Investigations on the mycoflora and processing effects on the nutritional quality of peanut (Arachis hypogea L. VAR. TS 32-1).

Abstract

The microbiological and nutritional characterization of peanut (Arachis hypogea L
var. TS 32-1) was investigated. Bacteria and fungi were isolated from this product. The fungal
isolates were Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus Speare,
Aspergillus ochraceus Wilhelm and Fusarium poae. The respective mean moisture content
and total acidity in samples were 8.19 ± 0.01% and 1.2 ± 0.02%. Nutritional analysis showed
that peanut (Arachis hypogea L var. TS 32-1) has interesting nutritional potential.
Carbohydrate content (7.84 ± 0.3%), protein content (33.88 ± 0.1%), fat (47.48 ± 0.01%) and
the presence of minerals such as calcium (0.25 ± 0.05g/kg), potassium (5.21 ± 0.02g/kg) and
magnesium (1.92 ± 0.03%) allowed its application as supplement in infant feeding in rural
areas. Anti-nutritional factors such as oxalate and phytate were also detected in samples. This
nutritional potential is significantly affect by thermal processes which can reduce essentially
protein and carbohydrate contents and also anti-nutritional factor levels. However, values
were lower than established toxic level. Finally, more attention should be made to its
microbial quality in order to preserve children’s health.
Keywords:

Full text

Journal of Microbiology,
Biotechnology and Adjou et al. 2012/13 : 2 (3) 1025-1039
Food Sciences
1025
REGULAR ARTICLE
INVESTIGATIONS ON THE MYCOFLORA AND PROCESSING EFFECTS ON THE
NUTRITIONAL QUALITY OF PEANUT (ARACHIS HYPOGEA L. VAR. TS 32-1)
Euloge S. Adjou; Edwige Dahouenon-Ahoussi*; Mohamed M. Soumanou
Address: Laboratory of Research and Study in Applied Chemistry, Polytechnic School of
Abomey-Calavi, University of Abomey-Calavi, 01P.O.B: 2009 Cotonou, Bénin.
*Corresponding author: ablawad2000@yahoo.fr
ABSTRACT
The microbiological and nutritional characterization of peanut (Arachis hypogea L
var. TS 32-1) was investigated. Bacteria and fungi were isolated from this product. The fungal
isolates were Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus Speare,
Aspergillus ochraceus Wilhelm and Fusarium poae. The respective mean moisture content
and total acidity in samples were 8.19 ± 0.01% and 1.2 ± 0.02%. Nutritional analysis showed
that peanut (Arachis hypogea L var. TS 32-1) has interesting nutritional potential.
Carbohydrate content (7.84 ± 0.3%), protein content (33.88 ± 0.1%), fat (47.48 ± 0.01%) and
the presence of minerals such as calcium (0.25 ± 0.05g/kg), potassium (5.21 ± 0.02g/kg) and
magnesium (1.92 ± 0.03%) allowed its application as supplement in infant feeding in rural
areas. Anti-nutritional factors such as oxalate and phytate were also detected in samples. This
nutritional potential is significantly affect by thermal processes which can reduce essentially
protein and carbohydrate contents and also anti-nutritional factor levels. However, values
were lower than established toxic level. Finally, more attention should be made to its
microbial quality in order to preserve children’s health.
Keywords: Peanut, proximate analysis, processing effects, anti-nutritional factor

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INTRODUCTION
Peanut is a major annual oilseed crop and a good source of protein. Oil and protein
content, fatty acid and amino acid composition, taste and flavour are important quality traits
(Asibuo et al., 2008). Protein of peanut is increasingly becoming important as food and feed
sources, especially in developing countries where protein from animal sources is not within
the means of majority of the population. Vegetable oils are in high demand due to diseases
associated with fat from animal origin. The seed has several such as peanut butter, oil, and
other products. The groundnut cake has several uses in feed and infant food formulations
(Asibuo et al., 2008). The literature has reported many health benefits associated with
consumption of peanuts including cancer inhibition (Awad et al., 2000). This benefit is
mainly attributed to micronutrients such as α-tocopherol, folate, minerals and health
promoting phytochemicals, particularly resveratrol, ferulic acid and other phenolic
compounds (Yu et al., 2004). From a microbiology point of view, several in vitro and in vivo
investigations demonstrated the contamination of peanut by fungi (Novas and Cabral, 2002).
Peanut is also very important in the vegetarian diet. It has as much or more protein than meat
and contains no uric acid or cholesterol (Pamplona-Rogers, 2006). Locally, consumption of
peanut is very popular among the population. It is mostly eaten in the roasted form throughout
the year. A substantial part is also eaten in the cooked form, while fewer people indulge in the
eating of the raw seeds (Ogunsanwo et al., 2004). However, peanuts contains some
antinutritional factor such as phytates, condensed tannins, trypsin and amylase inhibitor, that
may limit their usage and nutritional value. According to Njintang et al. (2001), traditional
processing methods such as germination or roasting could improve the nutritional value of
legume seeds. Offem et al. (1993) have reported changes on the chemical composition as a
result of processing. However, few information on the effect of traditional processing on
peanuts quality was reported. The aim of this study was to investigate the efficiency of
processing methods on nutritional composition and the reduction or elimination of
antinutritional factor in peanuts. An emphasis has been placed on identifying the most
effective methods to reduce antinutritional factor in peanuts at domestic level.

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MATERIAL AND METHODS
Materials
Peanuts (Arachis hypogea L var. TS 32-1) were obtained from Agronomical Research
Center (CRA/Savè) of National Institute of Agronomical Research in Benin (INRAB). The
whole seeds (raw) were divided into three (3) portions, one portion being designated as
control and stored in a cold room à 4°C. The remaining seeds were processed by boiling or
roasting.
Experimental procedures
About 0.5 kg of shelled peanuts seeds (harvest in 2011) were thoroughly sorted out
and cleaned of stones, bad seeds, and other foreign objects and roasted in an aluminum
saucepan. For the boiling process, about 0.5 kg of shelled peanuts seeds (2011 harvest) was
washed and then heated with addition of water. After boiling, water was discharged and
boiled peanuts were collected.
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, grinded and suspended in10 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,
1995).

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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 using 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). Ash was determined according to the standard methods described by the
Association of Official Analytical Chemists (AOAC, 1995). 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 potassium content 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).

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Microbiological analysis
To 25 g of each sample (shelled peanut), 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. 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 (shelled peanut) 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 milliliter 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).
Statistical analyses
Treatments were conducted in three repetitions. Macronutrients, micronutrients and
antinutritional factors analysis were performed in triplicate for each treatment repetition. The
data generated from these studies were analyzed using 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).

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RESULTS AND DISCUSSION
The results of physicochemical parameters and proximate composition of peanut
(Arachis hypogea L var. TS 32-1) are shown in Tables 1 and 2. The moisture content, pH and
acidity were respectively 8.19 ± 0.01%, 6.3 ± 0.2 and 1.20 ± 0.02%. Ash, protein, fat and
carbohydrate content were 3.80 ± 0.06%, 33.88 ± 0.12%, 47.48 ± 0.01% and 7.84 ± 0.30%,
respectively. All samples analyzed were also rich in minerals such as calcium, magnesium
and potassium, with a higher content of potassium (5.21±0.02%) (Table 3). The analysis of
anti-nutritional factors (Table 4) also revealed the presence of oxalate (0.108 ± 0.040 %) and
phytate (0.51 ± 0.09 %). The total flora count of unprocessed peanut sample (Arachis hypogea
L var. TS 32-1) was 2x101 ufc/g. The enumeration of total coliforms and fecal coliforms was
less than 10 cfu/g with an absence of spores of anaerobic sulfite reducers (ASR) (Table 5).
Fungal flora was high (9x101 cfu/g) with the presence of fungi such as Aspergillus niger,
Aspergillus flavus, Aspergillus parasiticus Speare, Aspergillus ochraceus Wilhelm, Fusarium
poae (Table 5). Compared to the raw seed, results obtained from the proximate analysis of
processed peanut samples depicted remarkable variation in the chemical composition of seeds
(p < 0.05), depending on the type of process. On roasting of peanuts, ash, mineral content and
fat levels did not change significantly, but there was a significant decrease in protein, total
carbohydrate, moisture and antinutritional factor such as oxalate and phytate. On boiled
peanuts, mineral content, protein, total carbohydrate and also antinutritional factors levels
were significantly decreased. The microbiological analyses revealed that microbial
contamination of all processed peanuts was very low with the absence of pathogens.
The high nutritional potential of peanut such as its proteins, fat, carbohydrates and its
mineral contents (Tables 2 and 3), justified its uses as supplement in infant feeding in Benin.
These findings are in agreement with those of Asibuo et al. (2008) who also underlined the
interesting nutritional potential of peanut from Ghana.
The elevated fat content was in line with the observation of Dwivedi et al. (1994) who
reported that oil content of peanut ranged from 44 to 56%. This result suggested that it is an
interesting oleaginous crop for which the implementation of improved cropping systems
should result in the economic well-being of rural people. Several studies have shown that the
nutritional value of oil is mainly related to its content of fatty acids. Indeed, Asibuo et al.
(2008) reported that the oleic and linoleic acid content of the peanut accounted for 75.30 to
81.05% of the total fatty acids. High level of oleic acid implies high oil stability and better
shelf life of groundnut seeds and products because oleic acid is a monounsaturated fatty acid,

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being less prone to oxidative rancidity (Rahman et al., 2001). According to FAO (1994),
during the first years of life, fat is the main source of energy needed for proper growth and
physical activity. Where diets are low in fat (less than 15 % of total calories), it is difficult to
ensure the energy required, which partly explains the high prevalence of malnutrition in these
areas. Fats also play a structural role in providing fatty acids and cholesterol for the formation
of cell membranes in all organs. Moreover, important organs such as the retina of the eye and
central nervous system are mainly composed of fat. Much of fat necessary for the formation
of these tissues are essential fatty acids, which cannot be synthesized by the body and must be
supplied by the diet. Breast milk has fat composition very special, which explains its unique
role in infant feeding. In the low-income groups the fat intake is insufficient and that this
lower consumption has a negative effect on the nutritional status of children and adults in
these groups.
Seed protein content was higher than cowpea which contains about 24% seed protein
(IITA, 1989). Cowpea and peanut are the major protein sources to the poor and rural dwellers
and these results also demonstrate that peanut is a valuable source of protein for improving
the nutrition of humans. Indeed, 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.
Minerals are also 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. Peanut samples analyzed have good and nutritional valuable minerals
whose importance had already been emphasized (Bowen, 1966; Bender, 1992). However, the
availability of these nutrients after ingestion depends on the antinutritional factors present in
the food. The antinutrients tend to bind to mineral elements there by forming indigestible
complex. Oxalate for instance binds to calcium to form complexes (calcium oxalate crystals).
These oxalate crystals formed prevent the absorption and utilization of calcium. The calcium

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crystals may also precipitate around the renal tubules thereby causing renal stones (Ladeji et
al., 2004). Phytic acid (inositol hexaphosphate) is an organic acid found in plant materials
(Heldt, 1997). Phytic acid combines with some essential elements to form insoluble salts
called phytate. Phytates reduces the availability of many minerals like calcium, magnesium,
iron and zinc. The ability of phytate to form complexes with these mineral can make the
mineral content of a food inadequate especially for children (Ilelaboye and Pikuda, 2009).
Phytate are also found to inhibit the protease and amylase of the intestinal tract (Vaintraub
and Bulmaga, 1991). Sanberg (1991) reported that the minimum amount of phytate to cause
negative effect on iron and zinc absorption were 10-50 mg per meal. Thereby, anemia and
other mineral deficiency disorders are common in regions where the diet is primarily a
vegetarian (Erdman, 1979).
The results obtained from microbial analysis, show that shelled peanuts were
contaminated with microorganisms of public health concern. The most dominant flora was
fungi, especially Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus Speare,
Aspergillus ochraceus Wilhelm and Fusarium poae. These fungi species are known spore
formers and their growth can result in the production and accumulation of mycotoxins. The
moisture content of samples would also encourage microbial growth and so deterioration.
Peanut contamination by fungi does not only reduce its quality but may also lead to
mycotoxin production (Sultan and Magan, 2010). According to Pittet (1998), the
mycotoxins produced by Aspergillus spp. of greatest significance in peanuts include
aflatoxins and ochratoxin A (OTA). Several studies have reported the contamination of
peanuts or peanut products by fungi (Pildain et al., 2008; Fagbohun and Faleye, 2012), and
by mycotoxins, especially aflatoxins and ochratoxin A (Ediage et al., 2011, Adjou et al.,
2012). The high susceptibility of peanuts contamination is mainly due to their nutritional
content, useful to numerous fungi. If the hulls, which protect the seed against invasion by
fungi, become damaged, the underlying cotyledons become susceptible to attack. This
contamination, mainly due to the injury of the hulls, is favored by insect attack, drought
occurring at the end of the vegetative cycle and poor harvesting practices.
Although some studies have been made on chemical change of peanut after
germination (Offem et al., 1993), roasting (Soliman et al., 1985) or during storage
(Fagbohun and Faleye, 2012), the present study constituted a comprehensive investigation
on the influence of thermal process on the chemical composition, nutritional value and
microbiological quality of peanut. Results obtained from the proximate analysis of processed

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1033
peanut samples depicted remarkable variation in the chemical composition of seeds compared
to the unprocessed seeds.
The increase in moisture content of processed samples could be due to the increased
imbibitions of water with boiling time. Muller (1988) explained that during boiling, cellulose
is little affected but the middle lamella gets broken down by heat, thus making vegetables to
take up water as the starch gelatinizes. The decrease in crude protein content could be
attributed to leaching effect. Gernah and Ajir (2007) also reported a decrease in protein
content with the boiling of cassava leaves. However, at the protein content of 27.02 %
(roasted peanut) and 22.26 % (boiled peanut) processed peanuts are considered a very good
protein source as compared to other everyday foods like eggs (12.00%), white bread (7.80%),
rice (6.50%), milk (3.30%), and potatoes (2.10%) as reported by Gamman and Sherrington
(1990). The significant decrease of protein, carbohydrate and antinutritional factor observed
after processing of peanut seeds can also resulted in the effect of heat or some migration of
substances from peanuts to boiling water. According to Ejigui et al., (2005), heat processing
may have destroyed some of heat-labile components in food. Indeed, significant decreases
were observed in antinutritional factor levels after processing. Their reduction was greater in
boiling than roasted processes. Alonso et al. (2000) also observed a decrease of phytate in
kidney beans after processing. This reduction may be due to the hydrolysis and heat
destruction of some molecules of inositol hexaphosphate to penta, tetra and triphosphate
(Alonso et al., 2000). These findings indicated that moist heat is more effective in reducing
antinutritional factor levels in food and foodstuffs than dry heat. This is in accordance with
the report of Ejigui et al., (2005). However, the values obtained for peanut (Arachis hypogea
L var. TS 32-1) were below the established toxic level.
Table 1 Physicochemical parameters of processed peanuts
Item Moisture (%) pH Acidity (%)
Control
(Unprocessed) 8.19 ± 0.01a 6.3 ± 0.2a 1.20 ± 0.02a
Roasted peanut 7.10
±
0.06
b
6.4
±
0.1
a
1.40
±
0.05
a
Boiled peanut 10.90
±
0.02
c
6.1
±
0.5
a
1.10
±
0.02
a
Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly
different according to ANOVA and Tukey’s multiple comparison tests.

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Table 2 Nutritional content of processed peanuts
Item Carbohydrate (%) Protein (%) Fat (%) Ash (%)
Control (Unprocessed) 7.84
±
0.30
a
33.88
±
0.10
a
47.48
±
0.01
a
3.80
±
0.06
a
Roasted peanut 5.42
±
0.50
b
27.02
±
0.30
b
47.33
±
0.03
a
3.70
±
0.03
a
Boiled peanut 4.21
±
0.10
c
22.26
±
0.50
c
47.39
±
0.07
a
3.80
±
0.01
a
Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly
different according to ANOVA and Tukey’s multiple comparison tests.
Table 3 Minerals content of processed peanuts
Item Magnesium (g/kg) Calcium (g/kg) Potassium (g/kg)
Control (Unprocessed) 1.92
±
0.03
a
0.25
±
0.05
a
5.21
±
0.02
a
Roasted peanut 1.91±0.02
a
0.24 ±0.01
a
5.23±0.04
a
Boiled peanut 1.87±0.05
b
0.19 ±0.03
b
5.18±0.01
b
Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly
different according to ANOVA and Tukey’s multiple comparison tests.
Table 4 Antinutritional factors content of processed peanuts
Item
Percentage composition
Oxalate Phytate
Control (Unprocessed) 0.108
±
0.040
a
0.51
±
0.09
a
Roasted peanut 0.064
±
0.080
b
0.15
±
0.08
b
Boiled peanut 0.043
±
0.060
c
0.14
±
0.04
c
Values are mean (n = 3) ± SE. The means followed by same letter in the same column are not significantly
different according to ANOVA and Tukey’s multiple comparison tests.
Table 5 Microbial count of processed peanuts (cfu/g)
Item Total bacterial
count
Total coliforms
count
Faecal
coliforms count
A.S.R spores
count
Mould and
yeast count
Control (Unprocessed) 1x10
2
06 02 00 9x10
1
Roasted peanut 00 00 00 00 00
Boiled peanut 00 00 00 00 00
European union Criteria
(2005)
- 10 10 Absence/10 g Absence/10 g
A.S.R: Anaerobic Sulfito-Reducer

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CONCLUSION
This work underlined the nutritional potentiality of peanut (Arachis hypogea L var. TS
32-1) and important effect of thermal processes in reducing of antinutritional factor levels and
also affected the mycoflora associated with peanut. However, more attention should be paid to
their secondary metabolites (mycotoxins) which are sometimes heat-resistant.
Acknowledgments: The authors are grateful to the Food Engineering Technology
Department of Polytechnic School of Abomey-Calavi University (UAC) for their financial
support. Authors wish to express their gratitude to Mrs. Brice Atrevy and Syntyche Zohounvo
for the technical assistance.
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