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Physicochemical and in vitro antimicrobial activity of the oils and soap of the seed and peel of Citrus sinensis

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Citrus sinensis seed and peel oils were extracted by solvent extraction using n-hexane, after air drying and grinding. Soaps were formed by saponification methods. Fatty acid composition of the oil samples were analyzed using Gas Chromatograph-Flame Ionization Detector (GC-FID). Physicochemical properties of the oils and soaps were determined following standard methods. Antimicrobial activities were assessed by the agar disc and hole-in plate methods. The seed and peel oil yield were 38 and 30%, respectively and the colors were golden yellow and brownish-yellow, respectively. Physicochemical properties of the oil samples determined were: refractive index (RI): 1.46 and 1.47, smoke point: 140 and 149, flash point: 150 and 160, pH: 5.2 and 4.2, acid value (AV): 23.6 and 25.1 mgKOH/g, free fatty acid (FFA): 11.86% as oleic acid and 12.61% as oleic acid, iodine value (IV): 78.83I 2 g/100 g and 120.10I 2 g/100 g, peroxide value (PV): 18.00 mgKOH/g and 5.40 mgKOH/g, saponification value (SV): 222.58 and 41.25 mgKOH/g, ester value (EV): 178.24 and 28.96 mgKOH/g for the seed and the peel oil respectively. Inhibitory antimicrobial activities were assessed for the two oils and the soap produced at concentrations of 40 mg/ml and below, against most of the gram positive and gram negative bacteria as well as the two candida strains, screened as compared with streptomycin (1 mg/L) and acriflavin (6.3 mg/ml) standard controls. Seed oil demonstrated better activities than the peel oil with growth inhibitions obtained against Staphylococcus aureus and Candida albicans at a concentration as low as 2.5 mg/ml. This study has shown that the results obtained for the physicochemical and antimicrobial properties of the oils provide a synergy for the oil samples as suitable raw materials for the cosmetic and pharmaceutical industries.
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Vol. 10(8), pp. 245-253, 28 February, 2016
DOI: 10.5897/AJMR2015.7797
Article Number: 47718C457467
ISSN 1996-0808
Copyright © 2016
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Physicochemical and in vitro antimicrobial activity of
the oils and soap of the seed and peel of
Citrus sinensis
Olabanji I. O.1*, Ajayi S. O.1, Akinkunmi E. O.2, Kilanko O.1 and Adefemi G. O.1
1Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.
2Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.
Received 6 October, 2015; Accepted 9 February, 2016
Citrus sinensis seed and peel oils were extracted by solvent extraction using n-hexane, after air drying
and grinding. Soaps were formed by saponification methods. Fatty acid composition of the oil samples
were analyzed using Gas Chromatograph-Flame Ionization Detector (GC-FID). Physicochemical
properties of the oils and soaps were determined following standard methods. Antimicrobial activities
were assessed by the agar disc and hole-in plate methods. The seed and peel oil yield were 38 and 30%,
respectively and the colors were golden yellow and brownish-yellow, respectively. Physicochemical
properties of the oil samples determined were: refractive index (RI): 1.46 and 1.47, smoke point: 140 and
149, flash point: 150 and 160, pH: 5.2 and 4.2, acid value (AV): 23.6 and 25.1 mgKOH/g, free fatty acid
(FFA): 11.86% as oleic acid and 12.61% as oleic acid, iodine value (IV): 78.83I2 g/100 g and 120.10I2 g/100
g, peroxide value (PV): 18.00 mgKOH/g and 5.40 mgKOH/g, saponification value (SV): 222.58 and 41.25
mgKOH/g, ester value (EV): 178.24 and 28.96 mgKOH/g for the seed and the peel oil respectively.
Inhibitory antimicrobial activities were assessed for the two oils and the soap produced at
concentrations of 40 mg/ml and below, against most of the gram positive and gram negative bacteria as
well as the two candida strains, screened as compared with streptomycin (1 mg/L) and acriflavin (6.3
mg/ml) standard controls. Seed oil demonstrated better activities than the peel oil with growth
inhibitions obtained against Staphylococcus aureus and Candida albicans at a concentration as low as
2.5 mg/ml. This study has shown that the results obtained for the physicochemical and antimicrobial
properties of the oils provide a synergy for the oil samples as suitable raw materials for the cosmetic
and pharmaceutical industries.
Key words: Physicochemical properties, antimicrobial activity, soap, seed oil, peel oil.
INTRODUCTION
Citrus sinensis (sweet orange) is one of the natural staple
food of man, containing essential nutrients in adequate proportion. The nutritional and medicinal values of the
fruit juice has made it essential and important part of
*Corresponding author. E-mail: ioolabanji@yahoo.com
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
246 Afr. J. Microbiol. Res.
human diet for ages (Okwu and Emenike, 2006; Ezejiofor
et al., 2011). Generally, citrus are excellent sources of
minerals, vitamins and enzymes. They have been
reported to be free from fat and cholesterol, but contain
important mineral elements such as potassium, calcium,
phosphorus, magnesium and silicon (Assa et al., 2013).
They are easily digested and bring about a cleansing
effect on the blood and the digestive tract. Orange fruits
have been discovered to have anti-scurvy property
(Rapisararda et al., 1999). Furthermore, they are rich in
vitamin C, folic acid and fiber; these contribute to the
prevention of degenerative processes, particularly
reducing the incidence and mortality rate of cancer as
well as cardio- and cerebro-vascular diseases
(Rapisararda et al., 1999; Cushnie et al., 2005; Pultrini et
al., 2006).
C. sinensis belongs to the race var. sinensis, of the
family Rutaceae. It is an hybrid between Pomelo (C.
maxima) and Mandarin (C. reticulata) originating from
Southeast Asia. The fruit size varies with cultivar and
crop load, but most often measures between 2.5 to 4 .0
inches in diameter (Manthey, 2004). The shape of the
fruit is spherical to oblong, with a peel thickness between
that of grape fruit and tangerine, and is either smooth or
roughly pebbly (Hilditch et al., 1950). It is usually very
closely adhered to the flesh of the fruit. Its colour tints
from green to light orange, depending on the cultivar. The
presence and amount of seed depends also on cultivar,
starting from 15 to 25 seeds per fruit (Nwobi et al., 2006).
Of all the citrus fruits, C. sinensis is the commonest in the
forest zone of Western Nigeria, Middle Belt, Eastern and
some part of South-south Nigeria (Odbanjo and
Sangodoyin, 2002).
The yield of orange juice is about half of the fruit weight
thereby generating a very high amount of waste annually
(Bovili, 1996). Citrus waste as huge as 36 metric tons are
produced annually with Florida citrus industry generating
3.5 to 5 tons, used and sold as feed stock for cattle, and
Nigeria generating about 0.3 million tons with potential to
generate more annually (Ezejiofor et al., 2011). These
agro wastes are common in Nigeria along major roads
where retailers peel and sell to motorists and others. The
wastes in market places constitute menace, causing
environmental pollution.
Citrus fruit peels are also known to have flavonoids, an
anti-oxidant (Bocco et al., 1998; Cushnie et al., 2005;
Ghasemi et al., 2009). Essential oil had been generated
in sweet orange and grape fruit (C. paradisi) peels
(Ezejiofor et al., 2011; Okunowo et al., 2013) and the
antimicrobial activities of grape peel oil had been
documented (Okunowo et al., 2013). Essential oils in
plant products have tremendous applications in food,
cosmetic and aromatherapy (Ramadan et al., 1996;
Haddouchi et al., 2013; Narmadha et al., 2013). Research
in medicinal chemistry have also shown that screening
plant products for antimicrobial activities have led to
detection and development of new potential anti-infective
agents (Ordonez et al., 2003; Arias et al., 2004; Rasool et
al., 2008). The peel of citrus fruits is a rich source of
flavones and many polymethoxylated flavones which are
very rare in other plants (Ahmed et al., 2006). The
antimicrobial abilities of essential oils from citrus plants
have shown to be of particular interest for applications
within the food industries (Caccioni et al., 1998).
In this study, the physicochemical properties and fatty
acid compositions of the fixed oil from the seeds and
peels of sweet orange were determined. Alkali generated
from the peel and seed oil were used to prepare soaps.
The antimicrobial properties of these oils and the soap
were also determined with a view to investigate their
suitability as possible alternative to the orthodox
antibacterial soaps. The results of this investigation is
expected to contribute to information on the usefulness of
C. sinensis in the cosmetic industries for the health
benefit of man and to reduce the menace of pollution
caused by the peel wastes in the environment.
MATERIALS AND METHODS
Collection and preparation of sample
C. sinensis were collected mainly from Oje market in Ibadan, Oyo
State, Nigeria (Specimen ID: 006653. Herbarium: PTBG). Its seeds
and peel were manually removed and were then air dried to remove
the moisture content. The dried seeds and peel were then grinded
to particles with the aid of an electric grinding machine.
About 1430 and 2350 g of the ground C. sinensis seed and peel
were weighed separately and were transferred into a porous
thimble and kept in the Soxhlet apparatus for extraction. Anti-
bumping granule was dropped into the flask to prevent the build of
pressure in the flask and n-hexane was added as the extracting
solvent. The oil was recovered from the mixture by evaporating the
residual extracting solvent using a rotary evaporator. The weight of
oil was noted (Soxhlet, 1879, Laurence et al., 2012).
After the extraction, the oil was transferred into a weighed round
bottom flask. The weight of the oil was determined by weighing the
oil and the flask and subtracting the weight of empty flask. The
percentage yield was determined.
Physical properties of the oils
The specific gravity of the seed and peel oil were determined by
measuring 10 mL of the oil samples into a pre-weighed measuring
cylinder. The values obtained were used to determine the specific
density of the oil. The pH of the oils were determined using Hannah
instruments, pH 210 Microprocessor pH meter while the refractive
index was determined at room temperature using the Abbey
refractometer at the Department of Pharmaceutical Chemistry,
Obafemi Awolowo University, Ile-Ife, Nigeria.
Other physical parameters such as flash and smoke points, cloud
and pour points and viscosity test were carried out using ASTM D56
(2001), TCWI (2009) and ASTM D445 (1965).
Chemical properties of the oils
The chemical properties such as the acid value (AV), free fatty acid
(FFA), Iodine value (IV), saponification value (SV) and peroxide value
(PV) of the seed and peel oil were determined by standard method
of AOAC (1990).
Determination of fatty acid composition
Fatty Acid composition of the oil samples were analyzed using
PERKIN Elmer Clarus 500 Gas Chromatograph employing the
following conditions: capillary column (RT-2560, 50 m x 0.25 mm
ID, 0.25 micron dry film); Nitrogen was used as a carrier gas, a
flame ionization detector and a sample volume of 1.0 L was
employed. The temperature programming of the instrument: Initial
temperature was 50°C held for 5 min, with an increase of 4/min to
190, then 0.8/min to 212, then 0.4°C /min to 220. The total GC-FID
running time was 85.49 and 78.90 min for the orange peel oil and
orange seed oil respectively.
Preparation of orange peel ash
The oranges were peeled and the peels were washed with double
distilled water and dried in an oven at (105°C ± 2) for two days to
constant weight. The dried peels were ashed in a porcelain crucible
placed in a Gallenkamp muffle furnace for 6 h by stepwise increase
of the temperature up to 500°C. The ashed samples were
homogenized in porcelain mortar and pestle and sieved. Sixty (60)
g of the sample were weighed into poly ethylene buckets of 2 L
capacities and one liter of water was added (Onyegbado et al.,
2002; Olabanji et al., 2012). The buckets were covered to prevent
contamination and extractions were done for 24 h. The extracts
were carefully decanted and double distilled water were added in
ratios of 1:4 of sample to double distilled water and were analyzed
by atomic absorption spectrophotometer (AAS) Buck Model 205 at
the Center for Energy Research Development, Obafemi Awolowo
University, Ile- Ife. These extracts were alkaline to litmus paper and
methyl orange.
Determination of molarity of orange peel ash alkali
Primary standard (Na2CO3) of known molarity was prepared and
used to standardize the acid (HCl) which was titrated against the
derived alkali using methyl orange indicator to determine its
molarity.
Saponification reaction using the ash-extracts
Two hundred milliliter of the ashed peel extract was concentrated to
50% by heating in a beaker (Babayemi et al., 2011); excess of
alkali is usually recommended in order to ensure complete
saponification of the oil/fat and to retain the antibacterial effect of
the alkalis (Kirk et al., 1954). The concentrated extract was heated
to 60 to 70°C and 15 g of oil was gradually charged into the pot.
The temperature was maintained at 70°C and 5 ml of double
distilled water was added intermittently with continuous stirring until
the mixture was semi solid and creamy in color, 10 ml of brine was
charged into the beaker content and the soap was homogenized.
The soap was scooped from the upper layer when the content of
the beaker had cooled and the lye discarded. The soap was
washed by pouring water on it.
Analysis of soap produced
Determination of total fatty matter (TFM)
The TFM was determined by the petroleum spirit extraction method.
Soap (1 g) was dissolved in 10 ml of warm water and transferred to
Olabanji et al. 247
a separating funnel. Two drops of methyl orange indicator were
added, followed by 4N H2SO4 until the indicator color changed from
orange to pink. Petroleum spirit 1mL was added and the separating
funnel shaken vigorously for 30 s. The solution was then allowed to
settle for a few minutes until the fatty acid liberated from the soap
formed a clear layer on top. The soap was skimmed off, washed
with distilled water and dried to constant weight in an oven at 60°C.
The percent total fatty matter was determined from the weight
obtained for the fat and the soap.
Determination of total alkali
The total alkali was determined by titrating excess acid contained in
the aqueous phase with standard volumetric NaOH solution. Five
millilitre of ethanol was added to 1g of finished soap after which 0.5
ml of 1N H2SO4 solution was added to the mixture and heated till
the soap sample dissolved. Test solution was titrated against 1 N
NaOH using phenolphthalein as indicator. The total alkali was
obtained following AOAC (1990).
Foamability test
About 0.5 g of the soap was added to a 100 ml standard flask
containing 100 ml of double distilled water. The mixture was shaken
vigorously two minutes to generate foams. The flask was allowed to
stand for 10 min. The height of the foam in the solution was noted.
Antimicrobial activity assays
Test organisms
Microorganisms used include reference and clinical isolates
comprising of Gram positive and Gram negative bacteria and fungi
strains. These include Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 27853, Pseudomonas fluorescens (clinical
strains), Shigella flexinerii (clinical strain), Klebsiella pneumonia
(clinical strain), Staphylococcus aureus ATCC 29213 and Bacillus
subtilis NCIB 3610. Candida albicans ATCC 24433 and Candida
pseudotropicalis NCYC 6 were the fungi strains used. The strains
were from stocks of culture collections maintained in the
Pharmaceutical Microbiology Laboratory of the Department of
Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University
where the experiments were performed.
Agar diffusion tests: Disc diffusion and cup plate methods
The disc diffusion test was used for the pure oils and the soaps
while the cup plate test was used for their dilutions. The oil samples
and their soap preparations were dissolved in MeOH/H2O to give
varying concentration of 2.5, 5.0, 10.0, 20.0 and 40.0 mg/ml.
Surface plating of the organisms were done for the 20 ml oven
dried Mueller Hinton Agar used for the overnight grown bacteria
and the Sabouraud Dextrose Agar used for the fungi strains. For
the dilutions, holes of diameter 9 mm were made in the agar plates
using a sterile metal cup-borer. Two drops of each dilution and
control were put in each hole under aseptic condition, kept at room
temperature for 1 h to allow the agents to diffuse into the agar
medium and incubated accordingly. For the pure oil and soap, each
of these were used to soak sterile 6 mm Whatman paper discs and
subsequently placed on the agar plates, allowed for diffusion and
incubated. Streptomycin (1mg/ml) and acriflavine (6.3 mg/ml) were
used as positive controls for bacteria and fungi respectively.
MeOH/H2O and Tween 80 were the negative controls. The plates
were incubated at 37°C for 24 h for the bacterial strains and at 25°C
248 Afr. J. Microbiol. Res.
Figure 1. Seed oil and peel oil.
for 72 h for the fungal strains. Antimicrobial activity was evaluated
by noting the zone of inhibition against the test organisms.
RESULTS AND DISCUSSION
The percent yield of the oils was 38 and 30% for seed oil
and peel oil, respectively. Using same extraction process
but different solvent Nwobi et al. (2006) got 36% yield for
the orange seed oil, this is close to the value in this study.
The oil of orange seed and peel showed acidic pH
values (4.2 and 5.2). These values were however higher
than 3.69 reported by Nwobi et al. (2006) for the peel oil.
The pH values indicated that the seed oil is more acidic
than the peel oil probably due to presence of more fatty
acids in the seed oil.
The seed oil has a golden-yellowish color (Figure 1);
similar result was obtained by Nwobi et al. (2006) while
the peel oil has a brownish-yellow color, similar to the
yellow color obtained in Okunowo et al. (2013) grape
peels oil. The yellow color may be an indication of
carotenoids, a fat, soluble in humans due to the presence
of long unsaturated aliphatic chains as in some fatty
acids. Carotenoids are known as provitamin A. They act
as precursors to the production of vitamin A in the body
which performs several biological functions within the
body. They also act as antioxidants (Sommer and Vyas,
2012).
Refractive Index decreased with unsaturation and
molecular weight of the fatty acids. The refractive index of
the seed oil and peel oil was 1.46 and 1.47, respectively.
This corroborates the findings of Nwobi et al. (2006) and
Ezejiofo et al. (2011). This indicates that the seed oil,
compared with the peel oil, has a lower unsaturation and
a lower molecular weight of fatty acids. The lower
molecular weight of fatty acid is suggestive of its higher
saponification value since saponification value is
inversely proportional to the mean molecular weight of
fatty acids (Dimberu et al., 2011). The smoke point, flash
point and free fatty acid content of the oils have a linear
relationship. The higher the free fatty acid content of an
oil, the lower the smoke point. The smoke point and flash
point of the seed oil were 140 and 150°C, respectively
while that of the peel oil were 149 and 160°C,
respectively. Nwobi et al. (2006) obtained 149°C in
orange seed oil which is still within the values found in
seed and peel oils of this study.
The seed oil has a specific density of 0.997 g/cm3 while
the peel oil has a value of 0.788 g/cm3. The value
obtained from Ezejiofor et al. (2011) study lies within the
range of density of the oils in this study. Density of seed
oils depends on their fatty acid composition, minor
components and temperature (Table 1).
Acid value accounted for the presence of free fatty
acids in the oils as an indicator of the presence and
extent of hydrolysis of lipolytic enzymes and oxidation
and it is used as an indicator of edibility of an oil. The
values indicated that the oils were non edible because it
was above the limit of 10 mg KOH/g of oil and found to
be unsuitable for dietary purposes (Barkatullah et al.,
2012) and 0.6 mg KOH/g FAO/WHO (1993), as the peel
oil contain higher fatty acid contents.
The free fatty acid content in seed oil which is 11.86%
(as oleic acid) is lower than that of peel oil which is
12.61% (as oleic acid). This indicates that the oils could
readily react with metal salt to generate soaps since the
FFA were far above the 2.5 and 1.376% FAO/WHO
recommended for coconut and palm oil respectively
(FAO/WHO, 1993). High FFA nullified their edibility.
Peroxide value serves as a common indicator of lipid
oxidation. Orange seed oil has a peroxide value of 18.00
millieq/kg while the peel oil has a value of 5.40 millieq/kg.
This indicates that the seed oil has undergone primary
oxidation than the peel oil since peroxide value gives a
measure of the extent to which an oil sample has
undergone primary oxidation. The peroxide value of the
peel oil is within the acceptable range of 10.00millieq/kg
FAO/WHO (FAO/WHO, 1993) while that of seed oil was
above indicating that lipid oxidation had occurred.
The saponification value of the seed oil is found to be
222.58 mg KOH/g while it is 41.25 mg KOH/g for the peel
oil. The higher saponification value of the seed oil shows
the presence of lower molecular weight fatty acids in the
oil and it may therefore be regarded as more edible than
the peel oil.
The Iodine value of the seed oil is 78.00 gI2/100 g
which is lower than that of the orange peel oil which is
120.10 gI2/100 g indicating that orange peel oil is rich in
unsaturated fatty acid (70.05%). This implies that orange
seed oil has a lower amount of double bond (59.76%
unsaturated fatty acid) thus lowering the susceptibility of
such oil to oxidative rancidity. Triglyceride oils are divided
into three groups depending on their iodine values:
drying, semi-drying and non-drying oils. The iodine value
of a drying oil is higher than 130. This value is between90
and 130 for semi-drying oils. If the iodine value is smaller
than 90, oil is called non-drying oil (Guner et al., 2006).
This classifies orange seed oil as a non-drying oil and
Olabanji et al. 249
Table 1. Physico-chemical parameters of the oil samples.
Variable
Orange peel oil
pH
5.2
Colour
Brownish -yellow
Percentage yield
30%
Specific density
0.778 g/cm3
Refractive Index
1.47
Smoke point
149°C
Flash point
160°C
Cloud point
16°C
Pour point
10 °C
Viscosity 100°C
0.9622cst
Viscosity 40°C
1.9766cst
Acid value
25.1 mgKOH/g
Peroxide value
5.40 mgKOH/g
Free fatty acid
12.61% as oleic acid
Saponification value
41.25 mgKOH/g
Ester value
28.96 mgKOH/g
Iodine value
120.10 I2g/100 g
Table 2. Fatty acid composition of the seed oil.
Saturated fatty acid (relative
abundance, %)
Monounsaturated fatty acid (relative
abundance, %)
Polyunsaturated fatty acid (relative
abundance, %)
Palmitic acid C16:0 (31.1)
Palmitoleic acid C16:1 (0.34)
Linoleic acid C18:2n6c (35.13)
Stearic acid C18:0 (4.97)
Oleic acid C18:1n9c (24.95)
Dihomo-linolenic acid C20:3n6 (0.04)
Arachidic acid C20:0 (3.68)
-
-
Heneicosylic acid C21:0 (0.32)
-
-
Tricosylic acid C23:0 (0.16)
-
-
Total = 40.23
Total = 25.29
Total = 35.17
the peel oil as a semi-drying oil. The peel oil will be more
applicable in varnishes and paint industry while the seed
oil will be useful in soap industry. The seed oil contains
59.76% unsaturated fatty acid, peel oil has 70.05%
unsaturated fatty acid, 24.59% mono-unsaturated fatty
acid and 35.17% polyunsaturated fatty acid (Table 2)
while the peel oil has 70.05% unsaturated fatty acid,
31.83% mono-unsaturated fatty acid and 38.22%
polyunsaturated fatty acid (Table 3). This implies that the
peel oil is more unsaturated than the seed oil thereby
confirming the reason for its higher iodine value, smoke
point and higher refractive index. This also predicts the
more oxidation stability of the seed oil and its possibility
of serving as edible oil.
From the fatty acid profiles represented in Tables 2 and
3), it indicates that the peel oil has a high proportion of
fatty acids with high molecular weight and this explains its
low saponification value (Table 1). This is because they
have relatively fewer numbers of carboxylic functional
groups per unit mass of the oil. Thus it is regarded non-
edible and may not be suitable for soap making. The
higher percentage of unsaturation (mono and polyun-
saturation) in peel oil makes it more reactive and useful in
industrial application such as surface coating applications
for example, paints, vanishes, printing and writing inks.
The seed oil contains one out of the two families of
essential fatty acid which is linoleic acid (omega-6) and it
is the most abundant unsaturated fatty acid with a relative
abundance of 35.13%. The peel oil contains the two
families of essential fatty acid which is linoleic acid
(omega-6) 18.63% and -linoleic acid 3.62%. Palmitoleic
acid (omega-7) is the most abundant unsaturated fatty
acid with a relative abundance of 22.78% in the peel oil.
The peroxide value of the orange seed oil exceeds the
permitted maximum peroxide value for edible oil, which is
10 mequivalent of oxygen/kg of the oil (FAO/WHO, 1993)
and its high acid value, coupled with high percentage of
saturated fatty acid indicate that the orange seed oil may
not be good for consumption but useful in industrial
applications such as the cosmetics industry which
includes soap making, perfumes and unguents.
The metal analysis (Table 4) of the peel ash showed
metals of varying concentrations. Although the soap
produced from the ash-derived alkalis was softer than bar
250 Afr. J. Microbiol. Res.
Table 3. Fatty acid composition of peel oil.
Saturated fatty acid
Relative
abundance %
Monounsaturated fatty
acid
Relative
abundance %
Polyunsaturated fatty acid
Relative
abundance %
Undecylic acid C11:0
10.83
Palmitoleic acid C16:1
22.78
Linoleic acid C18:2n6c
18.63
Lauric acid C12:0
0.88
Oleic acid C18:1n9c
9.05
-Linolenic acid
3.62
Palmitic acid C16:0
0.75
-
-
-
-
Stearic acid C18:0
2.61
-
-
Dihomo-linolenic acid C20:3n6
7.46
Eicosanoic acid C20:0
1.69
-
-
Arachidonic acid C20:4n6
1.63
Behenic acid C22:0
0.48
-
-
Cis-13,16- docosadienoic acid C22:2
6.88
Tricosylic acid C23:0
8.41
-
-
-
-
Lignoceric acid C24:0
4.32
-
-
-
-
Total
29.97
-
31.83
-
38.22
Table 4. Concentrations and percentage compositions of ash
derived alkali from peels.
Elements
Concentration (ppm)
Composition of
elements (%)
K
151.97
68.77
Ca
39.7
17.96
Na
24.7
11.17
Mg
4.62
2.08
Total
220.97
Figure 2. Soap from ashed peel alkali and seed oil.
Table 5. Physicochemical analysis of the soap produced.
Colour of soap
Yellow
pH
9.79
Total fatty matter
41.0%
Total alkali
4.65%
Foam height
7 cm3
Solubility in water
Soluble
Texture
Soft
Figure 3. Soap foamability test.
soap in the market it could still be described as soft solid
soap (Figure 2). This is expected as the percentage
concentrations of K, Ca, Na, Mg in the peel were 68.77,
39.7, 24.7 and 4.62% respectively (Table 4) of the total
metal ions analyzed in the sample. The solubility of soap
in water increased with the size of the monovalent cation
(base); an increase in the size of a divalent cation (Mg,
Ca) results in a decrease in the foamability. Potassium
soaps are more soluble in water than sodium soaps;
hence, the soap produced was soluble and lather very
well (Figure 3, Table 5). Potassium soaps in concentrated
form are called soft/liquid soap. Potassium soaps require
less water to liquefy because of their softness and
greater solubility; thus can contain more cleaning agent
than liquefied sodium soap and can be used as
shampoos, shaving creams, cleaning of dirty floors and
cooking utensils, in emulsion polymerization processes
used in rubber and plastic industries and in such other
Olabanji et al. 251
Table 6. In-vitro antimicrobial activity of the oil and soap of the seed and peel of C. cinensis.
Agent
Organisms
Concentration (mg/ml)
Diameter of zone of inhibition (mm)**
Peel oil
P. mirabilis (clinical strain)
Pure oil
16.0
K. pneumonia (clinical)
Pure oil
2.0
P. fluorescence (clinical strain)
40
4.0
S. aureus (ATCC 29213)
20
6.0
40
8.0
Shigella flexinerii (clinical)
40
5.0
C. albicans (ATCC 24433)
20
4.0
40
7.0
C. pseudotropicalis (NCYC 6)
40
10.0
Seed oil
E. coli ATCC 25922
Pure oil
6.0
40
2.0
B. subtilis (NCIB 3610)
Pure oil
3.0
20
2.0
40
8.0
Proteus mirabilis (clinical strain)
Pure oil
14.0
K. pneumonia (clinical strain)
Pure oil
5.0
40
4.0
Ps. aeruginosa ATCC 27853
10
2.0
20
3.0
40
5.0
Ps. fluorescence (clinical strain)
40
4.0
S. aureus (ATCC 29213)
2.5-5.0
6.0
10
7.0
20
8.0
40
14.0
Shigella flexineri (clinical strain)
20
4.0
40
11.0
C. albicans (ATCC 24433)
2.5
2.0
5.0-10.0
4.0
20
6.0
40
10.0
C. pseudotropicalis (NCYC 6)
10
4.0
20-40
10.0
Seed oil soap
Proteus mirabilis (clinical strain)
Pure Soap
4.0
Klebsiella pneumonia (clinical strain)
Pure Soap
9.0
Diameter of zone of inhibition of streptomycin (1 mg/ml) for each organism was: E. coli ATCC 25922, 14.0 mm; P. aeruginosa ATCC 27853, 14.0
mm; P. fluorescence (clinical strain), 14.0 mm; S. aureus ATCC 29213, 14.0 mm; B. subtilis NCIB 3610, 10.0 mm; K. pneumonia (clinical), 12.0
mm; S. flexinerii (clinical) 10.0 mm; P. mirabilis (clinical strain), 10.0 mm. Acriflavin (6.3 mg/ml) inhibition for the fungi was: C. albicans (ATCC
24433),18.0 mm, C. pseudotropicalis (NCYC 6), 21.0 mm. *The agents showed activities only against the organisms indicated. **Zone of inhibition
less cup size.
similar uses. The presence of 24.7% sodium out of the
total percent of the alkali increases the firmness of the
soap which ought to be liquid or semi-solid. Calcium is
the major ion that limits its foam ability because of 39.7%
composition.
The yellowness of the oil was considerably reduced by
bleaching, which gave the soap a cream colour.
Spectrophotometry analysis of the metallic ions present
in ashed samples solution (Table 4) showed that the
alkali consist of ions that are essential diet components
by contributing sodium, calcium, potassium and other
essential nutritional elements.
Results of antimicrobial evaluation show that the two oil
samples possess useful antimicrobial activities as anti-
bacterial and antifungal inhibitory activities were obtained
at concentrations of 40 mg/ml and below (Table 6). The
252 Afr. J. Microbiol. Res.
antimicrobial activities of grape fruit (C. paradisi) and
grape peel oil had earlier been documented (Okunowo et
al., 2013). Furthermore, the presence of metabolites with
documented antimicrobial effects such as alkaloids,
saponins, flavonoids, tannins and phenolic compounds in
C. sinensis peel extract has been reported (Bocco et al.,
1998; Hussain et al., 2015). Thus the antimicrobial
activities obtained in this study have known scientific
basis. The antimicrobial activities are broad spectrum
against a wide range of Gram positive and Gram
negative bacteria and the two candida strains, C.
albicans and C. pseudotropicalis, screened. These
organisms have been implicated in skin and mucous
membrane infections with reports of morbidity and
mortality (Mahmoud, 2001). Seed oil demonstrated better
activities than the peel oil indicating that antimicrobial
constituents are more concentrated in the seed oil.
Further studies are therefore needed to elucidate these
constituents and their contributions to the antimicrobial
effects. These results also indicate that free fatty acids,
obtained at a higher content in the peel oil compared with
the seed oil, do not contribute to the antimicrobial effects
of C. sinensis. Inhibition zones were obtained for the
seed oil against S. aureus and C. albicans at a
concentration as low as 2.5 mg/ml. In some cases the
activities of these oils were observed to be comparable to
that obtained for the standard antibacterial agent,
streptomycin, at the tested concentration. These cases
include inhibitory activities obtained for the pure peel and
seed oil against Proteus mirabilis (16 and 14 mm,
respectively) compared with that for streptomycin [1
mg/ml] which was 10 mm.
Seed oil at 40 mg/ml also demonstrated similar
inhibitory activity with streptomycin at 14 mm zone of
inhibition. The activity of the seed oil against P. aeruginosa
at a concentration as low as 10 mg/ml is especially
noteworthy as this organism is notorious for its intrinsic
resistance to most standard antibacterial agents. For the
soap, antimicrobial activities were obtained only for the
seed oil soap with activities demonstrated against P.
mirabilis and K. pneumonia at 4.0 and 9.0 mm zone of
inhibition respectively.
The antimicrobial activities of the pure seed oil and peel
oil showed its usefulness in cosmetic and pharmaceutical
industries in preparation of topical cream/gel against both
gram -positive and gram negative bacteria and fungi
infection. The activities of the seed oil soap further
strengthen the usefulness of the seed oil for potential use
in soap formulation against susceptible organisms. The
peel oil will also find good use as antimicrobial agent in
many infectious diseases especially against infections
caused by S. aureus. It also has great potential as
antifungal agents against the candida strains (Table 6).
Conclusions
From the physicochemical parameters and fatty acid
composition of C. sinensis seed and peel oil analyzed,
both oils are recommended for industrial applications,
specifically the cosmetics industry. High composition of
unsaturated fatty acid such as palmitoleic acid, linoleic
acid, cis-13, 16- docosadienoic acid, alpha-linoleic acid
and arachidonic acid in the peel oil makes it reactive and
to have a semi-drying property as confirmed by its iodine
value. Thereby making it suitable in the production of
paints, inks and vanishes.
The presence of fatty acids such as linoleic acid,
palmitoleic acid, oleic acid and other unsaturated fatty
acid in the seed oil could function as emollient and
thickening agents. They also serve as fragrance
ingredient and cleansing agents. Linoleic acid is an anti-
oxidant which could prevent ageing. Saturated fatty acids
such as palmitic acid, stearic acid and arachidic acid fulfill
the role of a fragrance ingredient, thickener or hardener
when the oil is used in soap making.
The broad spectrum activities of the seed oil against
strains of organisms responsible for many infectious
diseases together with the favourable physicochemical
properties obtained for this oil, which support its use in
cosmetic and soap making, are actually synergistic and
make this oil of tremendous potential for these industries.
This study has shown that C. sinensis seeds and peels
could be put to productive use in the cosmetic and
pharmaceutical industries rather than continuing to
constitute worrisome menace as environmental wastes
and pollutants.
Conflict of interests
The authors have not declared any conflict of interests.
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Citrus is a leading fruit crop of Pakistan. Among citrus cultivars, Kinnow is the most prominent and shares about 95% of total citrus production in Pakistan. The monopolized cultivation of Kinnow scion grafted over rough lemon needs a substituted rootstock for higher yield/return and for longer productive tree life. Trials for the selection of suitable rootstock for Kinnow mandarin under subtropical environmental conditions and highly alkaline rhizosphere were carried out in the experimental fruit orchard of the Institute of Horticultural Sciences, University of Agriculture, Faisalabad during 2003-2005. The studies revealed that Volkamer lemon, Brazilian sour orange and Citrumello 4475 were the reliable rootstocks for citriculture industry of the Punjab province.
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In the present investigation an attempt has been made to find out total free fatty acid and cholesterol content in some commercial edible oils in Ethiopia, Bahir Dar. Acid value, peroxide value, saponification value and cholesterol content were determined in a total of nine varieties of edible vegetable oils of which six are branded and three are non-branded. The analysis, performed using Liebermann-Burchard method, revealed varying levels of cholesterol content. Cholesterol was detected in seven of the vegetable oils while for two oil samples (Niger seed k-16 and Niger seed k-7) it was nil. Rapeseed branded vegetable oil has significantly highest cholesterol concentration (257.1 ± 0.42 mg/L) while branded palm oil has the least concentration (88.8 ± 0.85 mg/L) of cholesterol. Unlike the peroxide value, all the acid values and some saponification values show high values in comparison with the maximum permissibility level of codex standard for named vegetable oils (CODEX STAN210-1999). In conclusion, this study could be a guide line, to understand the quality of vegetable oils in Ethiopia, Bahir Dar. Consequently, these studies have been able to show that there is no cholesterol free oil in the market as opposed to what is claimed on the vegetable oil brand labels. Therefore, companies producing and marketing vegetable oils are enjoined to desist from misleading the public by labeling their products as "cholesterol free". They should indicate the amount of cholesterol present in the vegetable oil, no matter how small the quantity may be.