Nutritional and microbiological characterization of pulp powder of locust bean (Parkia biglobosa Benth.) used as a supplement in infant feeding in Northern Benin

Abstract

The microbiological and nutritional characterization of locust bean pulp powder (Parkia biglobosa) was investigated. Bacteria and fungi were isolated from this product. The bacteria isolated were essentially fecal coliforms. The fungal isolates were Aspergillus niger, Aspergillus ochraceus and Penicillium digitatum. The mean total plate count of samples was 2.8 × 103 cfu/g, while the mean coliform total count was lower than 10 cfu/g and the mean fungal count was 1.9 × 103 cfu/g. The respective mean moisture content and total acidity in locust bean pulp powder were 24.16 ± 2.45 and 2.10 ± 0.95%. Nutritional analysis showed that locust bean pulp powder has interesting nutritional potential. Carbohydrate content (6.28 ± 0.67%), protein content (4.129 ± 0.328%), carotenoid content (0.154 ± 0.03%) and the presence of minerals such as calcium (0.166 ± 0.005%), sodium (0.228 ± 0.006%), potassium (1.60 ± 0.071%) and magnesium (0.144 ± 0.002%) allowed its application as supplement in infant feeding in rural areas. Anti-nutritional factors such as oxalate and phytate were detected in samples, and values were lower than established toxic level. Finally, more attention should be made to its microbial quality in order to preserve children’s health.

Full text

African Journal of Food Science Vol. 6(9), pp. 232-238, 15 May, 2012
Available online at http://www.academicjournals.org/AJFS
DOI: 10.5897/AJFS12.016
ISSN 1996-0794 ©2012 Academic Journals
Full Length Research Paper
Nutritional and microbiological characterization of pulp
powder of locust bean (Parkia biglobosa Benth.) used
as a supplement in infant feeding in Northern Benin
Edwige Dahouenon-Ahoussi, Euloge S. Adjou, Evelyne Lozes, Leila L. Yehouenou,
Roméo Hounye, Nasif Famy, Mohamed M. Soumanou and Dominique C. K. Sohounhloue*
Laboratory of Research and Study in Applied Chemistry, Polytechnic School of Abomey-Calavi, University of
Abomey-Calavi, 01P.O.B: 2009 Cotonou, Bénin.
Accepted 28 February, 2012
The microbiological and nutritional characterization of locust bean pulp powder (Parkia biglobosa) was
investigated. Bacteria and fungi were isolated from this product. The bacteria isolated were essentially
fecal coliforms. The fungal isolates were Aspergillus niger, Aspergillus ochraceus and Penicillium
digitatum. The mean total plate count of samples was 2.8 × 103 cfu/g, while the mean coliform total
count was lower than 10 cfu/g and the mean fungal count was 1.9 × 103 cfu/g. The respective mean
moisture content and total acidity in locust bean pulp powder were 24.16 ± 2.45 and 2.10 ± 0.95%.
Nutritional analysis showed that locust bean pulp powder has interesting nutritional potential.
Carbohydrate content (6.28 ± 0.67%), protein content (4.129 ± 0.328%), carotenoid content (0.154 ±
0.03%) and the presence of minerals such as calcium (0.166 ± 0.005%), sodium (0.228 ± 0.006%),
potassium (1.60 ± 0.071%) and magnesium (0.144 ± 0.002%) allowed its application as supplement in
infant feeding in rural areas. Anti-nutritional factors such as oxalate and phytate were detected in
samples, and values were lower than established toxic level. Finally, more attention should be made to
its microbial quality in order to preserve children’s health.
Key words: Parkia biglobosa, microbiological and nutritional characterization, food safety, Benin.
INTRODUCTION
According to the World Health Organization, malnutrition
is the cellular imbalance between the supply of nutrients
and energy and the body's demand for them to ensure
growth, maintenance, and specific functions (Tierney et
al., 2010). The term protein-energy malnutrition (PEM)
applied to a group of related disorders that include
marasmus, kwashiorkor, and intermediate states of
marasmus-kwashiorkor (Tierney et al., 2010). The most
common form of malnutrition in Africa is protein energy
deficiency affecting over 100 million people, especially 30
to 50 million children under 5 years of age (Jildeh et al.,
2010). Some legumes such as soybean, bean, and
peanut, are important sources of protein and can
therefore help to increase the protein intake of the diet of
*Corresponding author. E-mail: ksohoun@bj.refer.org.
population. However, the low-income group, especially in
rural areas, sometimes cannot afford these protein foods.
Parkia biglobosa is a legume forest tree crop belonging to
the family Mimosaceae which provides to West African
population, a range of products used in food and
traditional medicines. The main interest is the use of its
fermented seeds, known for their very pronounced flavor,
as condiment in Africa and designated by various names
Afitin, Iru or Sonru in Benin, Soumbala in Burkina Faso,
Mali and Niger and Iru in Southwest Nigeria. The
fermented seed contains about 30 to 40% protein, 10 to
15% carbohydrates, 15 to 20% fat and 4% minerals
(Codjia et al., 2003). In the northern regions of Benin, the
pulp is collected, stored and later sold in the market in
powder form. It is usually applied by these populations as
a supplement in infant feeding and is added directly in
infant porridges. Because of the vulnerability of child
health, the poor conditions and duration of storage of the

pulp, the precarious conditions of hygiene in the market,
the direct use of this powder pulp in infant feeding and
the risk of reoccurring of childhood diseases epide-
miological, microbiological and nutritional quality of pulp
powder of locust bean must be evaluated to ensure the
health of consumers who are mostly children and infants.
MATERIALS AND METHODS
Collection of samples
Samples (pulps of P. biglobosa) were purchased from local markets
(the major sales depot of P. biglobosa) in Ouenou, Tamarou,
Gbegouru, Bori, and Teme, all in N’Dali, (north of Benin) and
labeled A, B, C, D and E respectively. The samples were purchased
from four different points in each market and were mixed together
to give each composite sample which was used for the analysis.
Fresh fruits were also harvested after maturation at N’Dali and
taken to the laboratory where they were dried at laboratory
temperature (25°C). Husks were manually removed. The floury pulp
were grated, passed through a 25 mm sieve and kept in airtight
container for laboratory analysis.
Determination of physicochemical parameters
Moisture content of samples was determined by desiccation using
the method of De Knegt and Brink (1998). A clean platinum dish
was dried in an oven and cooled in a desiccator and weighed. From
each sample, 5 g was weighed and spread on the dish, the dish
containing the sample was weighed. It was then transferred into the
air oven at 105°C to dry until a constant weight was obtained and
the loss in mass was determined. In order to obtain the pH of the
samples, 5 g of each sample was weighed and suspended in 10 ml
of distilled water. The pH was determined with a digital pH-meter
(HANNA HI 98129). Acidity of samples, expressed as citric acid
content per unit of volume, was determined by titration with 0.01
mol/L of sodium hydroxide solution, using phenolphthalein as
indicator (AOAC, 1990).
Nutritional analysis
The carbohydrate was determined according to phenol sulfuric acid
method (Agbo and Ronald, 1996; Ezoua et al., 1999). A standard
curve was obtained using the following concentration of sucrose in
(mg/ml) 2.5 2.0, 1.25, 1.0, 0.5 g of each sample with 9 ml of
distillated water was measured into test-tube. 2 ml of phenol
solution (1%) and 1 ml of concentrated H2SO4 solution were added.
This was shaken for 15 min and boiled for 30 min. It was then
allowed to cool. The absorbance was then read off a
spectrophotometer (spectrum lab 22) at 700 nm. The sugar
concentration was then obtained by extrapolation from the standard
curve. Protein was analyzed by the Microkjedhal nitrogen method,
using a conversion factor of 6.25 and fat content was obtained by
Soxhlet extraction as described by Pearson (1976). Carotenoids
content was determined according to the method described by
AOAC (1995). Ash was determined according to the standard
methods described by the Association of Official Analytical
Chemists (AOAC, 1990). Fiber content was determined by the ISO
method (ISO, 1981). 2 g of finely ground defatted sample were
weighed and boiled with sulfuric acid solution (0.255 mol/L) for half
an hour followed by separation and washing of the insoluble
residue. The residue was then boiled with a sodium hydroxide
(0.313 mol/L) solution followed by separation, washing and drying.
Dahouenon-Ahoussi et al. 233
The dried residue was weighed and ashed in a muffle furnace at
600°C and the loss in mass was determined. Minerals were
analyzed by the method reported by Oshodi (1992). Minerals were
analyzed by dry-ashing 1 g of the sample at 550°C in a furnace.
The ash obtained was dissolved in 10% HCl, filtered with filter
paper and made up to standard volume with deionised water.
Flame photometer was used to determine sodium and potassium
contents of the samples, while calcium and magnesium were
determined using atomic absorption spectrophotometer (Perkin
Elmer, Model 403).
Anti-nutritional factors analysis
Total oxalate was determined as described by Day and Underwood
(1986). 1 g of sample was weighed into 100 ml conical flask. 75 ml
H2SO4 (3 mol/L) was added and stirred for 1 h with a magnetic
stirrer. This was filtered using a Whatman No 1 filter paper. 25 ml of
the filtrate was then taken and titrated while hot against 0.05 mol/L
of KMnO4 solution until a faint pink colour persisted for at least 30 s.
The oxalate content was then calculated by taking 1 ml of 0.05
mol/L of KMnO4 as equivalent to 2.2 mg oxalate (Ihekoronye and
Ngoddy, 1985; Chinma and Igyor, 2007). Phytate was determined
using the method of Reddy and Love (1999). 4 g of each sample
was soaked in 100 ml of 2% HCl for 5 h and filtered. To 25 ml of the
filtrate, 5 ml of 0.3% ammonium thiocyanate solution was added.
The mixture was then titrated with Iron (III) chloride solution until a
brownish-yellow color that persisted for 5 min was obtained. A 4:6
Fe/P atomic ratio was used to calculate the phytic acid content
(Okon and Akpanyung, 2005).
Microbiological analysis
To 25 g of each sample, 225 ml of peptone water was added and
homogenized. From the initial concentration, appropriate decimal
dilutions were prepared and aliquots were plated in duplicates on
various media. Plate count agar was used for the total bacterial
count. Plates were incubated at 30°C for 72 h. Desoxycholate was
used for the total Coliforms count and plates were incubated at
30°C for 24 h. Desoxycholate was also used for the Faecal
coliforms count. In this case, plates were incubated at 44°C and the
identification was made using EMB (Eosine Methylene blue).
Tryptone Sulfite Neomicin Agar was used for Anaerobic Sulfito-
Reducer (ASR) count and tubes were incubated at 37°C for 24 h.
After incubation, the number of colonies was tracked using a colony
counter. The number of bacteria expressed as Colony Forming
Units per gram (CFU/g) was then determined by calculation,
bearing in mind the factors of dilution (Singh et al., 1991). The
isolation of fungi from samples was performed using dilution plating
method. 10 g of each sample were separately added to 90 ml of
sterile water containing 0.1% peptone water. This was thoroughly
mixed to obtain the 10−1 dilution. Further 10-fold serial dilutions up
to 10−4 were made. One mililitre of each dilution was separately
placed in Petri dishes, over which 10 to 15 ml of Potato Dextrose
Agar with 60 μg/ml of chloramphenicol (PDAC) was poured. The
plates were incubated at 28 ± 2°C for 7 days (Rampersad et al.,
1999). The identification of the bacterial isolates was based on
cultural, morphological, and biochemical characteristics following
standard methods (Buchanan and Gibbons, 1974) while that of
fungi was also based on cultural and morphological characteristics
using standard taxonomic schemes (Singh et al., 1991; Bryce,
1992). Microbiological parameters were evaluated periodically
during 16 days.
Statistical analyses
The data generated from these studies were analyzed using

234 Afr. J. Food Sci.
Table 1. Physicochemical parameters of locust bean pulp powder from markets.
Sample
Moisture (%)
pH
Acidity (%)
A
23.54
4.2
1.68
B
22.58
4.3
1.34
C
26.61
3.1
3.06
D
22.09
4.7
1.42
E
25.98
3.2
3.02
Mean
24.16 ± 2.45
3.9 ± 0.8
2.10 ± 0.95
Table 2. Nutritional content of locust bean pulp powder from markets.
Sample
Carbohydrate (%)
Protein (%)
Carotenoid (%)
Ash (%)
Fiber (%)
A
6.89
4.457
0.187
7.85
22.84
B
6.95
4.228
0.173
7.62
22.56
C
5.83
3.892
0.137
7.54
21.34
D
6.41
4.238
0.145
7.83
22.57
E
5.34
3.832
0.128
7.34
21.82
Mean
6.28 ± 0.67
4.129 ± 0.328
0.154 ± 0.033
7.637 ± 0.214
22.226 ± 0.614
Table 3. Antinutritional factors content of locust bean pulp
powder from markets.
Sample
Oxalate (%)
Phytate (%)
A
11.17
3.19
B
15.10
4.12
C
14.18
2.13
D
17.19
4.75
E
9.17
2.34
Mean
13.36 ± 3.83
3.30 ± 1.45
Statistical Analysis Software (SAS) and SYSTAT 5.05. The
statistical analyses carried out were mean and standard deviation
and analysis of variance (ANOVA) (Alder and Roessler, 1977;
Ogbeibu, 2005).
RESULTS
The results of physicochemical characterization of
different samples of locust bean pulp powder from
markets (Table 1) showed that the moisture content of
different samples ranged from 22.09 to 26.61%, with an
average of 24.16%. The pH was between 3.1 and 4.7
with a mean acidity of 2.36%. The locust bean pulp
powders are rich in nutrients (Table 2) such as
carbohydrates (6.28%), proteins (4.29%), carotenoids
(0.154%), ash (7.63%) and fiber (22.23%). The analysis
of anti-nutritional factors revealed the presence of oxalate
(9.17 to 15.10%) and phytate (2.13 to 4.75%) (Table 3).
All samples analyzed were rich in minerals such as
calcium, magnesium, potassium and sodium, with a
higher content of potassium (1.5 to 1.7%) (Figure 1). The
result of proximate composition of fresh pulp of locust
bean is as shown in Table 4. The moisture content and
acidity were respectively 11.02 ± 0.41 and 2.10 ± 0.95%.
Ash, protein and carbohydrate content were 8.05 ± 0.22,
9.61 ± 0.43 and 47.63 ± 0.27%, respectively. Fat content
was very low and fiber was 23.68 ± 0.14%. All samples
analyzed were also rich in minerals such as calcium,
magnesium, potassium and sodium, with a higher content
of potassium (1.8 ± 0.32%) (Figure 1). The analysis of
anti-nutritional factors also revealed the presence of
oxalate (12.74 ± 1.74%) and phytate (3.21 ± 0.96%)
(Table 3). The total flora count of samples from markets
ranged from 6 × 101 to 9 × 103. The enumeration of total
coliforms and fecal coliforms was less than 10 cfu/g with
an absence of spores of anaerobic sulfite reducers
(ASR). Fungal flora was high (3 × 102 to 7 × 103 cfu/g)
with the presence of fungi such as Aspergillus niger,
Aspergillus ochraceus and Penicillium digitatum (Table
8). These bacteria and fungi were also able to growth in
the pulp stored at room temperature (Table 7). However,
the microbial contamination of fresh pulp was very low
with the absence of pathogens (Table 6).
DISCUSSION
The results obtained from microbial analysis of locust
bean pulp powder from markets show that they were
contaminated with microorganisms of public health
concern. The high total bacterial and coliform count may
be a consequence of the low level of hygiene maintained
during the processing and sale of locust bean pulp

Dahouenon-Ahoussi et al. 235
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Percentage
Figure 1. Minerals content of locust bean pulp.
Table 4. Proximate composition of fresh pulp of locust bean.
Item
Percentage composition
Moisture
Acidity
Ash
Protein
Fat
Carbohydrate
Fiber
Fresh pulp
11.02 ± 0.41
0.2 ± 0.06
8.05 ± 0.22
9.61 ± 0.43
0.01 ± 0.004
47.63 ±0.27
23.68 ± 0.14
Table 5. Anti-nutritional factors content of fresh pulp of locust bean.
Item
Percentage composition
Oxalate
Phytate
Fresh pulp
12.74 ± 1.74
3.21 ± 0.96
powder. During the sale, dirty hands are dipped into the
powder for product selection by both hawkers and
consumers. The exposure of products while they were
displayed for sale can also serve as a source of
contamination. The detection of coliforms may indicate
possible faecal contamination. Being enteric bacteria,
their presence indicate poor hygienic practice among
handlers of products. Their presence in samples could
pose a serious threat to food safety, due to the fact that
locust bean pulp powder is a ready-to-eat food which is
consumed without further processing. Great attention
should therefore be given to the microbiological safety of
these products. Similar results were found at the street
foods which are mostly marketed under the same
conditions (OMS, 2004). In Africa, several studies on
street food showed that their hygienic quality is very poor
and this is a clear risk on the health of consumers (OMS,
2004). These precarious hygiene conditions promote the
risk of fecal contamination and therefore the
reoccurrence risk of food borne illness. The contami-
nation also varied according to sampling zones. It may be
due to the environmental conditions in the markets or the
level of hygiene maintained during the processing and
sale. The fungal isolates: A. niger, A. ochraceus and P.
digitatium species are known spore formers. It therefore
means that they can easily contaminate locust bean pulp
powders which are usually exposed in market. Their
growth can result in the production and accumulation of
mycotoxins. The result of proximate composition of pulps
from markets indicates that the moisture content is quite
high. This high moisture content would encourage
microbial growth and so deterioration. However, the low
level contamination of fresh pulp, compared to those from
markets, confirmed that the important source of
contamination was the low level of hygiene maintained
during the processing and sale of locust bean pulp. The
low microbial count is a reflection of the low fat and
moisture contents of the fresh pulp which is an indication
that the pulp can be stored in a tight container for a long
time without spoilage (Gernah et al., 2007). This is in

236 Afr. J. Food Sci.
Table 6. Microbial count of locust bean pulp (cfu/g).
Sample
Total bacterial
count
Total coliforms
count
Faecal
coliforms count
A.S.R spores
count
Mould and
yeast count
Fresh pulp
03
00
00
00
00
A
7 × 102
00
00
00
5 × 102
B
6 × 102
04
03
00
3 × 102
C
4 × 103
04
02
00
2 × 103
D
6 × 101
03
01
00
4 × 101
E
9 × 103
07
04
00
7 × 103
European Union criteria (2005)
-
10
10
Absence/10 g
Absence/10 g
Conformity (%)
-
100
100
100
00
A.S.R: Anaerobic Sulfito-Reducer
Table 7. Microbial growth in locust bean pulp from markets stored at room temperature (cfu/g).
Days
Total bacterial
count
Total coliforms
count
Faecal
coliforms count
A.S.R spores
count
Mould and yeast
count
1
7.0 × 102
04
03
00
3 × 102
4
1.0 × 103
04
03
00
7 × 102
8
1.5 × 103
06
05
00
9 × 102
12
2.7 × 103
08
05
00
1.2 × 103
16
3.2 × 103
12
09
00
6.0 × 103
European Union criteria (2005)
-
10
10
Absence/10 g
Absence/10 g
Table 8. Prevalence of fungi isolated from locust bean pulp samples from markets.
Fungal isolated
Prevalence (%)
A
B
C
D
E
Aspergillus niger
43.5
40.2
42.4
39.6
44.6
Aspergillus ochraceus
28.3
23.4
29.3
31
27.3
Penicillium digitatum
25
29
22
24
23
Other
3.2
7.4
6.3
5.4
5.1
accordance with the report of Owoyale et al. (1987),
Omojola et al. (2011) and Compaoré et al. (2011).
The high nutritional potential of locust bean pulp such
as its proteins, carbohydrates, carotenoids (provitamin A)
and its mineral contents (Tables 2 and 4, Figure 1),
justified its uses as supplement in infant feeding in
northern Benin. Minerals are important in human
nutrition. It is well known that enzymatic activities as well
as electrolyte balance of the blood fluid are related to
adequacy of Na, K and Mg. Potassium is very important
in maintaining the body fluid volume and osmotic
equilibrium. Metal deficiency syndrome like rickets and
calcification of bones is caused by calcium deficiency.
Several studies on nutrition in developing countries have
shown that adequate nutrient intake (daily calories, daily
protein, daily fat, minerals and vitamins) is an essential
ingredient for improved well-being, economic growth and
development, since a healthy body enhances the
capacity to learn which in turn determines productivity
and economic growth (Flores, 2001; Smith and Haddad,
2001; Diao et al., 2007). According to Musgrove (1993)
and Benson (2008), adult productivity depends to a
considerable extent on the contribution of health and
nutrition during early childhood. From birth to age 4
months, all the nutritional needs of children are fully
covered in milk. But between 4 and 6 months breast milk
is not sufficient to cover the needs for energy and protein
of the child. This is the period during which nutrients
necessary for child growth must supplement the breast
milk slurry (Claeson et al., 2001). Quantitative protein
requirements are about 20 g per day between 6 months
and 3 years. Ideally, the amino acid composition of these

complementary proteins should be identical to that of
breast milk that is containing the same proportion of the
nine essential amino acids (Hedley et al., 2004).
Fortunately, it is possible to reconstruct a protein mixture
composition meeting the needs of the child by mixing
cereal flour with legume flour. Amino acids absent in
cereal proteins are then supplemented by the amino
acids present in legumes. Among the 12 vitamins
essential for the child, some are particularly important,
such as vitamin A. It is involved in vision, and especially
protects the conjunctiva of the eye and cornea against
infection. Its deficiency is a real public health problem.
Vitamin A is present in foods in the form of provitamin A
(carotenoids) which is present in the vegetables (Shi,
2000; Newborn-Cook et al., 2002) and fruits such as pulp
of locust bean (Tables 2 and 4). This is in agreement with
that obtained by Compaoré et al. (2011) in pulp of locust
bean commonly used in food fortification in Burkina faso.
However, according to Ladeji (2004), oxalate can bind to
calcium present in food thereby rendering calcium
unavailable for normal physiological and biochemical role
such as the maintenance of strong bone, teeth, cofactor
in enzymatic reaction, nerve impulse transmission and as
clotting factor in the blood. The calcium oxalate, which is
insoluble, may also precipitate around soft tissues such
as kidney, causing kidney stones (Oke, 1969). However,
the values obtained for locust bean pulp powder were
below the established toxic level. According to Oke
(1969), a phytate diet of 1 to 6% over a long period
decreases the bioavailability of mineral elements in
monogastric animals. Phytic acid can bind to mineral
elements such as calcium, zinc, manganese, iron and
magnesium to form complexes that are indigestible,
thereby decreasing the bioavailability of the element for
absorption (Erdman, 1979). Phytic acid also has a
negative effect on amino acid digestibility (Makkar and
Becker, 1998). However, values obtained from these
locust bean pulp powder samples are lower than
established toxic level.
Conclusion
This survey underlined the nutritional potentiality of locust
bean pulp powder (P. biglobosa) used as supplement in
infant feeding in northern Benin. However, due to the fact
that it is used for infant diet, more attention (in the
storage and selling methods) should be paid to its
microbial quality in order to preserve children health.
ACKNOWLEDGEMENT
The authors are grateful to the Food Engineering
Technology Department of Polytechnic School of
Abomey-Calavi University (UAC) for their financial
support.
Dahouenon-Ahoussi et al. 237
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