values of R2 at all temperatures are reasonably significant. It can be observed that there was no appreciable difference in the values of k and n in the temperature range of 35-65 °C (p > 0.05). However, a huge difference exists in the values of k and n for adsorption at 25 °C and the rest of the temperatures (35, 45, 55, and 65 °C). This means that the surface excess at 25 °C was higher than that at the rest of the temperatures studied, perhaps because adsorption, as an exothermic process, would result in a decreased concentration of the adsorbed phase with increased temperature at a given equilibrium pressure. This observation was seen earlier in Figure 8a. To verify the data reported in Figure 8, the Freundlich empirical equation was used to simulate these data. After a few iterations, the values of V obtained for adsorption at 25 and 65 °C were 0.434 and 3.950 mL/g, respectively. In comparison, therefore, the data calculated using the Freundlich equation fitted the experiments quite well, as shown in Figure 9. This observation therefore confirms the applicability of the familiar Freundlich equation in describing the absolute adsorption that relates to surface excess adsorption from concentrated solutions. However, it must be emphasized that the inability to exploit the multicomponent constitution of the cassava extract in the adsorption processes might have resulted in an oversimplification of the analysis, which will be further explored in the future. Conclusion A 0.2 µm membrane was used throughout the membrane study, and a decline in the flux and the concentrations of BJS was observed as a result of a high concentration of cassava extracts that reduced the membrane flux, which is in agreement with the literature. The gradual decline in membrane performance as observed during the process can be attributed to membrane fouling or the accumulation of retained solutes or particles on the membrane, as observed from the different initial concentrations of BJS used. The apparent adsorption of the constituents of BJS, which consisted of linamarin, A, as the solute and the solvent water, B, which, in turn, contains the impurities, such as the proximate materials, minerals, and antinutrients, followed a U-shaped isotherm. Integration of the results obtained from both 1H NMR and IR analyses indicated that the condensed formula of linamarin is C6H11O6C(CH3)2CN or C10H17NO6. The decomposition temperature of the purified product was found to be 138-140 °C, which corresponds to values reported in the literature. To verify the

The authors acknowledge the financial support from the H. E. Griffin Trust Foundation and Professor Herman Potgieter of the School of Chemical and Metallurgical Engineering, Professor Neuse of the School of Chemistry, and Dr. Davids Hajierah of the Department of Pharmacy and Pharmacology, University of the Witwatersrand, Johannesburg, South Africa, for technical support. Literature Cited (1) Culbert, M. What the Medical Establishment Won’t Tell You That Could Save Your Life. In World Without Cancer; Griffin, G. E., Ed.; Donning Co.: Virginia Beach, VA, 1983; p 24. (2) IIza, A. F.; Mario, H. P. P. Cyanogenic Glycosides in Plants. Braz. Arch. Biol. Technol. 2000, 43, 2. (3) Bradbury, J. H.; Egan, S. V.; Lynch, M. J. Analysis of Cyanide in Cassava Using Acid Hydrolysis of Cyanogenic Glucosides. Food Agric. 1991, 55, 277. (4) Yeoh, H. H.; Tatsuma, T.; Oyama, N. Monitoring the Cyanogenic Potential of Cassava: The Trend Towards Biosensor Development. Trends Anal. Chem. 1998, 17, 234. (5) De Bruijn, G. H. A study of cyanogenic character of cassava. In Proceedings of an Interdisciplinary Workshop; Nestel, B., MacIntyre, R., Eds.; IDRC: London, 1973; p 43. (6) Grindley, B. A. P.; Omoruyi, I. F.; Asemota, N. H.; Morrison, Y. S. E. Carbohydrate Digestion and Interstinal ATPases in StreptozotocinInduced Diabetic Rats Fed Extract of Yam (Dioscorea cayenesis or dasheen Colocasia asculenta). Nutr. Res. 2002, 22, 333. (7) Greenberg, D. M. The Case Against Laetrile: The Fraudulent Cancer Remedy. Cancer 1980, 45, 799-807. (8) Dorr, R. T.; Paxinos, J. The Current Status of Laetrile. Ann. Intern. Med. 1978, 89, 389-397. (9) Milazzo, S.; Ernst, E.; Lejeunes, S.; Schmitt, K. Leatrile Treatment for Cancer. Cochrane Database Syst. ReV. 2006, 19, CD005476. (10) Oboh, G.; Akindahunsi, A. A.; Ashodi, A. A. Nutrient and Antinutrient Contents of Aspergillus Niger-Fermented Cassava Products (Flour and Gari). Food Comp. Anal. 2002, 15, 617. (11) Kim, M. R.; Kim, W. C.; Lee, D. Y.; Kim, C. W. Recovery of Narirutin Adsorption on a Non-ionic Polar Resin from a Water of Citrus Unshiu Peels. Food Eng. 2007, 78, 27. (12) Kalies, G.; Brauer, P.; Messow, U. Prediction of Multicomponent Liquid Adsorption using Excess Quantities II. Calculations for the Liquid/ Solid Interface. Colloid Interface Sci. 2004, 275, 410. (13) Cooke, R. D. An Enzymatic Assay for the Total Cyanide Content of Cassava (Manihot esculenta Crantz). Sci. Food Agric. 1978, 29, 345. (14) Seigler, D. S. Isolation and Characterization of Naturally Occurring Cyanogenic Compounds. Phytochemistry 1975, 14, 9. (15) Idibie, A. C.; Davids, H.; Iyuke, S. E. Cytotoxicity of purified cassava linamarin to a selected cancer cell lines. Bioprocess. Biosyst. Eng., in press. (16) Srikanth, G. Membrane Separation ProcesssTechnology and Business. Chem. Eng. World 1999, XXXIV, 55. (17) Mohr, C. M.; Engelgan, D. E.; Leeper, S. A.; Charboneau, B. L. Membrane Technology ReView: Membrane Applications and Research in Food Processing; Noyes Data Corporation: Park Ridge, NJ, 1989. (18) Nakao, S.; Kimura, S. Effect of gel layer rejection and fractionation of different molecular weight solutes by ultrafiltration. In Synthetic Membranes: Hyperfiltration and Ultrafiltration Uses; Turbak, A. F., Ed.; ACS Symposium Series 154; American Chemical Society: Washington, DC, 1981; Vol. II. (19) Haris, J. L. Influence of Gel Layer Rheology on Ultrafiltration Flux of Wheat Starch Effluent. Membr. Sci. 1986, 29, 97. (20) Haris, J. L.; Dobos, M. Enhanced Ultrafiltration Flux Rates by Enzymatic Hydrolysis in Protein Recovery from Wheat Starch Effluent. Membr. Sci. 1989, 41, 87. (21) Treybal, R. E. Mass-Transfer Operations; McGraw-Hill: London, 1981.

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ReceiVed for reView November 27, 2006 ReVised manuscript receiVed May 4, 2007 Accepted May 8, 2007 IE061512A