Detoxification of Cassava Leaves by Thermal, Sodium Bicarbonate, Enzymatic, and Ultrasonic Treatments Sajid Latif , Sonja Zimmermann, Ziba Barati , and Joachim Müller Cassava leaves are a valuable source of protein but the cyanogenic potential limits their use as food and feed. Four different treatments were investigated to detoxify cassava leaves. Thermal (55 °C for 6 hr), sodium bicarbonate (0.4% R GC Extra, 4 hr), and ultrasonic treatments (500 W, 35 kHz, NaHCO3 , 55 °C for 6 hr), enzymatic (0.32% Multifect 55 °C, 0.25 hr) reduced the total cyanide (µg HCN equivalents per g fresh leaf or ppm) content by 90%, 93%, 82%, and 84% while the cyanide content reduction in the respective controls was 85%, 90%, 79%, and 84%, respectively. The sodium bicarbonate treatment was found to be the most effective treatment. Therefore, it was further optimized by varying time and temperature. A significant effect on the cyanide content was observed by changing the incubation time while no significant effect of temperature was noticed. Nevertheless, extended incubation time during sodium bicarbonate treatment reduced ascorbic acid content by 7% and 39% when leaves were incubated with sodium bicarbonate for 0.5 hr and 48 hr, respectively. Abstract: Keywords: cassava leaves, cyanogenic potential, detoxification treatments, nutrients Cyanogenic glucosides are the major toxic compound in cassava leaves, which limits their use as food and feed. The methods proposed in this study can be used to detoxify cassava leaves, which are generally considered as an inferior by-product. Hence, detoxified cassava leaves may contribute to fulfil world protein demand in an eco-sustainable way. Practical Application: Introduction while linamarase and HNL are localized in the cell walls (White et al., 1994). In order to allow these hydrolyzing enzymes to interact with the linamarin, a mechanical disruption or dissolution of the cell walls is inevitable. Various processing methods have been established in different countries and for different varieties of cassava (Latif & Müller, 2015). Pounding for about 15 min followed by 10 to 120 min boiling are the common steps in most detoxification methods (Bradbury & Denton, 2011). Lancaster and Brooks (1983) stated that boiling for only 10 min can reduce the vitamin C content by 60%. Diasolua Ngudi, Kuo, and Lambein (2003) reported that 30 min boiling of leaves decreased the protein and methionine content by 58% and 71%, respectively. Therefore, the valuable nutrients of cassava leaves are reduced by thermal treatment. Bradbury and Denton (2010a, 2010b, 2014) introduced methods to decrease nutrient losses in cassava leaves detoxification. Pounding the leaves for at least 10 min followed by washing with water (two times their weight) at room temperature reduced the cyanide content by 92%. In another study, the cyanide content was reduced by 93% when whole leaves were immersed in water (10 times their weight) at 50 °C for 2 hr and repeating the same procedure after changing the water (Bradbury & Denton, 2011). Bradbury and Denton (2014) established a method that includes three consecutive steps: (1) pounding, (2) spreading in the shade at 30 °C for 5 hr or in the sun at 50 °C for 2 hr, and (3) three times washing with water. These methods reduced JFDS-2018-2018 Submitted 12/11/2018, Accepted 4/17/2019. Authors Latif, the cyanide content by 72%, 88%, and 99%, respectively. It was Zimmermann, Barati, and Müller are with Inst. of Agriculture Engineering (440e), Tropics and Subtropics Group, Univ. of Hohenheim, 70599 Stuttgart. Germany. obvious that even without excessive thermal treatment, high contents of cyanide can be removed. However, in all of the methods Direct inquiries to author Latif (E-mail: [email protected]). large amount of water was used for washing. As a result, valuable Cassava (Manihot esculenta Crantz) is a perennial shrub, which grows widely in 105 tropical and subtropical countries with an estimated production of 292 million tons (Achidi, Ajayi, Bokanga, & Maziya-Dixon, 2005; FAOSTAT, 2017). Cassava is mainly known for its roots and the leaves are eaten as a vegetable in some countries as a source of protein and valuable nutrients. However, the consumption is nearly 0.5 to 0.7 million tons per year, which is far less than the consumption of cassava roots (Latif & Müller, 2015). This low consumption is due to high cyanogenic potential of cassava leaves, which may cause serious illness, or death of the consumers if consumed without proper detoxification. Casssava leaves’ toxicity is mainly due to cyanogenic glucosides, which are present in cassava plant tissues in three forms, that is, mainly linamarin (95%), lotaustralin, cyanohydrins and free cyanide (McMahon, White, & Sayre, 1995). Therefore, cyanogenic potential or total cyanide is the amount of HCN released from the above mentioned three cyanogen forms. Linamarin hydrolyse to glucose and acetone cyanohydrin in the presence of linamarase while acetone cyanohydrin spontaneously or with hydroxynitrile lyase (HNL) decompose to HCN and acetone at pH 5.0 or temperature 30 °C (White, McMahon, & Sayre, 1994). The resulting HCN evaporates at 25 °C (Achidi, Ajayi, MaziyaDixon, & Bokanga, 2008). Limamarin is located in the vacuole Toxicology & Chemical Food Safety 1986 Journal of Food Science r Vol. 84, Iss. 7, 2019 R C 2019 Institute of Food Technologists doi: 10.1111/1750-3841.14658 Further reproduction without permission is prohibited Detoxification treatments for cassava leaves . . . Materials and Methods Plant material Cassava leaves were collected from the cassava plant of toxic variety grown in the greenhouse of the Univ. of Hohenheim. The age of cassava plants was between 6 and12 months grown at 23 °C and 40% relative humidity with daily watering. The adult leaves were selected from the middle part of the plant and the middle section of the leaves was used during our experiments (Figure 1). Figure 1–Cutting positions of cassava leaves for sampling. incubated in a water bath at 25 °C for 0.5, 3, 36, and 48 hr to evaluate the effect of time. In order to study the effect of temperature, the homogenized samples were incubated in a water bath at 25, 35, 55, 75, and 85 °C for 36 hr. Sodium bicarbonate treatment Sodium bicarbonate (NaHCO3 ) solution was used for the chemical treatment. Approximately, 1 g of fresh leaves were ground in 5 mL of 0.4 % NaHCO3 solution with a homogenizer for 3 min, transferred to a 50 mL falcon tube and incubated in a water bath at 55 °C for 6 hr. Furthermore, the effect of incubation time was determined by incubating the homogenized samples at 25 °C for 0.5, 3, and 6 hr. As control for the NaHCO3 effect, leaves were ground with distilled water without adding NaHCO3 and incubated at the same temperature and time. Enzymatic treatment The enzymatic treatment of cassava leaves was performed by R using a mixture of plant cell wall–degrading enzymes (Multifect GC Extra, DuPont, Wilmington, DE, USA). The main enzyme of R the Multifect complex is cellulase, with side activities of hemicellulase, xylanase, and beta-glucanase. This enzyme is produced from strain of Trichoderma reesei. The manufacture of this enzyme claimed a specified activity of 6200 international unit (IU)/mL. Leaves were treated with an enzyme concentration of 8% of their dry weight by preparing a 0.32% solution of the liquid enzyme in 0.1 M Na3 C6 H5 O7 buffer at pH 5. Approximately, 1 g of fresh leaves were ground to 5 mL of buffer with enzyme solution with a homogenizer for 3 min, transferred to a 50 mL falcon tube, and incubated in a water bath at 55 °C for 4 hr. In the control, buffer without enzyme solution was added to the leaves, ground and incubated in the same way. Detoxification treatments Four treatments comprising thermal, chemical, enzymatic, and ultrasonic treatment were applied on fresh cassava leaves for detoxification. Each treatment was performed in triplicate. Cyanide content of fresh and treated cassava leaves was analyzed to quantify the detoxification. Ultrasonic treatment A laboratory ultrasonic bath (Transsonic 780/H, Elma GmbH Thermal treatment & Co KG, Singen, Germany) with a frequency of 35 kHz and For the thermal treatment, approximately 1 g of leaves were 500 W output was used for the ultrasonic treatment according to ground in 5 mL of distilled water with a homogenizer (Ultraturax Kwiatkowska et al. (2011). Approximately 1 g of fresh leaves were T25, IKA, Staufen, Germany) for 3 min and then transferred to ground in 5 mL of distilled water with a homogenizer for 3 min, a 50 mL falcon tube. These samples were incubated in a water transferred to a 50 mL falcon tube, and placed in the ultrasonic bath (1083, GFL Gesellschaft für Labortechnik mbH, Burgwedel, bath at 55 °C for 0.25 hr. In the control, a falcon tube with leaf Germany) at 55 °C for 6 hr with a control at 25 °C for 6 hr. paste was placed in a water bath without ultrasonic application and To evaluate the effect of time, the homogenized samples were incubated in the same way. Vol. 84, Iss. 7, 2019 r Journal of Food Science 1987 Toxicology & Chemical Food Safety minerals and water-soluble vitamins may have been leached from the leaves. Furthermore, a comparison of the nutrient contents for the untreated and treated leaves after detoxification is required to investigate the effect of these processing methods on the nutrients. The existing processing methods, showed a high reduction of the cyanide content of cassava leaves through several treatments, especially during prolonged boiling, which also has the disadvantage of high nutrient losses through thermal treatment, leaching of nutrients into the cooking water, or degradation by light. Consequently, there is a need to develop methods that can detoxify the leaves to a safe level while preserving the nutrients. Sodium bicarbonate (baking soda), which is used to tenderize vegetables, has already been used to detoxify cassava leaves, for example, in Congo for the preparation of cassava leaf dishes (Achidi et al., 2005). Sodium bicarbonate can disrupt the plant cells by reducing intercellular adhesion and subsequent cell separation (Varriano-Marston & De Omana, 1979), which facilitates linamarin to react with linamarase. Sodium bicarbonate also leads to an increase in pH, which may facilitate the spontaneous decomposition of acetone cyanohydrin. Furthermore, the application of cell-wall degrading enzymes may enhance detoxification of cassava leaves. Sornyotha, Kyu, and Ratanakhanokchai (2010) investigated the ability of two plant cell wall–degrading enzymes to hydrolyze cell walls of the cassava root cortex and the removal of linamarin by the released linamarase. Additionally, the application of ultrasound can affect the activity of enzymes and may lead to a physical disruption of the material treated (Kwiatkowska, Bennett, Akunna, Walker, & Bremner, 2011; McClements, 1995). Since a disruption of the cassava leaf cells is necessary for detoxification, ultrasound may facilitate the enzymatic breakdown of linamarin by enhanced liberation of linamarase and HNL. This study intended to investigate the potential of these four different processing methods (thermal, chemical, enzymatic, and ultrasonic treatments) to reduce the cyanogenic potential of cassava leaves, optimize the most promising method, and determine the effect of the treatment on the ascorbic acid content of the leaves. Detoxification treatments for cassava leaves . . . Determination of total cyanide content Total cyanide content was analyzed by picrate paper method according to Bradbury, Egan, and Bradbury (1999), Haque and Bradbury (2004), and Egan, Yeoh, and Bradbury (1998). For preparing the extract, approximately 1 g of leaves (fresh ones for initial content and treated ones for final content) were ground in 5 mL of distilled water and 2 mL of 0.4 M HCl with a homogenizer for 1 min to disrupt the cells in order to release linamarin and linamarase and thus enable enzymatic degradation. The solution was squeezed through a 10 × 10 cm cotton cloth via a funnel into a 15 mL falcon tube, which was centrifuged for 15 min at 20,980 rcf. The supernatant was removed and 200 µL was added to 1 mL of 0.1 M Na3 PO4 buffer at pH 6 into a 50 mL falcon tube. The falcon tube was immediately closed after adding 100 µL linamarase and carefully placing a plastic sheet with a picrate paper in the falcon tube without touching the liquid and the falcon tube. The sample was kept at 30 °C for 20 hr in an incubator. Afterward, the picrate paper was removed from the plastic sheet and immersed in 5 mL distilled water in a 15 mL falcon tube, which was occasionally shaken. After 45 min, the picrate paper was removed and the colored solution was centrifuged for 10 min at 20,980 rcf. The solution was transferred to a plastic cuvette and the absorbance was measured in a UV/Vis spectrophotometer (DR 6000, Hach Lange GmbH, Düsseldorf, Germany) at 510 nm. A blank and a standard were prepared for every experiment. The picrate papers from the blank and the standard were treated in the same way as the picrate papers of the samples. Determination of ascorbic acid content For the NaHCO3 treatment and the respective control, the L-ascorbic acid content was measured by using L-ascorbic acid standard. For fresh leaves, 3 g of leaves were cut into pieces with scissors. Then, 7.5 mL of extraction solution (10% w/v HClO4 and 1% w/v HPO3 ) was added to the leaves. The samples were homogenized with homogenizer for 1 min and the homogenizer was washed with another 17.5 mL of the extraction solution, which was added to the sample. For treated leaves, 3 g of leaves were ground with 15 g (five times cassava leaves weight) of 0.4% NaHCO3 solution and incubated in a water bath at 25 °C for 0.5 hr and 48 hr. The leaves were treated the same way in the control. Afterward, 7.5 mL of the extraction solution were added for homogenization, and 12.5 mL were used to wash off the residues from the homogenizer. All the samples were centrifuged for 0.25 hr at 4 °C and 20,980 rcf. Eight milliliters of the supernatant was transferred into a falcon tube, which was centrifuged again for 0.25 hr at 4 °C and 20,980 rcf. Three milliliters of the supernatant was transferred to a 10-mL volumetric flask, which was filled up with mobile phase. All the samples were filtered through 0.45 µm PTFE membrane filters into High Performance Liquid Chromatography (HPLC) vials and measured by reversed-phase HPLC with UV/Vis detector (SPD-M 20 A, Shimadzu, Kyoto, Japan). Samples were injected to a precolumn (10 × 4.6 mm ReproSil Pur C18-AQ) attached to a main column (250 × 4.6 mm ReproSil Pur C18-AQ). The temperature of both columns was kept at 40 °C. The mobile phase buffer consisted of 20 mM NH4 H2 PO4 and 0.015% HPO3 . HPLC was operated in an isocratic mode with a flow rate of 0.6 mL/min. Quantification of the ascorbic acid content was performed at 254 nm. Figure 2–Reduction in total cyanide content of cassava leaf pulp during 48 hr incubation at 25 °C. and analyzed using one-way analysis of variance (ANOVA) at a significance level of α = 5%. Results and Discussion Effect of time on detoxification at 25 °C Figure 2 shows a rapid decrease of the cyanide content directly after preparing the leaf pulp, which then slows down until 48 hr of incubation time. The cyanide content decreased considerably from 534 ppm to less than 50 ppm after 36 hr incubation time with more than 90% reduction in cyanide content. This may be due to fine grinding of the leaves, which may have partially broken the cell wall and enabled linamarase enzyme to hydrolyse linamarin. Thus, our results are in agreement with a previous study in which pounding was reported to reduce cyanogen content by 63% to 73% (Montagnac, Davis, & Tanumihardjo, 2009). The length of the incubation time had an obvious influence on the detoxification process. It was confirmed by the results of the ANOVA that the incubation time had a significant (p < 0.001) effect on the detoxification of cassava leaves. After 36 hr of incubation, the reduction of cyanide content was not substantial. Therefore, this incubation time was chosen for further investigation during this study. However, the cyanide content at 36 hr incubation time was still above the safe level (10 ppm) recommended by Food and Agriculture Organization/World Health Organization (FAO/WHO). Toxicology & Chemical Food Safety Effect of temperature on detoxification Our aim was to completely detoxify cassava leaves; therefore, we used long incubation time, that is, 36 hr for different temperatures. Figure 3 shows that there were no significant (p > 0.05) differences among the relative cyanide content when incubated at different temperatures for 36 hr. Although, 55 °C is the optimum temperature for linamarase activity (Achidi et al., 2008), it yielded no better results than 25 °C (ambient temperature in the tropics; Figure 3). Even high temperatures such as 85 °C showed no inhibitory effect on the detoxification treatment. In other studies, it was found that the reduction of the cyanide content in cassava leaves was increased by treatments above 60 °C with prolonged incubation time (Bourdoux et al., 1983; Padmaja & Steinkraus, 1995). Contrarily, in our study, the results show that there was a nonsignificant difference among the five selected Statistical analysis temperatures. The reason could be explained by long incubation SAS 9.3/9.4 software (SAS Inst., Cary, NC, USA) was used to time of 36 hr, which was long enough to detoxify the leaves at analyze the data. Treatments were classified as different variants any temperature. 1988 Journal of Food Science r Vol. 84, Iss. 7, 2019 Detoxification treatments for cassava leaves . . . Figure 4–Relative cyanide content of cassava leaf pulp with NaHCO3 treatment and control (leaves were ground with distilled water without adding Figure 3–Relative cyanide content of cassava leaf pulp after incubation NaHCO3) during 6 hr incubation at 25 °C. for 36 hr at different temperatures (the mean values with the same letter in clustered columns were not significantly different). To further investigate different treatments (thermal, chemical, enzymatic, and ultrasonic), a short incubation time (6 hr for thermal and chemical, 4 hr for enzymatic, and 0.25 hr for ultrasonic treatment) and the optimum temperature of 55 °C for linamarase activity were chosen. Comparison of thermal, sodium bicarbonate, enzymatic, and ultrasonic treatments Thermal treatment. The cyanide content of fresh cassava leaves and cassava leaves incubated at 55 °C for 6 hr and its control was 534, 50, and 78 ppm, respectively (Table 1). The reduction of the cyanide content was 90% and 85% for the thermal treatment and its control, respectively. In contrast to the longer incubation time of 36 hr, there was a significant difference between thermal treatment and its control for 6 hr incubation time. This could be explained by an increased enzyme activity at 55 °C in thermal treatments, which is the optimum temperature for linamarase. This incubation temperature at 55 °C is also favorable for (1) HNL because HNL can be stable at 60 °C for 45 min (White et al., 1994); (2) at higher than 30 °C, spontaneous decomposition of acetone cyanohydrin to HCN and acetone (Achidi et al., 2008); (3) evaporation of the HCN (Achidi et al., 2008). Sodium bicarbonate treatment. The cyanide content of cassava leaves treated with sodium bicarbonate (NaHCO3 ) and its control was reduced by 93% and 90%, respectively. However, there was nonsignificant difference in the cyanide content of leaves treated with NaHCO3 (30 ppm) and its control at 55 °C for 6 hr (43 ppm; Table 1). McGee (2007) reported that NaHCO3 may lead to a neutralization by reacting with organic acids present in cassava leaves. Consequently, there was no major increase of spontaneous decomposition of acetone cyanohydrin compared to the control treatment. Additionally, NaHCO3 can also have a negative impact on the activity of linamarase and HNL. The optimum pH range for linamarase is 6 to 7.3 reported by McMahon et al. (1995). On the other hand, HNL has an optimum activity at pH 5.0 but it does not lose its activity at pH ranging from 4.0 to 7.0 when incubated for 24 hr (White et al., 1994). If the pH increases to 8.3 (0.4% NaHCO3 solution that was measured), it might reduce the activity of linamarase, especially HNL, thus slowing down the detoxification process. It was observed that the NaHCO3 treatment had the highest reduction of cyanide content among the different treatments (Table 1) although the difference was not statistically significant. Therefore, the NaHCO3 treatment was selected for further investigation with the intention to reduce the incubation time, avoid higher temperatures, preserve ingredients, and save energy. The effect of treatments with NaHCO3 with short incubation time of 0.5 hr and ambient temperature of 25 °C on the reduction of the cyanide content was investigated. Figure 4 shows a clear difference in the relative cyanide content of cassava leaves treated with NaHCO3 and its control at 25 °C for less than 6 hr. It can be observed that the length of the incubation time had an obvious influence on the detoxification process. Although the same cyanide content was reduced by 90% in treatment and control after 6 hr, there was a significant (p < 0.05) difference in the relative cyanide content between the NaHCO3 -treated leaves and the control at an incubation time of 0.5 hr (Figure 4). It was determined that treatment with NaHCO3 accelerated the detoxification process especially, at a short incubation time of 0.5 hr. Moreover, treatment with NaHCO3 could be a practical method for cassava leaves detoxification due to the availability of NaHCO3 (baking soda) in households. Enzymatic treatment. The cyanide content of cassava leaves R treated with Multifect GC Extra and its control was 100 and Thermal ppm (%) Fresh Treated Control ± 30 (100) 50b ± 6 (10) 78c ± 8 (15) 534a Sodium bicarbonate ppm (%) ± 85 (100) 30b ± 2 (7) 43b ± 1 (10) 431a R Multifect GC Extra ppm (%) Ultrasonic ppm (%) ± 114 (100) 100b ± 1 (18) 118b ± 7 (21) 420a ± 24 (100) 66b ± 5 (16) 67b ± 10 (16) 559a Notes: Treatments and controls were incubated at 55 °C, except the control for thermal treatment, which was incubated at 25 °C. Incubation time for thermal and sodium R GC Extra treatment 4 hr, and for ultrasonic treatment 0.25 hr. Means in columns followed by the same letter are not significantly bicarbonate treatment was 6 hr, for Multifect different (p > 0.05). Vol. 84, Iss. 7, 2019 r Journal of Food Science 1989 Toxicology & Chemical Food Safety Table 1–Total cyanide content and relative cyanide content (in parentheses) of cassava leaves in thermal, sodium bicarbonate, enzymatic, and ultrasonic treatments (n = 3, mean ± SD). Detoxification treatments for cassava leaves . . . Toxicology & Chemical Food Safety 1990 Journal of Food Science r Vol. 84, Iss. 7, 2019 500 a,b 450 Ascorbic acid content (mg·100 g-1) 118 ppm, equivalent to a cyanide content reduction of 82% and 79%, respectively (Table 1). In contrast to our findings, Sornyotha et al. (2010) reported a linamarin content reduction of 90.3% after 1.5 hr of incubation at 50 °C by using xylanase and cellulase for detoxifying cassava root parenchyma. The authors reported that the cell wall–degrading enzymes enhance the release of linamarin and linamarase, hence promoting cyanogenesis. It might R be assumed that the Multifect GC Extra enzyme did not affect cassava leaves to the same extent as the cassava root parenchyma simply because of the structural differences. However, Sornyotha et al. (2010) did not include a control along with the enzymatic treatment, that is, incubation without enzyme. Therefore, it is not obvious whether the linamarin content was reduced directly by adding cell wall–degrading enzymes or by homogenization and incubation of the plant material. Ultrasonic treatment. Table 1 shows that the cyanide content of the cassava leaves with and without ultrasonic treatment was 66 and 67 ppm, respectively. Both, ultrasonic treatment and its control reduced the cyanide content by 84% (Table 1). High intensity (10–1000 W/cm2 ) with low frequency (20–100 kHz) ultrasonic waves are responsible for high temperature, shear gradient and pressure which can cause physical disruption and can facilitate detoxification (McClements DJ, 1995). On the other hand, high intensity ultrasound may lead to decrease enzyme activity (Kwiatkowska et al., 2011); therefore, ultrasonic treatment did not show a significant positive effect on the detoxification. The cyanide content reduction in this treatment may be due to the grinding of the leaves with water and incubating for 0.25 hr at 55 °C, which is the optimum temperature for linamarase activity. During detoxification, physical disruption of the cassava leaf tissue is necessary to get linamarin and linamarase in contact since both are located in different parts of the cell (McMahon et al., 1995). Furthermore, it was found that ultrasonic treatment can increase the activity of certain enzymes by mixing enzymes and substrates (Barton, Bullock, & Weir, 1996). However, no studies are available for the activation of linamarase by ultrasonic treatment. Contrarily, a decrease in enzymes activity by ultrasound has been observed (Islam, Zhang, & Adhikari, 2014; Lindsay Rojas, Hellmeister Trevilin, & Augusto, 2016). Hence, ultrasonic treatment effects may differ with the intensity and frequency, type of enzyme, and inhibiting or activating factors (Kwiatkowska et al., 2011). Effect of sodium bicarbonate treatment on ascorbic acid content. Among the described treatments, NaHCO3 treatment was the most effective one to detoxify cassava leaves. Therefore, it was chosen to further investigate the ascorbic acid content. Figure 5 shows the ascorbic acid content of fresh leaves, leaves treated with NaHCO3 for 0.5 hr and 48 hr and their controls. The grinding of the leaves may have increased the release of ascorbic acid from the cells. After 0.5 hr incubation with and without NaHCO3 , the ascorbic acid content was 420.8 and 440.2 mg/100 g, respectively. However, after a longer incubation time of 48 hr, the ascorbic acid content was 274.2 and 373.4 mg/100 g for NaHCO3 and the control, respectively. This result shows a significant (p < 0.05) difference between NaHCO3 treatment and the control at longer incubation times. According to Lancaster and Brooks (1983), long storage of cassava leaves leads to a reduction in vitamin C content, which would explain the significant decrease when leaves were incubated for 48 hr. The application of NaHCO3 as well as the long incubation time obviously reduced the ascorbic acid content of the leaves. Vitamin C is easily degraded since it is sensitive to light, high temperatures, a,b b Ascorbic acid content 100.0 Relative cyanide content d 400 80.0 350 c 300 60.0 250 200 40.0 150 100 20.0 50 0 0.0 NaHCO3 Fresh leaves Control 0.5 h NaHCO3 Control 48 h Figure 5–Ascorbic acid content and relative cyanide content of cassava leaves (fresh weight basis) subjected to sodium bicarbonate treatment with two different incubation times (0.5 hr and 48 hr) at 25 °C (the mean values with the same letter in each series of clustered column were not significantly different). and exposure to oxygen. Furthermore, it is stable in acidic conditions but unstable at pH ࣙ 7 (Bässler, Golly, Loew, & Pietrzik, 2002). The treatment with NaHCO3 increased the pH, which has been demonstrated in a preliminary experiment. Therefore, a pH increase could have negatively affected the stability of ascorbic acid. In contrast to traditional detoxification methods, no water was discarded in the treatment with NaHCO3 . Therefore, watersoluble vitamins such as ascorbic acid, B vitamins, as well as minerals, which may leach into the cooking or washing water (Sun, Yang, Bai, & Zhuang, 2013), could be preserved in this treatment. According to our results in Figure 5, the NaHCO3 treatment appears to be an effective method for reducing the cyanide content with moderate losses of ascorbic acid (Figure 5). Conclusion All the four treatments, namely, thermal, sodium carbonate, enzymatic, and ultrasonic treatments reduced cyanide contents of cassava leaves to a different extent. The chemical treatment with NaHCO3 was found to be the most effective detoxification method; nevertheless, the ascorbic acid level was decreased. Traditionally, a high temperature is considered as one of the parameters for cassava leaves detoxification; however, there was no significant effect while increasing temperature from 25 to 85 °C. On the other hand, the incubation time showed significant effect on cassava leaves detoxification. Conclusively, prolonged heating at high temperatures and repeated washing are not necessary for cassava leaves detoxification. By finely grinding the leaves with prolonged incubation, cassava leaves can be detoxified without negatively affecting the valuable nutrients such as ascorbic acid. Acknowledgments Authors are grateful to the Stiftung Fiat Panis, Germany, and BiomassWeb Project 031A258F financed by BMBF (Bundesministerium für Bildung und Forschung), Germany, for providing financial support. Author Contributions S. Latif designed the study, interpreted the results, and drafted the article. S. Zimmermann performed the analysis, collected test data, and interpreted the results. Z. Barati interpreted the results Detoxification treatments for cassava leaves . . . Conflict of Interest The authors declare no conflict of interest. References Achidi, A. U., Ajayi, O. 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