J. Agric. Environ. Sci. Vol. 5 No. 2 (2020) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 55
Effects of Different Treatment Methods on the Composition of Nutrients and Phorbol Ester
Levels of Jatropha (Jatropha curcas L.) Seed Meal Produced in Ethiopia
Kefyalew Berihun
1
*, Tegene Negesse
1
, Adugna Tolera
1
School of Animal and Range Sciences, Hawassa University, Hawassa, Ethiopia
*Corresponding author: kefyalewbr@gmail.com
Received: October 1, 2020 Accepted: October 29, 2020
Abstract: Seed meal of Jatropha that is obtained after extracting the oil has high nutritive value in terms of crude
protein that can be used as animal feed. However, Jatropha seed meal contains several anti-nutritive compounds
that can be probably toxic to animals. The aim of the present study was therefore to evaluate the effects of different
treatment methods on the composition of nutrients, anti-nutritional factors and metabolizable energy content of
Jatropha seed meal. The seed meal was collected from a biodiesel industry at Bati, Oromia Zone of Amhara Region,
Ethiopia and treated using sodium hydroxide, backing yeast and heat and compared with the control (untreated)
jatropha seed meal. The nutrient composition (Ash, Crude Protein, Ether Extract, Crude Fiber, Neutral Detergent
Fiber, Acid Detergent Fiber and Acid Detergent Lignin), Phorbol ester, metabolizable energy and non-fiber
carbohydrate contents were estimated using the standard formula. The result indicates that crude protein content
varied from 26.97% in sodium hydroxide to 38.83% in the control treatments on a dry matter basis. The ash content
of sodium hydroxide treated and the control were significantly higher (p<0.05) than yeast and heat-treated
treatments. The ether extract content of sodium hydroxide-treated was significantly lowest (p<0.05) when compared
to the other treatments. The crude fiber contents of sodium hydroxide (30.05) and yeast- treated jatropha seed meal
(33.67) were higher (p<0.05) than heat-treated (28.54) and the control (24.3) treatments. The highest neutral
detergent fiber and crude fiber contents were observed form yeast and sodium hydroxide treatments whereas the
lowest from the control. The metabolizable energy contents of the control was higher (P<0.05) than all other treated
jatropha seed meal. Sodium hydroxide and the control treatments had higher (p<0.05) non-fiber carbohydrate
content than the yeast treatment. The lowest (360.35) phorbol ester content was recorded from yeast-treated
jatropha seed meal followed by sodium hydroxide (872.15) treated while the highest (2285.9 µg TPA eq./g ) was
from untreated control. Treating the Jatropha seed meal with yeast improved the feed values, reduced the phorbol
ester content and increased the crude protein and ether extract than the other treatments, which can be
recommended as possible treatment option for JSM. Feeding trial of yeast-treated JSM is also recommended for
further research.
Keywords: Biological treatment, Chemical treatment, Nutrient composition, Physical treatment
This work is licensed under a Creative Commons Attribution 4.0 International License
1. Introduction
The productivity of poultry in the tropics has been
limited by scarcity and high prices of the
conventional protein and energy sources (Aberra et
al., 2011). The high cost of protein sources of poultry
feed, their restricted availability and the
unpredictability of their markets increase the need for
utilization of other inexpensive sources of plant
proteins (Yue and Zhou, 2009). Over the years there
have been several studies such as Adejimi et al.
(2011), Prasad et al. (2012), Abbas (2013) and
Aberra et al. (2013), where the non-conventional
protein sources like Jatropha Seed Meal, Moringa
oleifera leaf meal and Cocoa pod husks have been
used in poultry ration as replacement of the
conventional protein sources. Among the different
non-conventional protein sources Jatropha seed meal
has a great potential to complement and substitute
soybean meal as a protein source which can be
included in livestock diets (Makkar et al., 2012).
Jatropha (Jatropha curcas L.), commonly known as
physic nut, belongs to the Euphorbiaceae family
J. Agric. Environ. Sci. Vol. 5 No. 2 (2020) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 56
(Heller, 1996, Barros et al., 2015) and is cultivated
primarily for bio-fuel production. According to the
Ministry of Water and Energy (MoWE, 2012), the
production of jatropha has gained high attention in
Ethiopia as a potential alternative and renewable
source of energy due to rising oil prices globally.
Jatropha produces a large quantity of seed (Heller,
1996) with a high oil content of 40 to 60% (Makkar
et al., 1997).
Seed meal of Jatropha is obtained after extracting the
oil from the seeds. The meal has high nutritive value
in terms of crude protein. The protein content of
detoxified Jatropha kernel meal was estimated to be
665g/kg dry matter and was higher than soybean
(Glycine max) meal, which is 471 g/kg dry matter
(Kumar et al., 2010). However, Jatropha seed meal
contains several anti-nutritive compounds such as
lectin, trypsin inhibitor (anti-trypsin), saponin,
phytate, and phorbol esters (Makkar et al., 1997). Of
all the compounds, phorbol esters are considered as
the main toxic compound in animal feeds (Makkar
and Becker, 1998). The concentration of minerals
and anti-nutritional substances in jatropha seed and
kernel meal may differ with the soil and agro-
climatic conditions where jatropha is grown (Chikpah
and Demuyakar, 2012). The biological effects of
these compounds include tumors promotion, a wide
range of negative biochemical and cellular effects,
alteration of cell morphology, induction of platelet
aggregation and also serve as lymphocyte mitogensn
(Azzaz et al., 2011).
These anti-nutritional factors therefore need to be
inactivated or removed using different methods at a
different level of application to make a comparison
for the possible use of treated JSM as a feed (Makkar
et al., 1997). The removal of phorbol esters would
transform JSM into a highly nutritious and high-value
feed ingredient for monogastrics, fishes and
ruminants (Haas and Mittelbach, 2000). In this
regard, various researches have been conducted to
evaluate the effects of different treatment methods on
the nutritional values of Jatropha seeds.
A study conducted by Annongu et al. (2010) on the
nutritional value and effects of physically (boiled,
soaked, roasted) and bio-chemically (fermented and
soaked in ethanol) treated Jatropha seed meal on the
cockerel diet found non-significant difference in feed
intake and body weight gain compared to the control
group. Studies by Aderibigbe et al. (1997) also
indicated that Jatropha seed meal could be used as
animal feed through adequate detoxifications using
physical or chemical processes.
According to Martinez-Herrera et al. (2006), jatropha
seed meal could be detoxified and the residual
protein-rich seed cake remaining after extraction of
the oil could form a protein-rich ingredient in feeds
for poultry, pigs, cattle and even fish. Different
reports indicated that the nutritive values and phorbol
ester contents of jatropha seed meal and jatropha
kernel meal are varied based on agro-climatic
condition and treatment methods used to remove or
reduce the toxic substances. Martınez-Herrera et al.
(2006) reported high concentrations of Phorbol esters
(3.85mg/g) of jatropha kernel collected in
Coatzacoalcos region of Mexico. According to the
authors, treating Jatropha seed meal collected from
Coatzacoalcos with ethanol 90%, and ethanol 90%
+NaHCO3 at 1210C for 25 min decreased the
phorbol ester content by 97.9% and 95.8%,
respectively. Areghore et al. (2003) also reported that
jatropha seeds treated with heat and washed four
times with 92% methanol reduced Phorbol esters
content to 0.09 mg/g and contained 68% crude
protein which was higher than value of soybean
(47.7%). Phorbol ester is heat tolerant and withstands
roasting temperature as high as 160ºC for 30 minutes
(Chang et al., 2014). Hence heat treatment alone may
not be effective to remove phorbol ester from the
seed and seed meal. Thus the combination of
chemical (sodium hydroxide) and heat, and yeast and
heat treatment methods may be successful in the
reduction of toxicity of jatropha seed meal.
On the other hand, no information are available in the
use of baking yeast as biological inoculate for
treating jatropha seed meal to reduce the anti-
nutritional factor and other toxic substances in
Ethiopia. However, studies conducted by Celik et al.
(2003) showed that yeast (Saccharomyces cerevisiae
Meyen ex Hansen) additives can be beneficial in
reducing the toxic effects of Aflatoxin. Therefore the
present study was initiated to assess the efficacy of
different methods in detoxification of phorbol ester in
Jatropha seed meal produced in Ethiopia and evaluate
J. Agric. Environ. Sci. Vol. 5 No. 2 (2020) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 57
the nutritional composition of major nutrients and
different carbohydrate fractions.
2. Materials and Methods
2.1. Description of the study area
Jatropha seed meal was collected from a biodiesel
industry situated at Bati district in Oromo Zone of
Amhara Region, Ethiopia, 420 km Northeast of
Addis Ababa located between 11°11′N latitude and
40°1′E longitude. The detoxification process and the
chemical analysis were conducted in Nutrition
Laboratory of the School of Animal and Range
Sciences, College of Agriculture, Hawassa
University, Hawassa, Ethiopia, while the phorbol
ester analysis was carried out at the University of
Hohenheim, Stuttgart in Germany.
2.2. Detoxification methods used
2.2.1. Physical (moist heat) treatment
Jatropha seed meal was made from the de-husked J.
curcas seed and pressed using expeller machine made
for the extraction of diesel oil by pressing
mechanically in a biodiesel factory at Bati town. The
meal was grouped as treated and untreated. The
untreated jatropha seed meal was the meal directly
from the biodiesel unit as a by-product without any
further treatment (control) and the treated groups
went through sodium hydroxide + autoclaving, yeast
+ autoclaving and autoclaving treatments.
Accordingly, four treatments of JSM were used in the
present study.
Physical treatment of jatropha seed meal was carried
out using autoclaving described by Aregheore et al.
(2003). Three replicates of jatropha seed meal of
approximately 1000 g each were taken, covered with
aluminum foil and placed in an autoclave at 121 for
30 min. The autoclaved samples were removed and
placed in a bucket, washed with distilled water four
times and the water decanted. Then the samples
were dried in an oven at 60 for 48 hours and
ground to pass through 1 mm sieve size and stored in
sampling glass bottles with screw caps.
2.2.2. Chemical treatment
The chemical treatment of jatropha seed meal was
carried out using sodium hydroxide + heat as
described by Aregheore et al. (2003). A hammer mill
with a sieve size of 4 mm was used to grind about
1000 g of jatropha seed meal. The grounded meal
was placed in three laboratory trays made of stainless
steel to replicate and mixed with 5% sodium
hydroxide solution to form a paste. The paste was
heated in an autoclave at 121
º
C for 30 min. The
autoclaved samples were removed and placed in a
bucket, washed with distilled water four times and
decanted the water. The samples were then dried in
an oven at 60
º
C for 48 hours and ground to pass
through 1 mm sieve size and stored in sampling glass
bottles with screw caps for further chemical analysis.
2.2.3. Biological treatment
The biological treatment was conducted according to
the method of Sumati et al. (2010) using baking yeast
(Saccharomyces cerevisiae) purchased from the local
super market. Approximately 2000 g of the JSM was
grounded to pass through 4 mm sieve size and
steamed at 121
º
C for 15 min. The jatropha seed meal
samples were allowed to cool at room temperature.
The cooled samples were then mixed with the baking,
instant yeast (Jami-instant
®
commercial name
)
at
3g/kg jatropha seed meal up to about 60% moisture
content level to the original weight of the sample.
Then the sample was covered with plastic to make
anaerobic condition and was incubated for 24 hours.
After 24 hour of incubation the growth of yeast was
terminated by oven drying the jatropha seed meal at
70
º
C for 24 hours. The sample was the allowed to
cool at room temperature and grounded to pass
through 1 mm sieve size and stored in sampling glass
bottles with screw caps.
2.3. Determination of chemical composition of
Jatropha Seed Meal
The dry matter, crude fat, and an ash content of the
jatropha seed meal were determined using the
standard methods described by AOAC (1990).
Nitrogen (N) was determined by Kjeldhal procedure
and crude protein was calculated using the formula N
x 6.25 (Sosulski and Imafidon 1990). The
metabolizable energy content of JSM was estimated
according to the equation proposed by Wiseman
(1987) as indicated below.
     [1]
Where,
ME = Metabolizable Energy in kilocalorie per kg
dry matter
DM = Dry Matter,
J. Agric. Environ. Sci. Vol. 5 No. 2 (2020) ISSN: 2616-3721 (Online); 2616-3713 (Print)
Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 58
EE = Ether extract
CF = Crude Fiber
Kcal = kilocalorie
(
K
c
a
l
kg
-1
DM)
Kg = kilogram
The crude fiber, neutral detergent fiber, acid
detergent fiber and acid detergent lignin were
analyzed according to the method of Van Soest et al.
(1991) using ANKOM
220
Fiber technology
®
. Non-
fiber carbohydrate content was determined based on
the formula below as described by NRC (2001).
      [2]
Where,
NFC = Non-fiber carbohydrate
NDF = neutral detergent fiber,
CP = Crude protein
EE = Ether Extract
Nitrogen free extract was computed by the difference
of organic matter and the sum of crude fiber, ether
extract and crude protein. Hemicellulose was
estimated as the difference between neutral detergent
fiber and acid detergent fiber while cellulose
estimated as the difference between acid detergent
fiber and acid detergent lignin.
       [3]
Where,
NFE = Nitrogen free extract
CP = Crude Protein
EE=Ether Extract
CF= Crude fiber
2.4. Determination of the anti-nutritional
components
Phorbol ester was extracted by the method described
by Makkar et al. (1997) using HPLC-UV analysis
method. All samples were analyzed in duplicate at
the laboratory of University of Hohenheim, Stuttgart,
Germany.
2.5. Statistical analysis
The data were analyzed using the General Linear
Models (GLM) Procedure (SAS, 2002) and means
were separated by the Duncan’s Multiple Range Test
(Duncan, 1955) at p<0.05. The following model was
used for the analysis of the collected data.
   [4]
Where,
Yij= Response of variables
µ = Over all mean
Ti = Treatment effect on nutrient compositions
eij= Experimental error
3. Results
3.1. Chemical Composition of Jatropha Seed
Meal
The chemical compositions of treated and untreated
Jatropha seed meal are presented inTable1. The crude
protein content of the control (untreated) was higher
(p<0.05) than the sodium hydroxide and Yeast
treated jatropha seed meal (Table 1). Sodium
hydroxide treated and control (untreated) exhibited
the highest ash content (p<0.05). The ether extract
content of jatropha seed meal treated with Sodium
hydroxide was lowest (p<0.05). However, there were
no differences among Yeast and heat-treated and
control treatments.
The highest (p<0.05) neutral detergent fiber and
crude fiber contents were recorded for yeast and
Sodium hydroxide-treated jatropha seed meal while
the lowest (p<0.05) crude protein and neutral
detergent fiber contents were recorded from the
control. The neutral detergent fiber contents of
Sodium hydroxide, and Yeast-treated jatropha seed
meal were greater (p<0.05) compared with heat-
treated and control treatment. The lowest acid
detergent lignin content was recorded from the
control treatment compared to the other treatments,
which were statistically similar.
The composition of different carbohydrate fractions
of treated and untreated JSM is presented in Table 2.
There were differences in non-fiber carbohydrate
content, cellulose, hemi-cellulose and nitrogen free
extract content across the different treatments. The
findings indicated that the non-fiber carbohydrate
content varied across the treatments with higher
(P<0.05) values recorded in Sodium hydroxide
treated and control (untreated) treatments and lowest
in Yeast treated jatropha seed meal. The study also
showed that the hemicelluloses values varied across
the treatments with higher (P<0.05) values recorded
in Sodium hydroxide and Yeast treated jatropha seed
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Publication of College of Agriculture and Environmental Sciences, Bahir Dar University 59
meal while the control (untreated) had the least
hemicelluloses. The nitrogen-free extract values were
highest (P<0.05) in the Sodium hydroxide treated
samples with no differences recorded across the other
treatments including the control (untreated). The
metabolizable energy value was highest (P<0.05) in
the control treatment while the lowest (P<0.05) in
Sodium hydroxide-treated jatropha seed meal.
Table 1: Chemical compositions of treated and untreated Jatropha Seed Meal
Nutrient content
(% in DM)
NaOH-
treated
Yeast-
treated
Heat-treated
Control
(untreated)
SEM±
P-value
Ash
10.00
a
8.19
b
8.37
b
9.37
a
0.25
0.0025
CP
26.97
c
33.39
b
34.93
ab
38.83
a
1.42
0.0022
EE
3.3
b
9.14
a
9.09
a
8.67
a
0.75
0.0001
NDF
48.38
a
46.52
a
39.64
b
34.27
c
1.72
0.0001
ADF
25.62
a
24.97
ab
22.87
bc
20.49
c
0.69
0.0074
ADL
13.59
a
11.75
a
10.90
a
7.71
b
0.74
0.0089
CF
33.05
a
33.67
a
28.54
b
24.30
c
1.16
0.0001
SEM = standard error of the mean; JSM = Jatropha seed meal; DM = dry matter; CP = crude protein; EE = ether
extract; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; CF = crude fiber;
NaOH = sodium hydro oxide
Note: Means in the same row with similar letter/s are not significantly different at (p<0.05)
Table 2: Composition of carbohydrate fractions in treated and untreated Jatropha Seed Meal
NaOH-treated
Yeast-
treated
Heat-treated
Control
(untreated)
SEM±
11.28
a
2.75
b
7.96
ab
8.86
a
1.79
12.03
13.21
11.96
12.77
1.29
22.75
a
21.55
a
16.76
b
13.77
c
0.79
26.61
a
15.59
b
19.66
b
18.83
b
1.36
1202.73
d
1461.57
c
1914.48
b
2267.24
a
47.363
SEM = standard error of the mean; DM = dry matter
Note: Means in the same row with similar letter/s are not significantly different at (p<0.05)
3.2. Anti-nutritional components
The results presented in Table 3 indicate that the
Phorbol esters content was lowest in yeast-treated
jatropha seed meal. Conversely the highest (P<0.05)
Phorbol esters content was recorded in untreated
jatropha seed meal.
Table 3: Phorbol esters content Jatropha Seed Meal
Treatment
PE
SEM ±
p-value
NaOH-treated
872.15
c
11.05
0.0001
Yeast-treated
360.35
d
22.25
0.0001
Heat-treated
1553.6
b
91.9
0.0001
Control
(untreated)
2285.9
a
86.50
0.0001
PE = Phorbol esters (µgTPA eq./g); SEM = standard
error
Note: Means in column followed by the same letter/s
are statistically similar at p<0.05 probability level
4. Discussion
4.1. Nutritive values of JSM
The lower value of crude protein in sodium
hydroxide, yeast, and heat-treated jatropha seed meal
compared with the control could be due to denaturing
effect of the heat during the process of autoclaving
(Emiola et al., 2003; 2007). The higher crude protein
content of yeast treatment compared with sodium
hydroxide could be as a result of the addition of
microbial protein during fermentation (Belewu,
2008). The crude protein content of jatropha seed
meal in the untreated control (38.83%) was higher
than the values reported by Chikpah and Demuyakor
(2012) where the crude protein content of jatropha
seed meal collected from four different areas ranged
between 27.33-29.61%. The variation in crude
protein content with the previous study could be
attributed to differences in agro-climatic conditions
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Journal of the College of Agriculture & Environmental Sciences, Bahir Dar University 60
in which the Jatropha grew harvesting season, age of
the plant and methods of treatment used to remove or
reduce the toxic substances (Martınez-Herrera et al.,
2006). However, the crude protein content of sodium
hydroxide and yeast treated groups in this study is
similar to results of some studies (Abdel-Shafy et al.,
2011) where de-hulled Jatropha seeds had values
between 27-33%.
The ash content for untreated jatropha seed meal in
the present study was lower (9.37%) than that of
earlier reports, which ranged between 9.8 and 10.8%
in defatted jatropha seed meal collected from four
different regions in Mexico (Martinez-Herrera et al.,
2006). On the other hand, Ojediran et al. (2014)
reported the ash content of defatted jatropha seed
meal was about 6.13%. The higher value of ash
content in sodium hydroxide treatment in the present
study could be attributed to the addition of some
elements like sodium from the chemical used for
treatment.
The values of ether extract observed in the control
(8.67) and yeast treated (9.14%) jatropha seed meal
in the present study are slightly lower compared to
untreated J. curcass kernel (9.35%) and fungus-
treated (10.85%) as reported by Ojediran et al.
(2014). On the other hand Abdel-Shafy et al. (2011)
reported 4.38% ether extract from solvent-extracted
Jatropha seed meal. The reason for the higher value
of ether extract for untreated jatropha seed meal in
the current study could be attributed to the
mechanical expeller methods of extraction that might
have not extracted the oil exhaustively. The
mechanical presses have low extraction efficiencies,
about 8-14% of the available oil remain in the press
cake (Bamgboye and Adejumo, 2007).
The acid detergent fiber value (25.62%) in sodium
hydroxide treated jatropha seed meal was lower than
the value reported by Chivandi et al. (2004) in
unshelled Jatropha seed (34.4%). The value of crude
fiber content observed in sodium hydroxide treated
was similar to the findings of Chikpah and
Demuyakor (2012)for raw Jatropha seed meal
(24.72%) but much higher than reported for defatted
Jatropha seed meal (4.9 – 6.1%) from four different
regions in Mexico (Martinez-Herrera et al., 2006).
The nitrogen free extract content of treatment
methods recorded in the present study was ranged
from 18.83% to 26.61% which is generally lower
than the findings of Abo El-Fadel et al. (2011) which
ranged from 31.13% to 31.92%. However the values
of nitrogen-free extract obtained from untreated and
yeast-treated JSM in the current study were higher
than those reported by Ojediran et al. (2014) for
untreated JSM (11.09%) and by Belewu et al. (2010)
for yeast-treated JSM (4.73%). The difference in
crude fiber content with pervious study might be due
to the difference in treatment methods, levels of
application, agro-climate and harvesting season. The
observed metabolizabe energy value in this study was
lower than the value reported for Jatropha kernel by
Nessiem et al. (2017) possibly because of the lower
ether extract values.
4.2. Anti-nutritional components
The results in the present study indicated that the
methods used to detoxify the jatropha seed meal
influenced the value of phorbol ester contents. The
higher phorbol ester value in the heat-treated (moist
heat) jatropha seed meal could be attributed to the
capacity of phorbol ester to withstand high
temperature up to 160
0
C for 30 min as reported by
Kumar and Sharma (2008), Belewu and Sam (2010)
and Chang et al. (2014). The results revealed that the
phorbol ester contents recorded in all treatments in
the present study were lower than that of earlier
findings reported by Ojediran et al. (2014) where the
phorbol ester content of raw defatted Jatropha meal
was 2.49mg/100g (2490 µgTPA eq./g). The lower
phorbol ester in yeast treated jatropha seed meal in
this study indicates that this treatments is more
effective compared to other detoxifying methods
evaluated.
5. Conclusion
The study revealed that sodium hydroxide treatment
did not reduce the fiber content, neutral detergent
fiber, acid detergent fiber, acid detergent lignin and
crude fiber but increased the ash content. Except for
the control (untreated), the crude protein and ether
extract contents in sodium hydroxide-treated jatropha
seed meal were lower than the values in other
treatments. However, no differences between yeast
and heat-treated jatropha seed meal in the crude
protein and ether extract contents were observed. The
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Journal of the College of Agriculture & Environmental Sciences, Bahir Dar University 61
sodium hydroxide and yeast treatments reduced the
phorbol ester content in the present study. Generally,
yeast treatment increased crude protein, ether extract
and decreased phorbol ester content of jatropah seed
meal, which can be recommended for detoxification
of jatropha seed meal so as to be used as animal feed.
Further studies towards feeding trail of yeast treated
JSM is also recommended.
Conflict of Interest
The authors declared that there is no conflict of
interest
Acknowledgement
The authors acknowledge Hawassa University for the
financial support. Countless appreciation goes to
school of Animal and Range Sciences for the effort in
facilitating to get the laboratory support for Phorbol
ester analysis in University of Hohenheim, Germany.
The authors also acknowledge Amhara Rehabilitation
and Development Organization at Bate town and
YME Product Design and Manufacturing Company
for providing us Jatropha seed meal.
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