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Detoxification of Cassava Leaves by Thermal,

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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
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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
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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.
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and drafted the article. J. Müller advised, supervised the study, and
revised the article.
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