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agriculture
Article
Influence of Temperature and Screw Pressing on the Quality of
Cassava Leaf Fractions
Haimanot Hailegiorgis Ayele *, Sajid Latif and Joachim Müller
Tropics and Subtropics Group, Institute of Agricultural Engineering, University of Hohenheim, 70599 Stuttgart,
Germany; sajid.latif@yahoo.com (S.L.); joachim.mueller@uni-hohenheim.de (J.M.)
* Correspondence: haimanot.ayele@uni-hohenheim.de or info440e@uni-hohenheim.de; Tel.: +49-(0)711-459-23464
Abstract: In this study, the development of a mild processing method for cassava leaves to remove
cyanogenic compounds with minimum nutritional loss is evaluated. Fresh leaves were reduced in
size using a mixer at temperatures of 25 (room temperature), 55, 80, and 100 ◦ C for 1 min before
screw pressing to separate the juice and press cake fractions. Cyanide content in the fresh leaves
was reduced by 60% at 100 ◦ C and by 57% in the juice sample processed at 25 ◦ C. The press cake
cyanide content was low (210 ppm) in both the control and the sample that was processed at 55 ◦ C.
An increase in the temperature for processing cassava leaves to 100 ◦ C resulted in a loss of 5–13% of
the CP and 7–18% of the vitamin C content. The press-cake fraction had high beta-carotene, lutein,
and chlorophyll a and b content, and low values were registered for the juice fraction. Processing
fresh cassava leaves at 25 and 55 ◦ C resulted in fractions with high beta-carotene and lutein content.
The protein quality of press cake was better than that of juice for feed. Short thermal shredding with
pressing resulted in minimal loss of nutrients and a significant reduction of cyanide in the leaves.
Keywords: cassava leaves; nutrient; cyanide; press cake; juice; pressing
Citation: Ayele, H.H.; Latif, S.;
1. Introduction
Müller, J. Influence of Temperature
and Screw Pressing on the Quality of Cassava (Manihot esculenta Crantz) is an essential staple crop in tropical and subtropical
Cassava Leaf Fractions. Agriculture areas [1]. The crop was introduced to Ethiopia in the 1960s and regarded as a food crop
2022, 12, 42. https://doi.org/ after 1984, where it is of strategic importance for combating food insecurity caused by
10.3390/agriculture12010042 drought [2]. It is mainly grown in the southern region of the country as a food security
crop, and the roots are primarily used [3]. In Ethiopia, due to cultural bias and fear of
Academic Editor: Silvia Tavarini
toxicity, the leaves are not used for human consumption; instead, they are left in the field as
Received: 7 December 2021 green manure [2]. Cassava leaves are commonly considered as a byproduct of cassava root
Accepted: 28 December 2021 harvest and used for human and animal consumption in different parts of the world [4].
Published: 31 December 2021 Depending on the age, variety, and growth conditions of cassava plants, the leaves contain
Publisher’s Note: MDPI stays neutral
a relatively high amount of protein, vitamins, minerals, and phytochemicals that are
with regard to jurisdictional claims in
of nutritional and biochemical importance to humans and animals [5,6]. However, the
published maps and institutional affil- consumption of the leaves in these areas is also limited due to the high level of antinutrients
iations. and toxic compounds, such as cyanogenic glucosides [4]. These compounds reduce nutrient
absorption and might even lead to other adverse effects [7].
Recently developed cassava leaf processing and protein isolation methods have fo-
cused on cyanogen removal, reducing the levels of antinutrient compounds, and reducing
Copyright: © 2021 by the authors. nutrient loss [8–10]. On a household level, cassava leaf processing is usually performed
Licensee MDPI, Basel, Switzerland. by pounding and boiling the leaves in water for long time [11]. This process facilitates
This article is an open access article the rapid removal of cyanogen but also reduces the content of proteins, vitamins, and
distributed under the terms and sulfur-containing amino acids that are necessary to detoxify ingested cyanide. The loss of
conditions of the Creative Commons methionine is particularly unfavorable because it is necessary for the conversion of cyanide
Attribution (CC BY) license (https://
to thiocyanate in the body [8]. Cyanide content of cassava leaves can be decreased by more
creativecommons.org/licenses/by/
than 98% through solid-state fermentation [12], 81% by combination of blanching, dry
4.0/).
Agriculture 2022, 12, 42. https://doi.org/10.3390/agriculture12010042 https://www.mdpi.com/journal/agricultureAgriculture 2022, 12, 42 2 of 13
heating and wet heating [13], and 93% via chemical treatment with NaHCO3 [10]. The loss
of ascorbic acid and protein content can reach 38–75 g 100 g−1 DM for different cassava vari-
eties and leaf processing methods [10,14,15]. Other milder cassava leaf processing methods
such as pounding and sun or shade drying of leaves can reduce the cyanogen content
but result in dull-colored products as well as a reduced water-soluble vitamin, protein,
and methionine contents [8]. Leaf processing and fractioning have also been performed
by chemical, thermal, and mechanical actions using screw pressing [16,17]. Processing
of leaves by screw pressing is commonly used to separate the liquid fraction from the
fiber and to concentrate protein [18,19]. However, the challenge remains to find a suitable
processing method to produce cassava leaves with high nutrition, low cyanide content, and
low fiber content for human consumption [20].
Cassava leaves also have the potential to be used as a major protein source in animal
feed for ruminants and monogastric animals, but the high cyanide and fiber contents limit
such use [21]. The development of a mild processing method to remove cyanogens and
preserve the nutritional content of cassava leaves will play a significant role in its wider
use [8]. Therefore, in this study, a method for cassava leaf processing involving short-term
heat application followed by size reduction and pressing was assessed. The two fractions
obtained during processing with different temperatures were evaluated as food (juice) and
feed (press cake).
2. Materials and Methods
2.1. Plant Material
Cassava (Manihot esculenta Crantz) cultivar Chichu grown at Hawassa Agricultural
Research Center, Ethiopia, (6◦ 480 54.0500 N, 38◦ 160 55.5800 E, 1862 m.a.s.l) was used for the
experiment. Cassava stem cuttings were planted in the 2018/2019 growing season with a
spacing of 1 m × 1 m. The plants were cultivated under rainfed conditions with an annual
mean temperature of 22.08 ◦ C and mean annual rainfall of 887.4 mm. Cassava leaves with
the petiole attached were harvested by hand from 100 plants within the same plot at the
age of one year. All leaf samples, ranging from the first fully expanded leaf to the 15th leaf,
were collected on the same day. The leaves were cleaned, packed in polyethylene zip bags,
and placed in a box with ice for cooling. The leaves were transported within 24 h to the
laboratory at the University of Hohenheim for processing.
2.2. Treatments
After removing the petiole, the size of the leaves was reduced using a food processor
with a chopping and heating function (Thermomix TM5, Vorwerk, Cloyes, France). Chop-
ping was conducted for 1 min at a speed of 3100 rpm and at four different temperatures—25
(room temperature), 55, 80, and 100 ◦ C—i.e., temperature was set as the variable factor in
the experiment. The temperatures were set based on previous research recommendations
for cassava leaves processing [10,21–23]. Untreated leaves were taken as a control. Treated
leaves and control were kept at room temperature for 30 min before mechanical extraction
with a screw press. Screw pressing was done using a commercial lab-scale twin gear screw
stainless-steel press (AG- 8500S, Angel Juicers, Queensland, Australia). The press was
equipped with a coarse size screen (hole size 1 mm). The processing was conducted at room
temperature at a screw speed of 82 rpm. The double screw press was fed continuously
to collect the juice and press cake separately (Figure 1). After measuring moisture and
cyanide content, the samples were freeze-dried, packed, and stored at −20 ◦ C for further
analysis. Treatments were replicated twice.
2.3. Sample Analysis
2.3.1. Antinutritional Factors
The total cyanide content in cassava leaves and fractions was analyzed using the
picrate paper kit method [24,25]. Picrate paper was prepared by dipping 0.3 mm thick filter
paper into a 2.5% (w/v) picrate solution (Sigma-Aldrich, St. Louis, MO, USA) followed byAgriculture 2022, 12, 42 3 of 13
drying in a fume hood. The dried papers were cut into a 3 cm × 1 cm rectangle and attached
to the plastic strip (size 5 cm × 1 cm, 1 mm thickness). Linamarase was isolated according
to the method described by Yeoh et al. [26] involving the extraction of enzymes followed
by subsequent purification using gel filtration chromatography. A sample of 0.05 g, 1 mL
of 0.1 M Na-phosphate buffer, and 100 µL linamarase were placed in a vile and the strip
carrying a picrate paper was placed inside the vial, which was closed immediately with a
screw cap. The sample and solutions in the vial were gently mixed and left at 30 ◦ C for 24 h.
Then, the picrate paper was removed and soaked in 5 mL distilled water for 30 min. A
picrate paper suspended in a vial without a sample was used as a blank. The standard curve
for cyanide content was prepared from a series of linamarin (Sigma-Aldrich) concentrations
FOR PEER REVIEW (0.2–2.4 µM). The picrate papers from the blank and the standard were treated 3 ofthe
14 same
way as the picrate papers of the samples. The absorbance of the solutions was measured at
510 nm.
Figure 1. Cassava leaf processing at different temperatures.
Figure 1. Cassava leaf processing at different temperatures.
Phytate in the samples was analyzed according to the method described by Latta and
2.3. Sample Analysis Eskin [27]. Sample extraction was done by placing 0.5 g of the dried sample in a 10 mL of
3.5% HC1 solution. The solution was stirred for 1 h and centrifuged for 10 min at 3000× g.
2.3.1. AntinutritionalThe
Factors
aliquot was removed from the supernatant, filled into a 2 mL tube, and centrifuged
again
The total cyanide content in×cassava
at 10,000 g for 10 min.
leavesWadeandreagent was prepared
fractions by mixingusing
was analyzed 30 mgtheof FeC 13 ·6H2 O
pic‐
and 300 mg of sulfosalicylic acid in 100 mL of distilled water. Standard phytate solution was
rate paper kit method [24,25]. Picrate paper was prepared by dipping 0.3 mm thick filter
prepared by dissolving 2632 mg sodium phytate (Sigma-P8810, Merck KGaA, Darmstadt,
paper into a 2.5% (w/v) picrate
Germany) in solution (Sigma‐Aldrich,
1 mL of distilled water (2632 mg St.mL
Louis, MO, USA)
−1 ). Distilled water followed
(9 mL) was byadded to
drying in a fume hood. The dried
the solution papers
(dilution were
of 1:10). cut intocurve
A standard a 3 was
cm prepared
× 1 cm rectangle
with a rangeand at‐ mL
of 0.0–1.0
tached to the plasticandstrip (size 5 was
absorbance cm measured
× 1 cm, 1atmm 500 nmthickness). Linamarase was isolated
using a UV-spectrophotometer (DR6000, Hach
Lange, Düsseldorf, Germany).
according to the method described by Yeoh, et al. [26] involving the extraction of enzymes
The total phenolic content (TPC) of the samples was determined using the Folin–
followed by subsequent purification using gel filtration chromatography. A sample of 0.05
Ciocalteu reagent method [28]. A freeze-dried sample of 0.5 g was diluted in 5 mL of 80%
g, 1 mL of 0.1 M Na‐phosphate
methanol and buffer,
placed inand
a 60100 μL linamarase
◦ C water wereThe
bath for 20 min. placed
solutionin was
a vile and
centrifuged at
the strip carrying a 13,500
picrate rpmpaper
for 10was
min (Zplaced
326 K,inside
Hermlethe vial, which
Labortechnik wasWehingen,
GmbH, closed immedi‐
Germany). The
supernatant
ately with a screw cap. The sample was transferred, and the
and solutions inresidue waswere
the vial mixedgently
again with
mixed3 mL of 80%
and leftmethanol
at
and centrifuged. The supernatant was combined with the previously extracted solution
30 °C for 24 h. Then, the picrate paper was removed and soaked in 5 mL distilled water
and the volume was adjusted to 10 mL with 80% methanol. The extracted solution was
for 30 min. A picratekept
paperat 4suspended in a (max.
◦ C until analysis vial without
48 h). To aavoid
sample was used
a deviation as a blank.
of values from the The
standard
standard curve for cyanide content was prepared from a series of linamarin (Sigma‐Al‐
curve, the sample was further diluted with 80% methanol (press cake (1:20), leaves (1:20),
drich) concentrations and(0.2–2.4 μM).The
juice (1:40)). The picrate
sample (150papers
µL) wasfrom
mixedthewithblank
150 µLand theNstandard
of 0.25 Folin–Ciocalteu
were treated the same way as the picrate papers of the samples. The absorbancea further
reagent and 2400 µL of HPLC water and incubated for 3 min before adding of the300 µL
of 1 N sodium carbonate solution. The sample and standard were incubated for 2 h at room
solutions was measured at 510 nm.
temperature in the dark using 80% methanol as a blank. The absorbance of the standards
Phytate in the samples was analyzed
and the samples at 725 nmaccording
were measured to the method
using described by Latta
a UV spectrophotometer. Theand
standard
Eskin [27]. Sample extraction was done by placing 0.5 g of the dried sample in a 10 mL of
3.5% HC1 solution. The solution was stirred for 1 h and centrifuged for 10 min at 3000 g.
The aliquot was removed from the supernatant, filled into a 2 mL tube, and centrifuged
again at 10,000 g for 10 min. Wade reagent was prepared by mixing 30 mg of FeC13∙6H2OAgriculture 2022, 12, 42 4 of 13
calibration curve was prepared by measuring the absorbance of dilutions of a gallic acid
stock solution ranging in concentration from 0.005 to 0.1 mg mL−1 .
2.3.2. Micronutrients
The method described by Valente et al. [29] was used to measure the vitamin C content
of the fractions, with minor modifications. A sample of 1 g was transferred into a 50 mL
Falcon tube, and a 15 mL extraction solution (10% perchloric acid and 1% metaphosphoric
acid in ultrapure water) was added to stabilize the ascorbic acid and precipitate proteins.
The solution was homogenized for 1 min with a vortex and centrifuged at 20,000 rcf for
15 min at 4 ◦ C. A supernatant of 8 mL was transferred into a 12 mL tube and centrifuged
again under the same condition. A total 3 mL of the supernatant from each sample was
transferred into a 10 mL volumetric flask and filled up with a mobile phase (20 mM
ammonium dihydrogen phosphate, pH 3.5, containing 0.015% (w/v) of metaphosphoric
acid). The samples were filtrated into HPLC vials with 0.45 µm nylon filter membranes. The
separation and quantification were performed using a HPLC system (Shimadzu Co., Kyoto,
Japan) equipped with a column of 250 mm × 4.6 mm Luna 5u C18(2) 100A (Phenomenex,
Torrance, CA, USA). The quantification of the ascorbic acid components was performed at
254 nm. To calculate the recovery rate, samples were spiked with the main standard.
β-Carotene, lutein, and chlorophyll a and b levels were characterized using HPLC
(Agilent 1200, Agilent Technologies, Waldbronn, Germany) according to the method of Lee
et al. [30], with some modifications. Mixed analytical standards consisting of β-carotene,
lutein, and chlorophyll a and b were prepared in acetone at concentrations from 0 to 100
ppm. Extraction was performed by adding 1 g of the sample in 30 mL of acetone, which was
then placed in an ultrasonic bath at 35–40 ◦ C for 90 min. The mixture was filtered through a
syringe filter (PTFE, 0.45 µm) and evaluated by HPLC analysis. To separate pigments, a C30
column (stability 100, 5 µm, dimensions 250 mm × 4.6 mm) with guard column (stability
100 C30, 5 µm, 5 mm × 4.6 mm, Dr. Maisch, Ammerbuch-Entringen, Germany) was used.
The column temperature was maintained at 30 ◦ C. The mobile consisted of solvent A (75%
methanol) and B (100% ethyl acetate). The gradient at a flow rate of 1.0 mL min−1 was set
as follows: 0–15 min, 30–90% B; 15–20 min, 90–30% B, followed by a constant 30% B until
the end of the running time of 25 min. A 20 µL injection volume was used each time. The
peak area of a photodiode array detector was used at 450 nm to calculate the amount of
each pigment.
2.3.3. Moisture, Ash, and Crude Protein Content
Cassava leaves, press cake, and juice fraction moisture content was measured by
drying the samples in an oven at 105 ◦ C for 12 h [31]. The ash content was measured by
placing the oven-dried samples in a muffle furnace, as described in AOAC [31] official
method 923.03. Freeze-dried leaf, press cake, and juice crude protein (CP) contents were
measured using the Kjeldahl method and a Kjeldahl analysis system (Vapodest 500, C.
Gerhardt GmbH & Co. KG., Königswinter, Germany). A conversion factor of 6.25 was used
to calculate the amount of CP content from the total nitrogen content.
2.3.4. Acid Detergent Fiber, Acid Detergent Lignin, and Neutral Detergent Fiber
The acid detergent fiber (ADF), acid detergent lignin (ADL), and neutral detergent fiber
(NDF) contents of fresh leaf, press cake, and juice were measured according to the method
described by AOAC [31] official method 973.18 using an automated fiber analysis system
(FibreBag Analysis System FBS6, Gerhardt GmbH & Co. KG., Königswinter, Germany).
2.3.5. Protein Fractioning
The CP of cassava leaves, juice, and press cake was partitioned following the proce-
dures described by Licitra et al. [32]. Samples were analyzed in duplicate, and repetitions
were performed in cases where the variation coefficient was greater than 5%. The N con-
centrations were determined using the Kjeldahl procedure, and all N concentrations wereAgriculture 2022, 12, 42 5 of 13
converted to CP using a conversion factor of 6.25. The nonprotein nitrogen (NPN) con-
centration of the samples was determined using the tungstic acid method [32]. A sample
of 0.5 g was weighed into a 100 mL Erlenmeyer flask; then, 50 mL distilled water and
8 mL of 0.3 M sodium tungstate solution were added. The solution was mixed for 30 min
and continuously stirred, and the solution pH was reduced to 2.0 using a sulfuric acid
solution (0.5 M). The flask was covered and kept at room temperature overnight. Soluble
true protein concentrations were determined using a borate–phosphate buffer (pH 6.7–6.8).
In total, 50 mL of buffer and 1 mL of freshly prepared sodium azide were added to 0.5 g of
the sample in 100 mL Erlenmeyer flasks. The flasks were covered for 3 h before filtration.
NPN and soluble true protein filtration of the suspensions were followed by washing
both the residue and filter paper (Whatman paper N◦ 54, GE Healthcare Life Sciences,
Darmstadt, Germany) with 250 mL of cold distilled water. The washed filter paper with
residue was dried at 38 ◦ C for 1 h. The N value for the residue and filter paper was
analyzed. The NPN and soluble true protein concentration of the samples was calculated
by subtracting the N concentration in the residual material from the total N concentration
in the sample.
The neutral detergent-insoluble CP (NDICP) was determined following the procedure
of NDF analysis without using sodium sulfite. The sample (0.5 g) was boiled in 100 mL
of neutral detergent solution for 1 h. A 25 µL aliquot of alpha-amylase (Ankom Technol-
ogy, NY, USA) was added 1 and 30 min after the solution started boiling. The solution
was filtered through a filter paper (Whatman paper N◦ 54, GE Healthcare Life Sciences,
Darmstadt, Germany). The acid detergent-insoluble CP (ADICP) was determined in the
same way as the NDICP, except that the neutral detergent solution was substituted with an
acid detergent solution and alpha-amylase was not used. The residue with filter paper was
washed with 250 mL hot distilled water (80 ◦ C). Then, it was rinsed twice with 5 mL acetone
and dried at 38 ◦ C for 1 h. The filter paper with residue was then analyzed for N. The
concentrations of different CP fractions were then calculated according to Sniffen et al. [33].
These have been described as fractions NPN (A), soluble true protein (B1), insoluble true
protein (B2), protein that is insoluble in neutral detergent but soluble in acid detergent (B3),
and protein that is insoluble in acid detergent (C).
2.4. Statistical Analysis
One-way analysis of variance (ANOVA) was conducted to determine the effect of
the size reduction of leaves subject to different processing temperatures on the nutritional
and antinutritional content of cassava leaf, press cake, and juice. The significant difference
of sample means was identified using Tukey’s test at a significance level of p ≤ 0.05. All
analyses were performed using SAS statistical software (version 9.2, SAS Institute Inc.,
Cary, NC, USA) and two independent replicates.
3. Results and Discussion
3.1. Antinutrients
A general comparison of the antinutrient contents across the fractions—namely, leaves,
juice, and press cake—showed that mechanical extraction by screw pressing leads to
unequal partitioning of the original content, with higher contents in the juice and lower
contents in the press cake. Observing the controls after pressing revealed that the original
cyanide content of 1275 ppm in the leaves increased to 2543 ppm in the juice but decreased
to 211 ppm in the press cake. This means that pressing alone was effective for obtaining
a press cake with a low toxicity level; however, for the juice, the need for detoxification
was increased. The difference between control and chopping at 25 ◦ C showed the effect of
chopping alone, i.e., without additional heating. Regarding cyanide content, it decreased to
671 ppm in the leaves, which corresponds to a 47% reduction. In the juice, the reduction was
even more pronounced at 57%. The reduction in cyanide content caused by the disruption
of leaf tissue and juice through grinding was caused by the action of endogenous linamarase
on glucosides [21]. The positive impact of size reduction on lowering cyanide content in theAgriculture 2022, 12, 42 6 of 13
present experiment is similar to what was reported by Ravindran et al. [34]. Applying heat
during chopping further reduced the cyanide content in the leaves but to a smaller amount
and without significant differences between the temperatures. Already at a moderate
temperature of 55 ◦ C, the reduction was 56%. In the juice, heating at 55 and 80 ◦ C during
chopping slightly weakened the detoxification effect, and cyanide was reduced by 50%. In
the press cake, chopping with and without heating yielded negligible differences (Figure 2a).
The significant reduction of cyanide in leaves processed at 55 ◦ C can be explained by the
stability of linamarase enzyme being optimum at a temperature of 55 ◦ C [21]. The amount
of cyanide in all the fractions in the current study was higher than what was stated as a safe
level (10 ppm) by the FAO/WHO [35]. The remaining cyanide content in the leaves and
press cake fractions can be reduced further by drying [36] or using membrane filtration
Agriculture 2022, 12, x FOR PEER REVIEW 7 ofor
14
coagulation for the juice fraction [37]. It was observed by Bradbury and Denton [8] that
processing methods with longer heat application time can reduce up to 99% of the total
cyanogens in cassava leaves but at the expense of high nutritional loss.
a 2750 a Control
2500 25
55
Cyanide (ppm DM)
2250
80
2000 100
1750
1500 a b
b
1250 c c
1000
750 b c c
500
c
250 c a c b bc
0
b 50
a
45 bc ab cd
TPC (GAE mg g−1DM)
40 d
35
30 a a
a
25 a a a ab ab ab
b
20
15
10
5
0
8
c 7 a a a
Phytate (g 100 g−1DM)
6 a
a
a a a
5 a
a
4 a a a
a a
3
2
1
0
Control
Control
Control
100
100
100
25
55
80
25
55
80
25
55
80
Cassava leaves Juice Press cake
Figure 2. Influence
Figure2. Influenceof
of pressing
pressingat at different
differenttemperatures
temperatureson onthe
the(a)
(a)cyanide,
cyanide,(b)
(b)total
totalphenolic
phenoliccontent
content
(TPC), and (c) phytate contents of cassava leaves and fractions. Control represents
(TPC), and (c) phytate contents of cassava leaves and fractions. Control represents the the sample without
sample with‐
heat application
out heat andand
application sizesize
reduction.
reduction.Bars with
Bars thethe
with same
same letter
letterare
arenot
notsignificantly
significantlydifferent
differentfrom
from
other sampleswithin
othersamples withinthe
thesame
samefraction.
fraction.
After pressing,(Vitamin
the initial −1
3.2. Micronutrients C, TPC of 25 GAE
Beta‐Carotene, mg g Chlorophyll
Lutein, DM in thea,fresh cassava leaves
and Chlorophyll b) was
concentrated in the juice to 38 GAE mg g−1 DM while it was lowered to 21 GAE mg g −1
DM in
Pressing of the fresh leaves with vitamin C content of 1425 mg 100 g−1DM resulted in a
juice fraction with high (4077 mg 100 g−1DM) and a press cake with low (327 mg 100 g−1DM)
levels of vitamin C. Size reduction of the leaves leads to a loss of 3%, 18%, and 13% of
vitamin C in the leaves, juice, and press cake fractions, respectively. The increase of pro‐
cessing temperature to 100 °C led to a reduction in the vitamin C content of cassava leavesAgriculture 2022, 12, 42 7 of 13
the press cake fraction. The impact of size reduction on the TPC of the fresh leaves was not
significant, whereas it tends to slightly increase the TPC in the juice and press cake from 38
to 41 GAE mg g−1 DM and 21 to 22 GAE mg g−1 DM , respectively. Increasing the processing
temperature to 55 and 80 ◦ C tends to increase the TPC of the juice fraction. The slight
increase in TPC of the juice and press cake fraction after heat application can be explained by
the increase of free-radical scavenging activities or the inactivation of several enzymes. The
same result was observed on blanched Carica papaya L. leaf by Raja et al. [38]. In the juice
fraction, TPC was increased to some extent when processing temperatures were increased
and then decreased, similarly to tea leaf drying at different temperatures [39]. Leaf fractions
processed without heat application and size reduction showed a significantly low value
of TPC (Figure 2b). As previously reported, leaf processing can help dephosphorylate
phytate to release minerals and facilitate their absorption [40]. Even though the impact
of size reduction and application of temperature did not show a significant difference in
the phytate content in all cassava leaf fractions, higher concentrations were observed in all
juice fractions after pressing (Figure 2c). Phytate is relatively heat-stable during processing.
To minimize phytate in cassava leaf, longer heat application or fermentation is needed after
pressing, as suggested by Montagnac et al. [40].
3.2. Micronutrients (Vitamin C, Beta-Carotene, Lutein, Chlorophyll a, and Chlorophyll b)
Pressing of the fresh leaves with vitamin C content of 1425 mg 100 g−1 DM resulted
in a juice fraction with high (4077 mg 100 g−1 DM ) and a press cake with low (327 mg
100 g−1 DM ) levels of vitamin C. Size reduction of the leaves leads to a loss of 3%, 18%, and
13% of vitamin C in the leaves, juice, and press cake fractions, respectively. The increase of
processing temperature to 100 ◦ C led to a reduction in the vitamin C content of cassava
leaves by 73%, while for the juice fraction, the reduction was only 13% (Figure 3a). The
highest loss of vitamin C was recorded for the juice extracted after size reduction without
any heat application (18%). The reason for the loss is the sensitivity of vitamin C to light,
high temperatures, and exposure to oxygen [10]. In leafy vegetables, vitamin C content is
considered to be high, which was the case in cassava leaves and juice fractions compared
with the values reported for other vegetables [41,42]. The loss of vitamin C in the current
study is small compared with what was reported for other cassava leaf processing methods
(7–60%) [10,42].
The beta-carotene and lutein contents of cassava leaves after pressing were higher
in the press cake than the juice fraction. Grinding of fresh cassava leaves showed a
significant increase in beta-carotene of 18%. Pressing after size reduction resulted in low
content of beta-carotene and lutein in the press cake fraction (Figure 3b,c). The processing
temperature increase to 55 ◦ C resulted in a slightly higher amount of lutein in fresh leaves
(70.4 mg 100 g−1 DM ). In the juice fraction, the highest loss of beta-carotene and lutein
was recorded when the temperature was above 80 ◦ C (Figure 3b,c). The contents of the
important, nutritional, plant-derived carotenoids, namely beta-carotene (hydrocarbon
carotene) and lutein (oxygenated xanthophyll), was high in cassava leaves [43]. Lutein
is a major component of the human retina and is considered beneficial for eye health
through reducing macular degeneration [44,45]. The higher amount of chlorophyll a and b
in samples after processing was mainly caused by the occurrence of cell disruption during
size reduction and pressing, generating a more intense bright green color on the surface [46].
The change or loss of lutein and β-carotene due to an increase in temperature might differ
based on time of exposure and crop type. Increasing the processing temperature to above
55 ◦ C reduced the beta-carotene and lutein content of cassava leaves, similarly to what has
been observed for red pepper and green pepper treated at higher temperatures [46].
Pressing of cassava leaves resulted in higher amounts of chlorophyll a and b in the
press cake fraction, while the size reduction of fresh cassava leaves led to a 24% increase
in chlorophyll a and b. By contrast, pressing after size reduction resulted in a 9% loss of
chlorophyll a and 5% loss of chlorophyll b in the press cake fraction. In the juice fraction,
the highest losses of chlorophyll a (24.3%) and chlorophyll b (12%) were recorded at aAgriculture 2022, 12, 42 8 of 13
temperature of 80 ◦ C (Figure 3d,e). In the current study, the content of chlorophyll a is
more than three times that of chlorophyll b, which is similar to what was reported by
Sánchez et al. [46] for different vegetables. The chlorophyll content presented a negative
correlation with vitamin C, which indicates that the highest antioxidant levels might be
found when the plant presents low chlorophyll levels [41]. In all fractions, the effects of
temperature increase are more pronounced on chlorophyll a than on chlorophyll b. This
can be explained by the greater stability of chlorophyll b to increases in temperature [46].
3.3. Macronutrient (Ash, Crude Protein, Acid Detergent Fiber, Acid Detergent Lignin, and Neutral
Detergent Fiber)
The ash content was not significantly affected by a size reduction in the leaves and
fractions. The ash content was higher in the press cake fraction than in the juice fraction.
Ash content increased significantly when the processing temperatures of fresh cassava
leaves (9.6–10.4 g 100 g−1 DM ) and juice (12.4–13.3 g 100 g−1 DM ) were increased. The ash
content of the leaves in this study lies within the range reported by Ravindran [21] but
is higher compared with other findings [47,48]. These variations are attributed to the
differences in varietal, age, and processing methods in the experiments [9,49]. A positive
relationship of ash content with the processing temperature of cassava leaves and juice
fraction was observed in this study, and a similar trend was seen in the case of tea leaves
dried at different temperatures [39]. The lower ash content in the press cake is similar to
that reported by Latif et al. [19] for pressed frozen cassava leaves.
Cassava leaf pressing led to higher CP content in the press cake, which is similar to
what was reported by Latif et al. [19] for mechanical pressing of frozen cassava leaves
(Table 1). Tenorio et al. [50] also found a higher CP content in the press cake fraction in their
study of sugar beet leaf pressing. Mechanical pressing of leaves usually results in higher
protein content in the press cake, as most proteins are retained in the fiber structure [51,52].
The CP content of cassava leaves and press cake was unaffected by size reduction, which is
similar to what was reported by Achidi et al. [53] and Ravindran et al. [34] but a positive
effect was seen in the juice fraction. A temperature increase to 100 ◦ C for processing of
cassava leaves resulted in a loss of 5–13% CP content. The juice fraction processed at 80 ◦ C
had the lowest CP (25.8 g 100 g−1 DM ) content among the samples. In the current study,
there was less CP loss compared with what was reported in other studies [14,54].
The leaves processed at 25 ◦ C showed a significant increase in ADL and NDF content
in the press cake, whereas the ADL and ADF content of the juice fraction increased at
55 ◦ C. The NDF content in the juice fraction increased significantly as the temperature
increased during processing, while the press cake showed an opposite trend. The NDF
is normally associated with cell-wall-bound protein nitrogen, which also includes the
indigestible nitrogen found in the acid-detergent residue [32]. The NDF values in the
current study (23.0–25.7 g 100 g−1 DM ) are higher than what was recorded previously by
Ayele et al. [17] (19 g 100 g−1 DM ) and less than those reported by Paengkoum et al. [55]
(47.5 g 100 g−1 DM ). The reason for this discrepancy might be caused by the difference in
age and variety of plants used for the study. After processing, the NDF content in the
press cake increased, which is similar to what was reported for sun-dried and ensiled
cassava leaves [17]. Overall, among the three factions, the ADF and ADL contents were
higher in the press cake fraction (Table 1). Increasing the processing temperature resulted
in increased ADF in all fractions, which is caused by the production of artifact lignin via
the nonenzymatic browning reaction [32]. A similar result was observed for the ADF value
of cassava leaves by Paengkoum et al. [55].
3.4. Protein Fractions
SP (true protein) and NPN were very low in the press cake fraction. NDICP was high
in the press cake, whereas NDICP and ADICP were very low in the juice fraction. A low
amount of ADICP in all three fractions indicates the high protein quality of the fractions [56].
CP contains both SP and NPN compounds, including amides, amine peptides, nucleic acids,Agriculture 2022, 12, 42 9 of 13
free amino acids, ammonia, and nitrate [57,58]. Levels of SP, soluble in buffer at rumen
pH, were very low in the press cake fraction. This result is attributed to the separation
of the juice, which showed a higher value for SP after pressing. Contents of A, B1, and
C were higher in the juice and leaf fraction whereas B2 and B3 were higher in the press
cake (Table 2). NPN (A) (trichloroacetic (TCA) acid-soluble N) also showed a similar trend.
The NPN values of the three fractions were similar to those of cassava leaf meal but lower
than those reported for alfalfa hay (10 to 20%) [59]. To estimate feed degradation rates in
the rumen, classification of B is subdivided into B1 (rapidly degraded in the rumen), B2
(fermented in the rumen and, depending on the relative rates of digestion and passage,
some may escape to the lower gut), and B3 (associated with the cell wall and slowly
degradable in the rumen) [33]. The higher amount of B2 in the press cake fraction is an
indication of high feed quality for ruminants [60]. The values in this study are in a similar
range to those reported by Tham et al. [60] for cassava leaf meal. However, the values
are much higher than those of most roughage feeds [61] and lower compared with alfalfa
leaves that are also used as a forage [61]. Levels of C, the unavailable or bound protein that
cannot be degraded by ruminal bacteria and does not provide amino acids postruminally,
were very low in all three fractions.
Table 1. Nutritional content of cassava leaf, press cake, and juice processed at different temperatures.
DM Ash CP ADF ADL NDF
Temperature
Fraction (g 100 (g 100 (g 100 (g 100
(◦ C) (%) (g 100 g−1 DM )
g−1 DM ) g−1 DM ) g−1 DM ) g−1 DM )
Control 29.1 ± 0.1 a 9.7 ± 0.1 b 31.0 ± 0.5 a 23.8 ± 0.4 a 7.5 ± 0.1 a 25.7 ± 0.2 a
25 27.7 ± 0.2 b 9.6 ± 0.1 b 30.5 ± 0.4 a b 22.6 ± 0.9 a 7.0 ± 0.1 a 23.0 ± 0.5 a
Leaves 55 27.9 ± 0.1 b 9.9 ± 0.0 b 29.9 ± 0.3 a b c 21.8 ± 1.0 a 7.0 ± 0.1 a 23.7 ± 0.5 a
80 28.3 ± 0.4 a b 10.4 ± 0.2 a 29.4 ± 0.3 b c 24.3 ± 0.3 a 7.6 ± 0.3 a 25.1 ± 0.6 a
100 28.4 ± 0.4 a b 10.4 ± 0.0 a 28.9 ± 0.2 c 22.4 ± 0.6 a 7.6 ± 0.1 a 25.2 ± 1.3 a
Control 13.6 ± 0.0 a 12.4 ± 0.1 b 27.7 ± 0.2 b 3.2 ± 0.2 a b 1.1 ± 0.0 a 3.8 ± 0.3 c
25 13.1 ± 0.3 a 12.4 ± 0.3 b 29.6 ± 0.7 a 3.1 ± 0.1 a b 1.1 ± 0.0 a 5.2 ± 0.9 b c
Juice 55 12.9 ± 0.1 a 12.7 ± 0.3 a b 26.6 ± 0.5 b c 2.9 ± 0.1 b 0.9 ± 0.0 b 8.6 ± 1.9 a b
80 13.1 ± 0.3 a 13.1 ± 0.0 a b 25.8 ± 1.1 c 3.1 ± 0.0 a b 1.1 ± 0.0 a 11.5 ± 0.6 a
100 13.6 ± 0.1 a 13.3 ± 0.0 a 27.8 ± 0.0 b 3.5 ± 0.1 a 1.1 ± 0.0 a 12.2 ± 0.0 a
Control 52.5 ± 0.1 b 8.6 ± 0.3 a 31.0 ± 0.4 a 24.9 ± 0.4 a b 10.4 ± 0.6 a 28.7 ± 0.3 a b
25 54.8 ± 0.2 a 8.5 ± 0.0 a 31.2 ± 0.0 a 26.6 ± 0.2 a 9.0 ± 0.1 a 31.9 ± 1.0 a
Press cake 55 56.1 ± 0.8 a 8.6 ± 0.1 a 31.2 ± 0.2 a 25.1 ± 1.0 a b 9.5 ± 0.0 a 29.2 ± 1.1 a b
80 55.1 ± 0.3 a 9.1 ± 0.1 a 31.0 ± 0.1 a 23.9 ± 0.1 b 9.7 ± 0.3 a 29.4 ± 0.9 a b
100 54.5 ± 0.6 a 9.1 ± 0.1 a 29.5 ± 0.1 b 25.1 ± 0.6 a b 9.4 ± 0.4 a 28.2 ± 0.5 b
Note: Control represents the sample without heat application and size reduction. CP—crude protein, ADF—acid
detergent fiber, ADL—acid detergent lignin, NDF—neutral detergent fiber. All the results are expressed on a dry
matter basis; values in columns followed by the same superscript letters are not significantly different.
Table 2. Primary protein and fractions of cassava leaf, press cake, and juice.
CP (g 100 SP (g 100 NPN (g NDICP (g ADICP (g A (g 100 B1 (g 100 B2 (g 100 B3 (g 100 C (g 100
Fraction
g−1 DM ) g−1 DM ) 100 g−1 DM ) 100 g−1 DM ) 100 g−1 DM ) g−1 CP ) g−1 CP ) g−1 CP ) g−1 CP ) g−1 CP )
Leaves 31.0 ± 0.5 a 8.9 ± 0.3 a 7.4 ± 0.1 a 2.1 ± 0.0 a 1.3 ± 0.0 a 24.0 ± 0.2 a 4.7 ± 0.8 a 64.6 ± 0.6 b 2.5 ± 0.2 b 0.7 ± 0.0 a
Juice 27.7 ± 0.2 b 9.9 ± 0.5 a 8.3 ± 0.6 a 0.3 ± 0.0 b 0.1 ± 0.0 b 30.0 ± 2.5 a 5.8 ± 0.5 a 63.3 ± 2.4 b 0.6 ± 0.0 c 0.1 ± 0.0 b
Press cake 31.0 ± 0.4 a 2.3 ± 0.1 b 1.5 ± 0.1 b 2.6 ± 0.3 a 1.3 ± 0.1 a 5.0 ± 0.4 b 2.4 ± 0.1 b 84.2 ± 1.3 a 4.4 ± 0.6 a 0.7 ± 0.1 a
CP—crude protein, SP—soluble protein, NPN—nonprotein nitrogen, NDICP—neutral-detergent-insoluble CP,
ADICP—acid-detergent-insoluble CP, A—nonprotein nitrogen, B1—soluble true protein, B2—insoluble true
protein, B3—protein insoluble in neutral detergent but soluble in acid detergent, C—protein insoluble in acid
detergent. Values in columns followed by the same superscript letters are not significantly different.Agriculture
Agriculture 2022,
2022, 12,12,
42x FOR PEER REVIEW 910
ofof
1413
4500 a Control
Vitamin C (mg 100g−1 DM)
a 4000
d c
b c 25
55
3500
80
3000 100
2500
2000
1500 a ab ab a b
1000
500 a b b b b
0
a
Betacarotene (mg 100g−1 DM)
90
b 80
a
ab c bc bc b
bc c bc
70 a
a a
60
50 b
b
40
30
20
10
0
90
c 80 a ab ab b
Luteine (mg 100g−1 DM)
a b
70 b ab b a
b
ab ab
60
50 c bc
40
30
20
10
0
600
Chlorophyll a (mg 100g−1 DM)
550 a a
d 500 a a
b b b
a
450 b b a ab
400 c ab
350 b
300
250
200
150
100
50
0
175 ab
c bc ab a
Chlorophyll b (mg 100−1 DM)
e 150 a a a
a
a ab a ab
125 b
b
100
75
50
25
0
Control
Control
Control
100
100
100
25
55
80
25
55
80
25
55
80
Leaves Juice Press cake
Figure3.3.Impact
Figure Impactofoftemperature
temperatureandandprocessing
processingmethod
methodon on(a)
(a)vitamin
vitaminC,C,(b)
(b)beta-carotene,
beta‐carotene,(c)
(c)lutein,
lu‐
tein, (d) chlorophyll a, and (e) chlorophyll b content of cassava leaves and fractions. Control repre‐
(d) chlorophyll a, and (e) chlorophyll b content of cassava leaves and fractions. Control represents the
sents the
sample sampleheat
without without heat application
application and size reduction.
and size reduction. Bars with Bars
thewith
samethe sameare
letter letter
not are not sig‐
significantly
nificantly different from other samples within the same fraction.
different from other samples within the same fraction.
4. Conclusions
The present study advocates the possibility of using simple processing techniques
to minimize nutritional loss and produce cassava leaf products that are low in cyanide.
We found that the impact of size reduction on the antinutritional content of the differentAgriculture 2022, 12, 42 11 of 13
fractions was higher than that of temperature change. In this study, we observed that
short-term temperature increases do not lead to very significant nutritional losses but few
losses were observed in the leaves and juice fraction when the processing temperature
was 80 and 100 ◦ C Pressing of size-reduced samples alone can result in significant cyanide
detoxification of the juice fraction. Even though the nutritional content of the resulting
juice fraction was high, the antinutritional factors must be further reduced before it can be
considered suitable as food. The press cake cyanide content was low (210 ppm) in both
the control and the sample that was processed at 55 ◦ C as compared to the fresh leaves
(1275 ppm). The low cyanide content and protein quality of the press cake support its use
as a viable animal feed source. Further studies investigating the time of exposure of the
leaves at higher temperatures with screw pressing should be conducted. The results of the
current study will help in establishing methods for exploiting cassava leaves as food and
feed in Ethiopia in the long run.
Author Contributions: Conceptualization, H.H.A. and S.L.; methodology, H.H.A.; software, H.H.A.;
formal analysis, H.H.A.; investigation, H.H.A.; resources, J.M. and S.L.; data curation, H.H.A.;
writing—original draft preparation, H.H.A.; writing—review and editing, H.H.A., J.M. and S.L.;
supervision, J.M. and S.L.; project administration, S.L.; funding acquisition, J.M. and S.L. All authors
have read and agreed to the published version of the manuscript.
Funding: This publication is an output of a Ph.D. scholarship from the University of Hohenheim in
the framework of the project “German-Ethiopian SDG Graduate School: Climate Change Effects on
Food Security (CLIFOOD)” between the University of Hohenheim (Germany) and the Hawassa Uni-
versity (Ethiopia), supported by the DAAD and with funds from the Federal Ministry for Economic
Cooperation and Development (BMZ).): 57316245.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy.
Acknowledgments: The authors are appreciative of all the help from the lab team and colleagues in
the Institute of Agricultural Engineering, Tropics and Subtropics Group, University of Hohenheim,
Germany.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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