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Volume 10 No. 11
November 2010
4398
REDUCTION OF NITRATES, OXALATES AND PHENOLS IN FERMENTED
SOLAR-DRIED STORED COWPEA (Vigna unguiculata L.) LEAF
VEGETABLES
Muchoki CN1*, Lamuka PO1 and JK Imungi 1
Charity Muchoki
*Corresponding author email: charity.muchoki@yahoo.com
1 Department of Food Technology and Nutrition, University of Nairobi, P.O. Box
30197-00100, Nairobi, Kenya.
Volume 10 No. 11
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ABSTRACT
This study was conducted to determine the effect of fermentation, solar drying and
storage duration on the levels of anti-nutrients: nitrates, oxalates and phenols, in
cowpea leaf vegetables. The rationale was reduction of the anti-nutrients. Reduction
of nutritional stress factors in plant foods increases bioavailability of nutrients, hence
improving their quality as foodstuffs. The cowpea leaves were purchased from the
local markets, sorted to remove blemished leaves and foreign materials, washed in
running tap water. Then, the vegetables were drained and divided into three batches of
16 kg each. One batch was heat-treated in hot water for 3 minutes and then cooled to
ambient temperatures, drained and solar-dried. The second portion was acidified to a
pH of 3.8, heat-treated, and solar-dried. The third portion was fermented for 21 days,
heat-treated, and solar-dried. The three batches of vegetables were spread at different
times on drying trays at the rate of 4 kg/m2 and dried in a solar drier to an
approximate moisture content of 10%. The dried vegetables were packaged in either
polyethylene bags or Kraft paper bags and stored for three months at 18oC, 22o- 26oC
or 32oC. Fermentation, heat-treatment and drying of vegetables led to significant (P <
0.05) reduction in nitrates compared to fresh cowpea leaves, but the reduction in
oxalates and phenols was not significant. Storage for three months led to significant
(P < 0.05) reduction in nitrates in the fermented sample compared to the other
samples. The acidified sample had significantly (P < 0.05) higher levels of phenols
after three months of storage than the other samples. Samples stored at 18oC had
higher levels of oxalates and phenols but lower levels of nitrates, compared to those
stored at higher temperatures. Packaging material had no significant effect on the
level of nitrates, oxalates and phenols. Data obtained in this study reveal a novel
technique for the reduction of anti-nutrients in cowpea leaf vegetables, namely;
fermentation followed by solar drying. The increased acceptability of these
fermented-dried vegetables would help rural communities in providing better
foodstuff with fewer anti-nutrients, thus alleviating micronutrient malnutrition. This
novel long-term storage technology can greatly help to deal with the issue of
seasonality and will increase food security, especially during the dry season.
Key words: Fermentation, solar drying, vegetables, anti-nutrients
Volume 10 No. 11
November 2010
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INTRODUCTION
Malnutrition due to nutritionally inadequate diets is a major concern in Kenya and
many other developing countries [1]. The prevalence rates of micronutrient
malnutrition remain high, with devastating consequences for health and productivity
[2]. In Africa, people have always depended on traditional leafy vegetables to meet
their nutritional needs. The vegetables represent cheap but quality nutrition for large
segments of the populations in both urban and rural areas. The vegetables are rich in
vitamins, especially A, B, and C, and minerals such as iron, zinc, calcium and
phosphorus [3].
Unfortunately, most plant species contain nutritional stress factors (anti-nutrients) that
increase the loss of essential nutrients from the body, interfering with the metabolism
of absorbed essential nutrients, decreasing the digestion of food, or decreasing food
intake. Reduction of nutritional stress factors in plant foods increases the
bioavailability of nutrients in the plant and thus improves its quality as a foodstuff.
The most commonly occurring antinutrients in plant foods include nitrates and
nitrites, phenols, cyanogenic glycosides, glucosinolates, oxalates and saponins [4].
Toxicity to humans is due to nitrites that arise from microbial reduction of nitrates in
the gastro-intestinal tract. This can cause methaemoglobinaemia or act as precursor in
the endogenous formation of carcinogenic nitrosamines. This reduction is more likely
in infants than in adults, due to low acidity in their digestive tract, which allows
coliforms and clostridial bacteria to survive [5]. The leafy vegetables are the major
vehicle for the entry of nitrates into the human system [6]. High concentrations of
oxalate may be of great nutritional disadvantage to both humans and animals. Oxalic
acid is a plant toxicant, which forms an insoluble salt with the essential nutrient
calcium, thus inhibiting its absorption [7]. It also inhibits the absorption of iron and,
to some extent, zinc [8, 9]. This manifests as calcium deficiency, even in diets with
adequate levels of calcium. This is more significant in growing children, with
developing bones and teeth than in adults [10]. In addition to potential toxicological
concerns, phenolic compounds have been implicated in influencing the functional,
nutritional and sensory properties of foods with which they are associated [11]. High
levels of phenolic compounds are undesirable for women trying to become pregnant,
since these compounds also decrease fertility, possibly by modulating hormone levels
and even by interfering with the critical early stages of pregnancy [12].
The cowpea (Vigna unguiculata syn Vigna sinensis) is one of the most important
legumes in Kenya. It is cultivated all over Kenya mainly for seeds, but the leaves are a
popular local vegetable. The main problem with traditional vegetables is their lack of
availability due to seasonality. However, in areas where seasonality is a critical factor
that limits availability, promoting home gardening and appropriate local preservation
technology can improve availability [13].
Fermentation of indigenous foods is considered an effective, inexpensive and
nutritionally beneficial household technology, especially in the developing world.
Likewise, sun drying has been a means of preserving food from earliest times [14].
Volume 10 No. 11
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The main problem with the conventional solar drying is huge nutritional losses. This
study aimed at reducing these nutritional losses and reducing the stress factors by
incorporating fermentation into solar drying. The study also considered the problem
of food security, which is devastating during the dry season. The levels of nitrates,
oxalates and phenols in fermented solar-dried cowpea leaf vegetables were assessed.
MATERIALS AND METHODS
Cowpea leaves
The fresh cowpea leaves were purchased from local markets in the morning and
transported quickly to the University of Nairobi’s Department of Food Technology
and Nutrition. For the fermentation trials, the stalks, withered and dried leaves, weeds,
stones and other foreign materials were sorted out from the rest of the vegetables. The
vegetables were then thoroughly washed and well drained. They were cut manually
with a kitchen knife into slices approximately 5mm thick.
Determination of optimal levels of salt and sugar for fermentation
To determine the optimal level for salt, the sorted cowpea leaves were divided into
seven portions and fermented in lots of 500g each. Each lot was mixed thoroughly
with 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0% concentration, respectively, of table salt,
followed by tight packing in 4-litre plastic beakers. Fermentation was carried out at
ambient temperatures (22o – 26oC). To determine the optimal level for sugar, each
sample was mixed with 3% salt (determined as the optimal level of salt for
fermentation) and varying percentages of glucose and sugar, that is, 2.5%, 3.0% or
3.5%. The fermentation was carried out for 16 days with three replicates. Sensory
analyses were performed on the fermented vegetables to determine the effect of added
sugar on their acceptability.
Product manufacture
The fermented-dried vegetables were prepared in comparative trials with control and
acidified samples as follows: Procurement and preparation of the raw materials were
similar to those carried out during the determination of optimal levels of salt and
sugar for fermentation. The amount of the cowpea leaves used was larger. The
vegetables were sliced and then divided into three equal portions each of 16 kg. One
portion was thoroughly mixed with 3% salt and allowed to stand for two hours, then
heat-treated. This was treated as control sample. The second portion was thoroughly
mixed with 3% salt and citric acid (EFF Chemicals Ltd, Kenya) to a final pH of 3.8
and allowed to stand overnight, then heat-treated. This was treated as an acidified
sample. This was done to see whether acid alone could lead to the same results or
different from fermentation. The third portion was thoroughly mixed with 3% salt and
3% sucrose, which were then tightly packed in a 60-litre plastic bucket. The salted
and sugared vegetable sample was allowed to stand for 10 minutes before a
polyethylene bag full of water was placed inside the bucket as a weight to ensure that
the vegetables were immersed in the brine and fermented for 21 days. After
fermentation, the sample was heat treated [15].
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Dehydration and Storage
The fermented, acidified and control vegetable samples were heat treated by boiling
in their own liquor at 90o – 95oC for 3 minutes. Each vegetable sample was cooled
and drained immediately after heat treating and loaded onto a solar drier with shade
provision [16]. The vegetables were spread evenly on trays (4kg/m2) and the trays
inserted into the drier. They were then dried until the weight was constant, which took
on average five days. The fermented dried vegetables were packaged in either Kraft
or polyethylene paper. Each package contained 50g of the fermented dried vegetables.
The packaged products were stored at: 32oC ambient temperatures (22o – 26oC) and
18oC in enclosed dry places for three (3) months. From each batch, one polyethylene
and one Kraft paper bag were opened each month and the vegetables analyzed for
ascorbic acid and beta-carotene. Two bags were used every month for sensory
evaluation. The fermented dried vegetables were prepared in comparative trials with
control and acidified samples as shown in Figure 1. All experiments were repeated
twice.
Volume 10 No. 11
November 2010
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RAW COWPEA LEAVES
Acidified sample Fermented sample Control
sample
Figure 1: Product manufacture flow diagram
WASHING
CUTTING
5mm thickness
SALTING (3% salt)
FERMENTATION
(Spontaneous, at 22o – 26oC for
21 days)
SOLAR DRYING
(To constant weight)
PACKAGING
(Polyethylene and kraft
paper)
SORTING AND DISTEMMING
STORAGE
(At 18o, 22o – 26oC and
32oC )
NO PROCESS
ACIDIFICATION
(With citric acid to
pH 3.8)
SUGARING (3% sugar)
HEAT TREATMENT
(Boiling in own liquor at
90o – 95oC for 3 min)
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November 2010
4404
Anti-nutrient Analyses
Nitrates were determined using the following method: A standard curve was prepared
using different concentrations of potassium nitrate, and nitrates were calculated as
equivalent milligrams/100 g fresh weight. The sample was ground and re-dried over-
night in a hot air oven at 70oC. A sample of 0.1 g was then suspended in 10 ml
distilled water in 100 ml beaker and incubated at 45oC for 1 hr, to extract the nitrates,
and then filtered through Whatman filter paper No. 1. An aliquot of 0.2 ml of the
filtrate was pippeted into a 50 ml beaker and 0.8 ml of 5% (w/v) salicyclic acid in
sulphuric acid was added and mixed thoroughly. The mixture was allowed to stand for
20 min at ambient temperatures. Sodium hydroxide (19 ml) of 2 N concentration was
added and the mixture allowed to cool for 30 min. The absorbance was measured at
410 nm against a common blank. The nitrate content was determined from a standard
curve and the nitrates content calculated as mg/100 g [17].
Oxalates were determined as follows: Standard sodium oxalate solution was prepared
by dissolving 3 mg of sodium oxalate in 10 ml of 0.5 M sulphuric acid. This was
followed by titration with 0.1 M potassium permanganate at 60oC, using a
microburette to a faint violet colour that was stable for at least 15 seconds and a
standard curve was plotted. A dried sample of 0.1 g was extracted with 30 ml of 1 M
hydrochloric acid in a boiling waterbath for 30 min. The sample was cooled, then
shaken and filtered through No. 1 Whatman filter paper. The filtrate was adjusted to a
pH greater than 8 with 8 M ammonium hydroxide followed by re-adjusting it to pH
5.0 – 5.2 with 6 N acetic acid. An aliquot of 10 ml was precipitated with 0.4 ml of 5%
calcium chloride, shaken thoroughly, allowed to settle at ambient temperatures for at
least 16 hrs, and centrifuged at 3000 rpm for 15 min. The supernatant was discarded,
rinsed twice with 2 ml of 0.35 M ammonium hydroxide and then the cake (pellet)
drip-dried. The pellet was dissolved in 10 ml of 0.5 M sulphuric acid followed by
titration with 0.1 M potassium permanganate at 60oC using a microburette to a faint
violet colour that was stable for at least 15 seconds. Oxalates content in the sample
was determined from the standard curve prepared earlier as mg/100 g [18].
Total phenols were determined as tannins by Folin-Denis method [19]. The Folin-
Denis reagent was prepared by mixing 100 g sodium tungstate, 20 g
phosphomolybdic acid and 50 ml phosphoric acid with 750 ml water. The mixture
was then refluxed for 2 hrs, cooled and diluted to 1 litre. Saturated sodium carbonate
solution was prepared by dissolving 35 g anhydrous sodium carbonate in 100 ml
water at 70o – 80oC, and allowed to cool overnight. The supersaturated solution was
seeded with crystals of hydrated sodium carbonate and filtered through glass wool
after crystallization. Tannic acid solution was prepared by dissolving 100 g tannic
acid in 1 litre distilled water. Fresh solution was prepared for each determination. A
standard curve was prepared by pippeting 1 – 10 ml aliquots of the standard tannic
acid solution into 100 ml flasks containing 75 ml of distilled water. Five ml Folin-
Denis reagent, together with 10 ml sodium carbonate solution were added. The
solution was diluted to volume with distilled water and mixed thoroughly. Optical
Volume 10 No. 11
November 2010
4405
densities were determined at 760 nm after 30 min and absorbance plotted against mg
tannic acid/100 ml, to obtain a standard curve.
A ground sample of 0.5 g was extracted in a mortar and pestle with 50 ml distilled
water, and filtered. One millilitre of the filtrate was pipetted into a 100 ml flask
containing 75 ml distilled water. Five milliliters of Folin-Denis reagent and 10 ml
sodium carbonate solution were then added. The solution was made to volume, mixed
thoroughly and then absorbance determined at 760 nm after 30 min incubation.
Milligrams of tannic acid per 100 g of sample were calculated from the standard
curve.
Data analysis
All experiments were designed as complete factorial with three main factors: storage
temperature, processing treatment and type of packaging. Storage temperature had
three levels: 18oC, 22o – 26oC and 32oC, which were fixed-effect treatments
representing various agro-climatic zones in Africa. Processing treatments had three
levels: fermentation, acidification (citric acid – positive control) and untreated
control; each followed by blanching and solar drying. The type of packaging had two
levels: polyethylene and Kraft paper, representing airtight and aerated packaging,
respectively. The experiments were laid on a completely randomized design with
three replicates. All experiments were repeated twice.
All data were then subjected to analysis of variance (ANOVA) and means were
separated by Duncan Multiple Range Test using Genstat 6th Edition and Costat
Statistical Software Programmes.
RESULTS
Levels of nitrates, oxalates and phenols in raw, fermented-, acidified- and control-
dried cowpea leaves are given in Table 1. The levels of nitrates in raw cowpea leaves
were significantly higher (P < 0.05) than those in the fermented-, acidified- and
control-dried samples. There was no significant difference among the raw cowpea
leaves and the fermented-, acidified- and control-dried samples (P < 0.05) in the
levels of oxalates and phenols.
There were apparent losses in nitrates, oxalates and phenols during storage for three
months compared with those before storage. The effect of fermentation and
acidification on the retention of nitrates, oxalates and phenols during the three months
of storage is given in Table 2. After three months of storage, the fermented-dried
sample had the lowest levels of nitrates, oxalates and phenols, as compared with the
other samples. This indicates that fermentation has a reducing effect on the levels of
nitrates, oxalates and the phenols during storage. After drying, the three processed
samples’ levels of nitrates were not significantly different in nitrates (see Table 1), but
after storage, the fermented dried sample had a significantly (P < 0.05) lower nitrate
level compared to the acidified and control dried samples (Table 2). The acidified
dried sample had a significantly higher level of phenols compared to the fermented
and control dried samples after storage, whereas before storage there was no
Volume 10 No. 11
November 2010
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significant difference. This suggests acidification has a significant retention effect on
phenols during storage.
The effect of storage temperature on the retention of nitrates, oxalates and phenols is
shown in Table 3. Samples stored at 18oC had a significantly higher level of oxalates
than those stored at either 22o - 26oC or 32oC. Level of phenols was significantly
lower for samples stored at 32oC compared to those stored at 18oC and 22o - 26oC.
This indicates that the higher the storage temperature the lower the retention rate of
oxalates and phenols.
There were apparent losses in the levels of nitrates, oxalates and phenols in samples
packaged in Kraft paper as compared to those in polyethylene, but the differences
were not significant.
DISCUSSION
The lower levels of nitrates in fermented-, acidified- and control-dried cowpea leaves
than in the raw leaves indicate that much of the nitrate leached into the blanching
water. Leaching of nitrates has been reported [20, 21, 22]. Reduction in nitrate
concentration represents added value for vegetable products rich in carotenoids,
vitamin C and E, selenium, dietary fiber, plant sterols and so on [23]. Blanching,
fermentation, acidification and dehydration resulted in minimal reduction of oxalates
and phenols in the three samples. It has been reported that oxalates and phenols could
change in form during food processing. However, the methods used for their
determination in this study could not differentiate these forms; hence their levels did
not change significantly with the treatments. Another researcher, when working with
fermented Uji (a traditional porridge consumed in Kenya, made out of maize, millet
and sorghum) reported that drum-drying directly, or in combination with fermentation
with or without boiling, did not affect the content of phenols [24]. It has also been
reported that fermentation, dehydration or storage of noni (Morinda citrifolia L.) fresh
juice resulted in minimal reduction of total phenols [25]. However, domestic
processing such as cooking in boiling water, seems to have a dramatic effect on
phenolic content on edible vegetables [26]. High levels of oxalate can be reduced or
eliminated by cooking, especially boiling [27, 28]. Unfortunately, in this study the
contents of oxalates and phenols of the cooked vegetables were not determined.
Generally, oxalates and phenols are easily vaporized organic compounds. Possibly
low storage temperature (18oC) hindered the vaporization of both oxalates and
phenols compared to the higher temperatures of 22o – 26o C and 32oC [29]. The
apparent lower levels in samples stored in Kraft paper could be due to vaporization
also, as opposed to those in polyethylene, which is impermeable.
It is, therefore, concluded that blanching, fermentation, solar-dehydration and storage
of cowpea leaf vegetables results in a more valuable food product due to the reduction
of anti-nutrients. This reduction effect is significant in the long run, since such
vegetables form the bulk of foods consumed by rural communities. The increased
Volume 10 No. 11
November 2010
4407
acceptability of the fermented-dried vegetables, as demonstrated in this study would
assist rural communities in providing a better foodstuff with lower levels of anti-
nutrients, thus alleviating micronutrient malnutrition. This novel technology;
fermentation followed by solar-drying, would ensure long-term storage and thus help
deal with issues of seasonality and increase food security, especially during the dry
season. It is, therefore, recommended:
1. Transferring this technology, which is cheap and effective, to local
communities and women groups to preserve and improve seasonal vegetables
like cowpeas.
2. Promoting increased acceptability and consumption of fermented and
dehydrated vegetables among rural communities.
Volume 10 No. 11
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Table 1: Levels of nitrates, oxalates and phenols in raw, fermented-, acidified-
and control-dried cowpea leaves expressed in mg/100 g edible portion
on dry matter basisa
Sample Nitrates Oxalates Phenols
Raw 771 ± 36a 1889 ± 98 a 2783 ± 88 a
Fermented- dried 217 ± 27b 1679 ± 84 a 1992 ± 115 a
Acidified -dried 166 ± 13b 1859 ± 67 a 2119 ± 89 a
Control - dried 352 ± 34b 1830 ± 103 a 1959 ± 96 a
L. s. d. 376.1 536.2 871.1
a Mean ± Standard Deviation (n = 4)
Means within columns superscripted by different letters are significantly different at
(P < 0.05)
Table 2: Effect of fermentation and acidification on the nitrates, oxalates and
phenols during storage for three months (mg/100 g solids)
Samples Nitrates Oxalates Phenols
Fermented-dried 96.2b 729.5 a 1438b
Acidified-dried 205.3
a
847.0
a
1712
a
Control- dried 227.3
a
819.7
a
1485
b
L. s. d. 51.2 276.5 167.6
Means within columns superscripted by different letters are significantly different at
(P < 0.05)
Volume 10 No. 11
November 2010
4409
Table 3: Effect of storage temperature on the nitrates, oxalates and phenols
during storage for 3 months (mg/100 g solids)
Storage Temperature Nitrates Oxalates Phenols
18oC 161.4 a 1035 a 1616 a
22
o
– 26
o
C 174.2
a
702
b
1596
a
32
o
C 193.2
a
659
b
1424
b
L. s. d. 51.5 276.5 167.6
Means within columns superscripted by different letters are significantly different at
(P < 0.05)
Volume 10 No. 11
November 2010
4410
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