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 (121C, 15 min), freezing (-20C, 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), hexane-ethanol 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 35C 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 anti-microbial 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 gram-negative 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 121C for 15 min, although activity was reduced to about 1/4 when heated at 121C 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 gram-negative 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.