Characterization of mango kernel extracts for use as antibacterial and
bacteriostatic agents
Abstract:
Aims: The antimicrobial properties of four extracts obtained from Mexican mango seed
kernel: ethanol (EKE), Hexane-Ethanol (EKHE), water (EKW) and Hexane-water
(EKHW)) were investigated in terms of their composition, antibacterial activity and
stability, with a view to selecting the best performing extract as a natural preserving agent.
Methods and Results: All extracts were separated by reverse phase HPLC with 10-30%
acetonitrile linear gradient to characterize antimicrobial compounds. The FITR spectra of
the four kernel extracts showed the characteristic bands related to the presence of
polyphenolic compounds. Minimum inhibitory concentrations (MICs) of the EKE, EKHE,
EKHW, EKW were tested against 4 species, of 5 strains, containing food-borne pathogenic
bacteria. This MICS were determined using the agar dilution method. Kernel extract
spectra obtained using hexane-ethanol and hexane-water, did not show a substantial
difference compared to EKE, while the EKW spectra exhibited a larger percentage
polyphenolic compounds, in a more bioavailable form, and moreover this last extract is also
the simplest and cheapest extract to obtain. All these extracts show substantial antibacterial
action with inhibition rates of more than 80% both on gram-positive and gram-negative
bacteria. Antimicrobial activity was stable against heat (121C, 15 min), freezing (-20C,
16 h) and pH treatment (pH 3-9) normally used in food processing. Furthermore, these
extracts show better characteristics than more commonly used chemicals such as sodium
benzoate, which is effective only in an acidic environment with an DL50 in rats of 2gr/kg of
body weight, while the extracts reported here show a DL50 rate in rats of 100g/kg of body
weight in environments ranging from a pH of 3 to 9. Chemical analysis showed that the
EKW was composed of 11.1 % polyphenols and 77 % carbohydrate. There were two
fractions having antimicrobial activity; both peaks had maximum absorbance at 275 nm.
Conclusions: We can conclude that Mexican mango kernel extracts obtained possess a
strong antibacterial actino against both gram-positive and gram-negative bacteria. EKW
extract showed the best performance compared to the other extracts, since it showed
inhibiting percentages comparable to those of the other extracts, while the method for its
production is both the simplest and most economical.
Significance and Impact of the Study: These extracts show very little variation in
chemical composition, their toxicity level is very low, with a very simple and low-cost
variation in the EKW extract, and therefore EKW particularly can be used as a natural
preserving agent, derived from agricultural surplus material, thereby helping the
environment, satisfying customer demand for natural products and increasing the
profitability of a very important crop in many less developed nations.
Introduction
Mango (Mangifera indica) family Anacardiaceae trees are tropical fruit bearing plants of
Asia and Africa. However, the fruits are popular worldwide and are relished for the tasty
fleshy mesocarp ( Arogba 1997 & Bassam, 2004). For several decades, the seeds (shell plus
kernel) have been discarded as waste in Africa whereas in Asia, interest has mainly been on
the lipid component of the kernel because of its potential application in the confectionery
industry as a source of cocoa-butter substitute (Arogba 1997, Beuchat 1994, Puravankara
2000, Hara 1989, Arogba 2000). The composition of several varieties of mango kernel has
been reported. However, underutilization of the whole mango kernel could be partly due to
the limited knowledge of its toxicological properties but it has been reported (Soong &
Barlow, 2004) that toxicity was not evident in rats fed with diets containing 100 g/kg of
mango kernel crude fat. The consumption of 0.4 kg of the kernel or 0.8 kg of the processed
flour per 70 kg body weight is deemed safe. Based on the toxicological evaluations
conducted on the mango seed kernels in the past and the availability of huge amounts of
kernels, they seem promising as a possible source of safe antioxidants (Skerget et al., 2005,
Berardini et al., 2004). In recent years, there has been a dramatic increase in number of
reported poisoning outbreaks caused by food-borne pathogenic bacteria. In 1996, one of the
serious outbreaks caused by Escherichia coli O157:H7 occurred in Sakai, Japan.
Consequently, there has been considerable interest in preventing food contamination by
food-borne pathogens. Traditionally, various methods, such as heating, reducing water
activity, smoking, fermentation, and adding antimicrobial agents, have been used to prevent
spoilage of foods (Beuchat, 1994). The addition of antimicrobial agents has been a
particularly effective method for controlling microbial contamination.
Mexico is the word’s largest mango exporter and the fourth largest producer. Local
producers currently have a very low rate of return on their crops, as most of the profit
margin is obtained by the downstream trading activity, and therefore desperately need to
improve profitability by making use of the up to 50% surplus or low quality fruit, which
otherwise represents an environmental hazard that has to be resolved at a substantial further
cost. Threfore, there is an ample cheap supply of raw materials for this type of products, as
well as a growing market demand.
Unlike chemically synthesized antimicrobial agents, those from natural sources are
attractive to consumers. Thus, one area of research is the development of the new agents
from natural sources. Antimicrobial agents from spices and their essential oils food plants,
vegetable and fish oils, and bacteriocin, antimicrobial peptides produced by bacteria have
been reported. Parmar & Sharma (1986) reported that mango seed kernel enhanced
oxidative stability of fresh-type cheese, ghee.
Here, we report the polyphenolic content and IR characteristics and antimicrobial activity
of some extracts of Mexican mango seed kernel: ethanol kernel extract (EKE), hexaneethanol kernel extract (EKHE), hexane-water kernel extract (EKHW) and water kernel
extract (EKW) that exhibit antimicrobial properties against food-borne pathogenic bacteria.
MATERIALS AND METHODS
The hexane and ethanol used were reagent grade and obtained from Sigma (St. Louis,
MO, U.S.A.); The water used throughout was of 18.3 Micro-Siemens and obtained from an
Easy Pure instrument from Barnstead.
Fruit samples of Mangifera Indica used
The samples employed were collected from a collection of trees of the Tommy variety in
the State of Nayarit, Mexico during the months of June to August 2005 and processed soon
after.
Seed kernel extraction
The mango pulp was separated from the seed and the kernel from the seed, which in turn
was milled to a flour in a Retz milling machine at a 1.5 mm mean diameter.
Kernel powder analysis
An analysis of the kernel powder raw material was carried out, including the moisture
content of the original kernel and after drying, proteins, fats, ash, fiber and polyphenols.
EKE and EKW extract Preparation
Kernel flour (26g) were extracted using a homogenizer at 150 rpm for 5 min, 100ml de
ethanol (99.5%) were added, and extraction was carried out in a cold and dark place
overnight while shaking. The resulting ethanol extract solution was filtered through a filter
paper Watman No 4 to remove insoluble substances. Then the filtrate was concentrated to a
syrup under vacuum in a rotary evaporator. The residue, suspended in sterilized Milli Q
water, was used as the EKE for the following experiments. The EKW extract was prepared
in the same manner, except water was employed as the solvent.
EKHE and EKHW Extract Preparation
Kernel (26g) were extracted using a homogenizer at 150 rpm for 5 min, 100ml de hexane
(99.5%) were added, and extraction was carried out in a cold and dark place overnight
while shaking la solution was filtered and the solids were suspended in water during 18hr.
The resulting hexane-water extract solution was filtered through a filter paper (Watman No
4 to remove insoluble substances. Then the filtrate was dried to a solid under vacuum in a
rotary evaporator. The residue, suspended in sterilized Milli Q water, was used as the
EKHE for the following experiments. The EKHE extract was prepared in the same
manner, except hexane and then ethanol were employed as solvents.
EKE Extract Characterization
Folin–Ciocalteu reagent assay
The Folin–Ciocalteu reagent assay was used to determine the total phenolics content (see
also Seigler, 1986 & Turkmen et al., 2005). A total of 0.125g of the EKE extract are
dissolved in 250 ml of water, a sample of 1ml is then taken and diluted in a 1:100 ratio. An
aliquot of the samples (2ml) was mixed with 2 ml of Folin–Ciocalteu reagent previously
diluted with distilled water (1:100). The solution was allowed to stand at 25 ªC for 5 min
before adding 2 ml of 15% sodium carbonate solution in distilled water. The absorbance at
765 nm was read after initial mixing and up to 90 min until it reached a plateau. Gallic acid
was used as a standard curve. Total for the calibration polyphenol concentration was
measured using the method of Charrier (1992).
Total nitrogen content was determined by the Kjeldahl method according to AOAC
methods 955.04 (Association of Official Analytical Chemists [AOAC]1995) described by
Peterson, 1979. Total sugar content was measured by the phenol-H2SO4 method with
glucose as a standard Dubois, et al, 1956. Fatty acid and ash contents were determined by
the method of Southgate, 1971 and AOAC methods 942.05 (AOAC, 1995). All quantitative
analyses were performed in duplicate and the value was calculated as a mean.
Antimicrobial assay
In order to study the antibacterial effects of the extracts germane to this paper, the
cultures shown in Table 1 were employed. The method employed was a standard method
as specified in the Mexican pharmacopea methodology MGA0305 for measuring the
effectiveness of bacteriostatic and antibacterial agents.
Starting from a 24 hr. growth basic culture, each bacteria was inoculated into a Roux
bottle containing a modified brain/Heart infusion broth, and incubated at 35C for 24 hrs.
The bacterial cultures were then harvested with a peptid saline solution, the microbian
concentration of the resulting suspension was estimated using a plaque-counting method.
Extract samples of 0.0015gr, 0.005gr and 0.05gr of each extract were transferred into 20 ml
of sterile peptid saline solution, thus concentrations of 75, 250 y 2500 ppm were obtained.
The bacterial cultures were inoculated with the extracts at the abovementioned
concentrations, taking the initial microbian concentration prior to the addition of the
extracts as an control, complemented by a control growth culture where no extract was
added. All antimicrobial experiments were performed in duplicate and were run with
bacterial counts by the agar meted after zero, 24hr and 7 days in order to more reliably
obtain an determine the effectiveness of the extracts.
HPLC analysis of extracts
The EKE was dissolved in 10% (v/v) acetonitrile and separated by an HPLC (HPLC
system, model 1050, Hewlett Packard, Waldbronn, Germany) attached to a reversed-phase
ODS column (Agilent HP model 1100, U.S.A.). Linear gradient elution was performed
starting with 10% (v/v) acetonitrile in Milli Q water, and ending with 30% (v/v) acetonitrile
at a flow rate of 1 ml/min at room temperature. Eluates were detected at 280 nm and
fractionated. They were dried with a centrifugal evaporizer (model, ER120, Buchi Co.
Switzerland). Each fraction was dissolved in sterilized Mill Q water to analyze the antimicrobial activity. The antimicrobial activities were assayed as described above.
Effect of pH and temperature on stability of the extract
The pH stability was measured with three kinds of buffers: a 50 mM sodium acetate
buffer (pH 3.0-5.0), 50 mM sodium phosphate buffer (pH 6.0-7.0) and 50 mM tris-HCl
buffer (pH 8.0-9.0). The extracts were exposed to these buffers for half an hour. After
treatment, the extracts was diluted appropriately with PBS (-) and antimicrobial activity
was measured by the agar dilution method as described previously, using Bacillus cereus
IFO 14160 as an indicator strain. The heat stability was measured following corresponding
treatments for half an hour.
FTIR analysis
The EKE, EKHE, EKHW and EKW samples (6mg each), in duplicate, were mixed with
solid crystalline KBr (spectroscopic grade) and pressed into a 1-mm pellet. The FTIR
spectra were recorded in the absorbance mode at a resolution of 4 cm−1 with wave number
range 400–4000cm−1, using a Perkin-Elmer FT-IR system spectrum GX, Norwalk, CT,
U.S.A.).
Results
Chemical analysis of the extracts
The EKE, EKW, EKHW y EKHE formed intense blue-violet precipitation by qualitative
analysis with alkaline solution and ferrous sulphate, respectively, which indicated that the
extracts contained polyphenols. All the extracts formed two layers precipitation by the lead
acetate precipitation method; the white upper layer indicated tannins, while the yellow
bottom layer suggested flavones. They also produced hydrogen gas, characteristic of
flavones reacted with MgCl . The chemical composition of the extracts is shown in Table 2.
Chemical analysis for the extracts
Extract fractionation was performed by reverse phase HPLC to identify active
compounds. The results obtained were similar to those previously reported for EKE
(Kabuki et al. 2000, Puravankara et al. 2000). However, our Mexican kernel extracts show
a higher amount of polyphenols than previously reported, with EKHE having the highest
polyphenol content, followed by EKHW, EKW, and finally EKE. Carbohidrate content was
calculated on a difference basis.
Antimicrobial activity
The antimicrobial activity results of the EKE, EKW, EKHW and EKHE extracts against
5 strains (4 species) determined by the agar dilution method are shown in Tables 3 and 4.
All the extracts studied displayed substantial antimicrobial effectivenes of more than 80%,
both for gram-positive and gram-negative bacteria, which is higher than those previously
reported for instance by kabuki et al. 2000. Moreover, MICs for these extracts were under
250 ppm for gram-positive bacteria Bacillus subtilis y Bacillus cereus and for gramnegative bacteria Salmomella typhimorium, Pseudomonas aeruginosa the amount was only
75 ppm. Concentrations as high as 2500 ppm were also tested for all bacteria and the
results were in fact very similar to those shown in Table 4 for 250 y 75 ppm.
The EKE extract shows a lower MIC than that reported by Toshihide et. al. for a similar
extract, where for Bacillus subtilis 500 ppm are reported , E. Coli 2500 ppm, Salmomella
typhimorium 2500 ppm, said results showing a higher effectiveness against gram-positive
bacteria. Out of the extracts herein reported, the EKW extract shows strong activity against
both gram-positive and gram-negative bacteria, even though its polyphenol content is the
second lowest, its inhibition performance is comparable to EKHE although this extract has
50% more polyphenols. The remarkable performance of the KW extract can be explained
by the fact that EKW posseses a larger proportion of bioavailable polyphenols, as shown by
the 1700,1600,1550 y 1450 cm-1 bands in the IR spectrum of a lower intensity than those of
the other extracts, suggesting that the gallic acid is more readily available to interact.
pH and temperature stability
Table 5 shows heat and pH stability of the EKW. The extract antimicrobial activity as
a function of pH was stable at pH 3.0-9.0. All extracts showed teperature stability, being
stable when heated at 121C for 15 min, although activity was reduced to about 1/4 when
heated at 121C for 60 min., while being stable against freezing, as shown in Table 5.
FTIR analysis
The results for the EKW extract are shown in Figure 1 where we can see the
characteristic bands related to polyphenolic content. A band at 3383 cm –1 characteristic of
OH bonds and a weak peak at 2925 cm –1 corresponding to CH2, the intensity of this band
being related to the solvent employed to obtain the extract. The spectrum shows a band at
1032 cm-1 indicating the presence of carbohydrates, while the presence of polyphenols is
indicated by the 1240 and 1032 cm–1 bands. The bands observed at 1700,1600,1550 y 1450
cm-1 are characteristic of the elongation of aromatic rings in the gallic acid molecules.
Bands observed at 718-773 cm-1 indicate H-aromatic links. These results are also similar to
those previously reported by Prasanna, 2004. The results of the extracts obtained with
other solvents are consistent with those of the EKW extract, as shown in Figure 1.
Discussion
The study of the Mexican mango kernel extracts obtained here shows that their main
active ingredients are polyphenols, as shown by the HPLC research where 2 fractions with
antimicrobial activity were isolated, both with a peak absorbance at 275nm indicative of
polyphenols, many of which are known for their antimicrobial activity such as gallic acid.
All extracts showed a substantial effectiveness against both gram-positive and gramnegative bacteria, with the EKW extract being the best performing. Our MIC values were
very much lower than those previously reported for the same bacteria for the EKE extract,
which in the literature appears to be more effective against gram-positive bacteria unlike
our extract.
The results obtained for the EKE extract were compared to crude catechins in green tea
and crude theaflavins in brown tea, with the result that our extract has a higher antibacterial
effect as shown by the MICs values in Table 6, where they are lower for our extract than
for catequines and theaflavins (Sakanaka et al., 1997). The difference suggests that the
molecular structures of the polyphenols, as active components in the EKE, differ from
those in green or brown tea.
The antimicrobial peptide, nisin, produced by Lacto cocci lactis, has a similar
antimicrobial spectrum to our Mexican EKE extract. Nisin generally has a bacteriostatic
effect against only gram-positive bacteria. The extracts reported here however, show better
performance since they have a wider effective spectrum since they are effective against
both gram-positive and gram-negative bacteria.
The primary site of the action of nisin against vegetative cell is considered to be the
cytoplasmic membrane, because nisin interacts with the phospholipid of cytoplasmic
membranes and forms a nisin phospholipid complex. To argue the difference of
susceptibility of our EKE extract against gram-positive and gram-negative bacteria; the
mode of action must be studied in detail. More detailed characterization and biochemical
studies are being carried out in our laboratory.
Conclusion
Based on these results, we can conclude that the Mexican mango kernel extracts obtained
possess a strong antibacterial actino against both gram-positive and gram-negative bacteria.
The EKW extract showed the best performance compared to the other extracts, since it
showed inhibiting percentages comparable to those of the other extracts, due to its higher
polyphenol content and bioavailability, while the method for its production is both the
simplest and most economical.
Since these extracts are stable at cooking temperatures and remain active over a large
range of 3-9 pH, they can be utilized in a wide range of foodstuffs. These results also show
that EKW extract possesses better performance than current ingredients, such as sodium
benzoate, since this ingredient is only effective in acid conditions and has a very much
smaller recommended dose.
More generally, all the extracts herein described can be used as additives to markedly
improve the shelf life of many foodstuffs, particularly those that do not require heating such
as salads. These extracts also show very little variation in chemical composition, their
toxicity level is very low, with a very simple and low-cost variation in the EKW extract,
and therefore EKW particularly can be used as a natural preserving agent, derived from
agricultural surplus material, thereby helping the environment, satisfying customer demand
and increasing the profitability of a very important crop in many less developed nations.
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Table 1. Bacteria Strains
1.-Bacillus cereus (IFO-14160)
2.-Bacillus subtilis (IFO-137199)
3.-Escherichia coli (IFO-3301)
4.-Salmonella typhimurium (ATCC-13311)
5.-Pseudomona aeroginosa ATCC 27853)
Table 2 Extracts’ chemical composition
Extract
%Carbohydrates
%Total
Nitrogen
%Ashes
% Humidity
% fat
Polyphenols %
(dry weight)
EKE
78.7
7.0
0.9
5.6
7.8
8.8
EKW
77.0
6.8
1.2
10
5.0
11.1
EKHW
75.2
6.0
0.8
12
6.0
14.2
EKHE
74.6
6.0
0.9
12
6.5
16.4
Table 3 Antimicrobial activity and MICs of EKW, EKE, EKHE and EKHW
Bacterial strain
MIC ppm
% Reduction
EKW
%Reduction
EKE
% Reduction
EKHE
% Reduction
EKHW
1.-Bacillus cereus (IFO14160)
250
85
80
99
99
2.-Bacillus subtilis (IFO137199)
250
83
83
93
90
3.-Escherichia coli (IFO3301)
75
100
98
99
99
4.-Salmonella typhimurium
(ATCC-13311)
75
81
80
85
80
5.-Pseudomona aeroginosa
ATCC 27853)
75
82
80
83
81
Table.4 Antimicrobial activity and MICs of EKW, EKE, EKHE and EKHW
Bacterial strain
MIC ppm
% Reduction
EKW
% Reduction
EKE
% Reduction
EKHE
% Reduction
EKHW
1.-Bacillus cereus (IFO14160)
2500
80
80
99
99
2.-Bacillus subtilis (IFO137199)
2500
83
80
93
90
3.-Escherichia coli (IFO3301)
2500
100
99
99
99
4.-Salmonella typhimurium
(ATCC-13311)
2500
82
80
83
80
5.-Pseudomona aeroginosa
ATCC 27853)
2500
87
81
82
80
Table 5. pH and heat stability of the EKW extract
Condition
MICs (ppm)
pH 3
250
pH 6
250
pH 9
250
121C, 15 min
250
121C, 60 min
500
No treatment
250
Table 6. Comparison of the antimicrobial activity (MIC) of the EKE and Polyphenols
Indicator strain
EKE (ppm)
Crude catechins from
green tea (ppm)
Crude theaflavins from
tea (ppm)
Escherichia coli
75
>1000
>1000
Bacillius subtilis.
250
600
500
Salmonella sp.
75
>1000
>1000
B Kernel-Hexane-Ethanol (EKHE)
D kernel-Hexane-water (EKHW)
F kernel-Ethanol (EKE)
H kernel-water (EKW)
4000
3500
3000
2500
2000
-1
Cm
1500
1000
500
Figure 1. Mango kernel extract spectra for the following solvents: ethanol (EKE), water
(EKW), Hexane-water (EKHW) and Hexane-Ethanol (EKHE)