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Journal of Food, Agriculture & Environment Vol.6(2) : 251-255. 2008

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Proximate composition and cyanide content of cassava peels fermented with


Aspergillus niger and Lactobacillus rhamnosus
C. E. Okpako, V. O. Ntui *, A. N. Osuagwu and F. I. Obasi
Department of Genetics and Biotechnology, University of Calabar, Calabar, Nigeria.
*e-mail: ntuival@yahoo.com

Received 14 December 2007, accepted 28 March 2008.

Abstract
Cassava (Manihot esculenta) is an important root crop in the tropics, providing energy for more than 500 million people. Cassava and its products
are widely used in tropical areas to feed farm animals. The effects of Aspergillus niger and Lactobacillus rhamnosus on the proximate composition
and cyanide content in peels of cassava variety ADP3(4) were investigated using solid media fermentation. The objective of the study was to
increase the nutritive value of cassava peels in order to make them good component for animal feeds. Six treatment samples viz: T1 (peels fermented
with a mixture of A. niger and L. rhamnosus), T2 (peels fermented with A. niger), T3 (peels fermented with L. rhamnosus), T4 (naturally fermented
peels), T5 (soaked and unfermented peels) and T6 (non-soaked and unfermented peels) were used in the study. The experimental design was the
completely randomized design with 3 replications. Results obtained showed significant (P<0.05) increase in protein (24.40.46%), moisture
(10.340.20%), ash (7.520.5%) and crude fibre (10.620.12%) with a subsequent reduction in cyanide level (7.35 0.81 mg/kg) in cassava peels
fermented with a mixture of A. niger and L. rhamnosus (T1). Non-soaked unfermented cassava peels (T6) produced significantly (P<0.05) lower
protein content (5.50.22%) and higher carbohydrate (72.53.03%) and cyanide (40.332.8 mg/kg) contents. Simple linear correlation coefficients
were also calculated to determine the relationship between the components. Significant (P<0.001) positive relationship between cyanide and
carbohydrate (r = 0.9917), and between moisture and fibre (r = 0.9907) were obtained. Cyanide correlated significantly and negatively with
moisture (P < 0.01, r = -0.9332), ash (P<0.05, r = -0.8682) and fibre (P<0.05, r = -0.9134). It is suggested that cassava peels could be engineered
by microorganisms to increase nutritive value and then used to feed livestock.
Key words: A. niger, L. rhamnosus, cassava peels, proximate composition, cyanide, fermentation.

Introduction
Cassava (Manihot esculenta) is an important root crop in Africa,
Asia and South America, providing energy for about 500 million
people. It counts as one of the leading crops with respect to the
energy produced per hectare per year 1. It is a staple food for
many countries and is a source of raw material for agricultural
development 2. Nigeria today stands as the foremost producer of
cassava in the world with about 26 million tons 3.
The peels and leaves, which are the by-product of harvesting
and processing, constitute 25% of the whole plant. These byproducts and flour constitute a potential source of livestock feeds
ingredients. The utilization of cassava and its by-products for
livestock feeding has long been realized. Various authors have
reported the use of cassava peels for feeding livestock 4, 5.
The major limitation in the use of cassava for feeding livestock
is its low protein content. The flour for example contains about
3.0% protein and the peels about 1.66% proteins. The tubers
constitute about 20 to 25% starch but very limited quantities of
protein, fats, vitamins and minerals 6, 7. Additionally, peels contain
considerable quantities of the antinutritional factor cyanide.
Cassava pulp and peels vary widely in the cyanide content
although most varieties contain 15-400 mg/kg fresh weight 8.
Cyanide doses of 50-100 mg are reported to be lethal to livestock.
Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008

Several diseases are associated with the consumption of


inadequately processed cassava pulp and peels, such as tropical
ataxic neuropathy, endemic goiter, spastic paraparesis and konzo9.
Because of the low protein and high cyanide content of cassava
peels their use in animal feeding requires protein supplementation
of such diet and a subsequent reduction in the cyanide content.
Protein enrichment of cassava peels through less expensive means
such as fermentation is therefore desired for the optimal utilization
as livestock feeds 10-13.
Fermentation is an important process in preparation of many
cassava products in Africa, although a limited number of
techniques are used. Fermentation has been reported to be
responsible for product stability, flavour development and cyanide
elimination 14. Fermentation enhances the nutrient content of food
through biosynthesis of vitamins, essential amino acids and
protein by improving protein quality and fibre digestibility. It also
enhances micronutrient bioavailability and aids in degrading antinutritional factors 15.
In this study, Aspergillus niger and Lactobacillus rhamnosus
were examined for their potential to increase the nutritive value of
cassava peels with the hope of making them a good source of
animal feeds as well as the effect of fermentation on the cyanide
251

content of the peels. Such base line information will be useful for
animal breeders.
Materials and Methods
Materials: Cassava tubers of sweet variety ADP 3(4) was obtained
from the Cross River State Agricultural Development Programme
(ADP).The microorganisms were collected from Federal Institute
of Industrial Research Oshodi (FIIRO) Lagos, Nigeria.
The microorganisms used were Aspergillus niger and
Lactobacillus rhamnosus. These microorganisms were
subcultured in nutrient agar and MRS media in the Medical
Microbiology Laboratory, University of Calabar Teaching Hospital,
Calabar, Nigeria.
Treatment preparation: Six treatment samples were prepared
following the methods of Oboh 13 with some modifications.
For treatments 1, 2, 3 and 4 freshly harvested cassava tubers of
the variety ADP 3(4) were peeled, washed and soaked in water 16
for 2 days (initial fermentation), after which they were removed
and grated. Four kg of the processed pulp were spread in 4 trays,
each tray containing 1 kg of the pulp.
Tray 1 (Treatment 1) was mixed with 10 g of a mixture of freshly
subcultured pure strains of Aspergillus niger and
Lactobacillus rhamnosus. Tray 2 (Treatment 2) was mixed with
10 g of Aspergillus niger and Tray 3 (Treatment 3) had 10 g of
Lactobacillus rhamnosus. Tray 4 was not inoculated with any
microorganism. These were allowed to ferment for 7 days.
The incubation temperature and relative humidity of air were 30C
and 90-93% respectively 13.
In Treatment 5, peels which were soaked for 2 days were grated
and used for analysis without fermentation. Treatment 6, which
served as the control contained peels which were not soaked in
water but grated and analyzed without fermentation.
Proximate composition analysis of samples: Proximate
composition (moisture, ash, protein, fat, crude fibre and
carbohydrates) of the 6 treatments was determined using standard
analytical methods 17, 18 and official and standard method of
analysis 19. The moisture content of the sample was determined
gravimetrically by drying 5 g of the sample in a crucible to a
constant weight at 120C. The ash content of the sample was
determined gravimetrically by ashing 2 g of each sample in a clean
pre-weighed crucible in a furnace at 550C for 24 hours.
The protein content was determined using Kjeldahl method of
nitrogen (N) analysis. Approximately 2 g of each sample was
digested with concentrated H 2SO 4 using K 2SO 4 catalyst.
The ammonia in the digested sample was then distilled into a
standard boric acid and titrated with 0.1 M HCl. The crude protein
of the sample was obtained using the formula: crude protein =

titre value x 1.4 x 50 x 100 x 65/(1000 x 10 x 1)N2.


The crude fibre was determined using an acid alkaline hydrolysis
method involving boiling 2 g of the sample with 0.1 M H2SO4 and
0.1 M NaOH in a beaker. The content of the beaker was filtered
through a Bchner funnel, dried and ashed at 550C. The total fat
content of each sample was determined gravimetrically by Soxhlet
solvent extraction technique and the residue was dried to a
constant weight and calculated as % of ether extract = (wt of
extract/wt of sample) x 100. Carbohydrate content was determined
as the difference: 100 (moisture + ash + protein + fat + crude
fibre).
Determination of cyanide content: For cyanide content, 10.0 g of
the sample was weighed into a round bottle flask and left to soak
for about 4 hours. It was then steam distilled into 20 ml of 2.5%
(w/v) NaOH contained in an Erlenmeyer flask. The distillation
continued until 250 ml of the distillate was collected. Then 8.0 ml
of 6 N NaOH and 2 ml of 5% (w/v) potassium iodide were added to
the distillate and the distillate was titrated with 0.02 N AgNO3 until
there was a faint but permanent turbidity.
Statistical analysis: All data were subjected to analysis of variance
test using completely randomized design with 3 replications.
Significant means were separated using least significant difference
(LSD) test at P< 0.05. Simple linear correlation coefficients were
determined for proximate composition parameters and cyanide
level. A linear regression model which fitted into the equation;
y = a+bx, where y = proximate composition parameters, x = cyanide
level, was used to predict the relationship between significant
proximate composition parameters and cyanide level 20.
Results
The results of the proximate composition of the different treatments
are presented in Table 1. The moisture, ash, protein and fibre
contents of cassava peels fermented with a mixture of
Aspergillus niger and Lactobacillus rhamnosus were significantly
(P < 0.05) higher than those fermented with either of the
microorganisms, naturally fermented or unfermented peels.
Non-soaked unfermented cassava peels recorded the lowest
moisture, protein and fat contents (Table 1). Cassava peels
fermented with either of the microorganism did not differ
significantly (P > 0.05) from each other in most of the parameters
studied. A significant (P <0.05) drop in carbohydrate (44.620.211%)
value was recorded for cassava peels fermented with a mixture of
the microorganisms, while the non-soaked unfermented peels had
higher carbohydrate content of 72.53.03%. The fat content
(Table 1) ranged from 2.50.2% in peels fermented with
a mixture of the microorganisms to 3.520.3% in the naturally
fermented peels.

Table 1. Proximate composition of fermented and unfermented cassava peels (%).


Sample
Moisture
T1
10.34d0.2
T2
9.09c0.01
T3
9.05c0.23
8.60b0.21
T4
T5
7.81a0.32
7.50a0.2
T6

Ash
7.52c 0.5
6.87b0.15
6.92b0.34
6.54a0.32
6.51a0.32
6.50a 0.2

Protein
24.40e 0.46
20.60d0.46
19.95d0.43
10.60c 0.33
8.61b0.25
5.50a 0.22

Fat
2.50a0.2
3.10b0.52
2.72a0.31
3.52c0.3
3.32b0.24
3.31b0.34

Crude fibre
10.62d0.12
7.80c0.3
7.52c0.51
6.23b0.2
4.90a0.45
4.69a0.4

Carbohydrate
44.62a1.211
52.54b4.31
53.84b2.26
64.51c 3.01
68.85d2.51
72.50e 3.03

Means followed by the same case letter on a given vertical array indicate no significant difference (p > 0.05). Key: -T1 (peels fermented with
a mixture of A. niger and L. rhamnosus), T2 (peels fermented with A. niger ), T3 (peels fermented with L rhamnosus), T4 (naturally fermented
peels), T5 (soaked unfermented peels), T6(non-soaked unfermented peels).

252

Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008

Cyanide contents of the different treatments are presented in


Table 2. Low cyanide level of 7.350.81 mg/kg was obtained in
cassava peels fermented with a mixture of A. niger and
L. rhamnosus while non-soaked unfermented cassava peels
produced significantly (P<0.05) higher cyanide level of 40.332.8
mg/kg. There were no significant (P>0.05) differences in the
cyanide levels of peels treated with either of the microorganisms.
Table 2. Cyanide content of fermented and
unfermented cassava peels (mg/kg).
Sample
T1
T2
T3
T4
T5
T6

Cyanide content
7.35a 0.81
12.34b1.02
11.63b1.51
26.52b1.9
32.64d2.6
40.33c2.8

Means followed by the same case letter are not significantly different
(P<0.05). Key: -T1 (peels fermented with a mixture of A. niger and
L. rhamnosus), T2 (peels fermented with A. niger), T3 (peels fermented
with L rhamnosus), T4 (naturally fermented peels),T5 (soaked
unfermented peels), T6(non-soaked unfermented peels).

The results presented in Table 3 show that cyanide level


correlated positively and significantly with carbohydrate at
P < 0.001 and negatively with moisture (P<0.01), ash and fibre
(P<0.05). Moisture had significant positive relationship with ash
(P<0.01), protein and fibre (P<0.001). No significant relationship
was observed between cyanide and protein.
Discussion
Proximate composition: Moisture is very essential for life
maintenance and analysis of it is one of the most widely used
measurements in food processing. Moisture has been found to
have a stable effect on livestock. As a result of this cassava peels
with high or considerable moisture content will be of great value
in livestock feeds.
From the results given in Table 1, the cassava peels fermented
with mixture of the microorganisms had the highest moisture
content of 10.30.20%. This increase in moisture content might
have been as a result of the combined activities of the
microorganism which had utilized this material during respiration
to release more water during the breakdown process 13, 21. Another
possibility is that the activities of the microorganisms may have
caused the pulp to clump together thereby reducing the process
of evaporation.
The ash composition of food samples is very important in
determining mineral contents. In our study, high ash content was
recorded in cassava peels fermented with a mixture of
microorganisms, suggesting that microbial fermentation increases
the ash content of cassava peel which could be useful in animal

feeds. These findings are in line with those of Oboh and


Akindahunsi 21, who reported an increase in ash content in cassava
products subjected to Saccharomyces cerevisae.
Protein is needed for normal body growth, repairs and
maintenance. A relatively high amount of protein is therefore
required during growth in livestock. In this study there was a
significant (P<0.05) increase in the protein level in cassava peels
fermented with the microorganisms (Table 1). This increase could
be attributed to the possible secretion of some extracellular
enzymes (proteins) such as amylase, linamarase and cellulose 21
into the cassava mash by fermenting microorganisms in an attempt
to make use of the cassava starch as a source of carbon 22. Also,
the increase in the growth and proliferation of the microorganism
growth complex in the form of single cell protein may possibly
account for the apparent increase in the protein content of the
peels fermented with the microorganisms 13, 23-24.
Carbohydrate level in the inoculated cassava peels was
significantly (P<0.05) lower than in the uninoculated peels. This
low level may be an indication that the microorganisms probably
hydrolysed starch into glucose and used it as a source of carbon
for their growth 13. It could also be possible that an increase in the
protein level in the inoculated cassava peels may have resulted in
the decrease in carbohydrate. This is further confirmed in Table 3
in which significant negative relationship was noted between
protein and carbohydrate. As the protein increases carbohydrate
decreases.
Cyanide content: Many authors 5, 10-12 reported that cassava peels
usually have higher concentration of cyanogenic glucosides than
the root meal which makes the peel unsuitable as animal feed.
Processing of cassava peels by fermentation significantly reduces
the cyanide content of the cassava peels.
In this research, cassava peels fermented with microorganisms
showed significant reduction in the cyanide level especially in
peels inoculated with a mixture of A. niger and L. rhamnosus.
This significant low cyanide value recorded in the inoculated peels
suggests that the microorganisms were capable of partially
degrading the cyanogenic glycosides in the peels, confirming
earlier reports 5, 21, 24, 25.
It was also noted that soaked unfermented peels produced lower
cyanide content (Table 2) than the non-soaked unfermented peels.
This is an indication that fermentation probably occurred during
the process of soaking, thereby reducing the cyanide content.
These findings are in line with those of Oyewole and Odunta 16
and Oluremi and Nwosu 26, who reported that soaking of cassava
and its products before processing stimulates the progressive
reduction in cyanide.
The significant positive relationship recorded between cyanide
and carbohydrate (Table 3) suggests that increase in carbohydrate

Table 3. Correlation matrix for proximate composition and cyanide content in cassava peels.
Moisture
Ash
Protein
Fat
Fibre
Carbohydrate
Cyanide

Moisture
1.00
0.964**
0.976***
-0.800
0.9907***
-0.9375**
-0.9332**

Ash

Protein

Fat

Fibre

1.00
-0.8432*
0.7163
0.7966

1.00
-0.9353** 1.00
-0.9134* 0.9917***

1.00
0.9589** 1.00
0.9441* -0.8162*
0.9006*
0.2186
-0.9105* -0.4314
-0.8682* -0.690

Carbohydrate

Cyanide

1.00

* significant at P<0.05, ** significant at P<0.01, *** significant at P<0.001.

Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008

253

content increases cyanide concentration (Fig. 1a). However, the


cyanide content was below the deleterious level of 30 mg/kg in
fermented peels (Treatments 1-4) indicating that fermented
cassava peels could be considered safe in terms of cyanide

poisoning 5, 13. It was also noted that increase in fibre content,


moisture content and ash content of cassava peels significantly
reduced the cyanide level (Figs 1b-d) as these correlated negatively
with cyanide (Table 3) suggesting that this by-product could be a
good supplement in compounding animal feed.

100

Carbohydrate %

80
y=39,65+0.861x

60

R=0.9917
y=39,65+0.861x
R=0.9917

40
20
0

10

20

30

40

50

60

Cyanide level (mg/kg)

a)

Conclusions
Cassava peels fermented with the microorganisms recorded
significant increase in the protein, ash, moisture and fibre contents
with a subsequent reduction in the cyanide level compared to the
naturally fermented and the unfermented peels. It is thus
suggested that cassava peels which are regarded as having no
economic value could be engineered by microorganisms to
increase nutritive value, reduce cyanide content and used as feed
for farm animals provided it is accepted and highly digestible.
Acknowledgement
The authors gratefully thank the Cross River State Agricultural
Development Programme for providing the cassava variety and
the Federal Institute of Industrial Research Oshodi for the
microorganisms.

12
10

Fibre %

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y=10.26-0.151x
R=-0.9134

4
2
0
0

10

20

30

40

50

60

Cyanide level (mg/kg)

b)

12

Moisture %

10
y=10.29-0.0715x
R=-0.9332

8
6
4
2
0
0

10

c)

20

30

40

50

60

Cyanide level (mg/kg)


12

Moisture %

10
8
y=7.355-0.025x
R=-0.8682

6
4
2
0
0

d)

10

20

30

40

50

60

Cyanide level (mg/kg)

Figure 1. Relationship between proximate composition parameters and


cyanide content: a) carbohydrate and cyanide, b) crude fibre and cyanide,
c) moisture and cyanide, d) ash and cyanide.
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