ANTIBACTERIAL ACTIVITY OF ASPONGOPUS VIDUATUS (MELON BUG) OIL

Aspongopus viduatus (melon bug) oil is insect oil used as famine food in western parts of Sudan and has traditional medicinal uses. The antibacterial activities of melon bug crude oil, silicic acid column purified oil and phenolic compounds‐free oil were determined by agar diffusion assay against seven bacterial isolates, four of them are food‐related bacterial species: Staphylococcus aureus, Salmonella enterica serovar Paratyphi, Escherichia coli and Bacillus cereus, and the three other isolates are: B. subtilis, Enterococcus faecalis and Pseudomonas aeruginosa. The main constituents of this oil were examined. The crude oil and the phenolic compounds‐free oil showed high antibacterial activities against some test species while the silicic acid column purified oil showed no antibacterial activity. The study highlights the possibility of using this oil in food preservation. PRACTICAL APPLICATIONS: In this study, the antimicrobial properties and main components of insect oil (Aspongopus viduatus) were identified. The oil has traditional medicinal uses in human and animal skin diseases and is also used for meat dressing before drying in western regions of Sudan. The oil has long stability against rancidity because of its high oleic acid content. This oil in its crude form or phenolic compounds‐free form could be used in meat and meat products preservation processes to control gram‐positive bacteria.

To preserve food from microbial deterioration, man used different preservation methods, among which are many natural inhibiting substances. Many natural antimicrobial extracts were obtained from plants and few from insects. Some of these can be polypeptides or fatty acids ([29]; [23]; [5]; [9]). The antimicrobial activity of plant extracts may reside in a variety of different components, including aldehyde and phenolic compounds ([18]). Naturally occurring combinations of these compounds can be synergistic and often result in crude extracts having greater antimicrobial activity than the purified individual constituents ([7]). Using antibacterial agents in food preservation is a relatively old practice since the discovery of nicin in the 1930s, which is a widely used natural antibiotic ([4]; [11]).

Aspongopus viduatus (melon bug) is a bug of 20 mm height, belonging to the order Hemiptera. It is widely distributed in Sudan, mainly in the western areas (Kordofan and Darfur states), where field watermelons are considered as one of the most important crops for the traditional rain‐fed agriculture. The bug is considered to be the main pest of watermelon plants ([20]).

In the remote territories of Sudan, oil from the bug A. viduatus (Pentatomidae) is used as sweet oil. It was not possible to point out a poisonous effect of this oil. This bug oil is similar in its main components to most animal oils ([30]). It contains high amount of palmitoleic acid (10.7%), which is unusual for commonly used edible oils ([22]). This high content is comparable to that of menhaden oil with 13.9%, herring oil with 8.0% or edible tallow with 8.8% ([10]). Melon bugs are edible, and in the last nymph stage, which is a relatively soft stage, the bugs are cooked and eaten. Many Namibians collect the adults and use them as a relish or as a spice in ground form for cooking meals (National Museum of Namibia Vindhoek Namibia, 1998, http://www.natmus.cul.na, biology of Insects in Biology 4FF3 Entomology, http://www.science.mcmaster.ca).

In the western part of Kordofan State of Sudan, the bugs are known locally as Um Buga and they are used as a food ingredient by collecting the oil from them after a hot water extraction. The oil is used in cooking during famine and shortage of food. Traditionally, the oil is also used in some parts of Sudan in meat preservation by dressing before drying and as skin lesion remedy in both humans and animals, e.g., camels ([20]).

The conventional uses of melon bug oil directed this study to investigate its antimicrobial activity against food poisoning bacteria (Staphylococcus aureus and Bacillus cereus), food‐borne bacteria (Salmonella enterica serovar Paratyphi and Escherichia coli) and other bacterial contaminants (B. subtilis, Enterococcus faecalis and Pseudomonas aeruginosa). The gram‐positive food intoxication organisms S. aureus and B. cereus produce several enterotoxins ([8]; [26]). The gram‐negative S. enterica serovar Paratyphi is still forming an emerging public health threat in developing countries ([12]). E. coli is a widespread gram‐negative bacterium that contains several pathogenic strains ([28]). P. aeruginosa is well‐known for its natural resistance to antibiotics ([19]). The gram‐positive B. subtilis is a common soil bacterium while E. faecalis is a gram‐positive commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals ([1]).

The study also examined melon bugs for tocopherol content, as they are important to protect food lipids against autoxidation, and thereby to increase their storage life and their value as wholesome foods ([16]). The mode of action of tocopherols as antioxidants is explained by their role in donating hydrogen from their phenolic group to radicals to stabilize them and to stop the growing formation of hydroperoxides ([21]). [33]) found that some tocopherols exhibited strong antibacterial activities against S. aureus and S. epidermidis.

A. viduatus insects were handpicked from watermelon fields in the Ghibaish province of western Sudan, and the oil was collected by using a local hot water extraction method. In brief, the collected bugs were killed by a sudden hot water shock and crushed using a local wooden mortar. The oil was extracted using boiling water, and the top oily layer was collected. The oil was heated again at 100C in an open cooking pan to remove water drops and was afterwards kept in a plastic container at 4C until used.

The purification was carried out following [2]) and [24]). In brief, the oil was purified by passing 300 g of oil through a chromatographic column (60 × 3 cm), packed with a series of 20 g activated silicic acid (100 mesh; Merck, Darmstadt, Germany), 10 g activated charcoal and celite 2:1 (Serva, Feinbiochemica, Hiedelberg, Germany) and 40 g powdered sugar. The silicic acid was activated following [25]) method where 300 g of silicic acid is suspended in distilled water and agitated. After 20–30 min of settling, all the suspended silicic acid is decanted and the process is repeated three times. Five hundred mL of methanol is then added with agitation, and the slurry is allowed to settle for 30 min. Supernatant methanol is then decanted, and the silicic acid is dried in three stages: first on a water bath at 100C for 4 h, then at 100C in an oven for 12 h, followed by activation at 200C for 12 h. Then the oil was purified using petroleum ether and the obtained eluant was evaporated using a rotary evaporator at 40C, and its traces were removed by flushing with nitrogen. Purified oil was kept at −18C for analysis.

Phenolic compounds‐free oil was obtained by extracting phenolic compounds from the crude oil following the method of [31]). In brief, 50 g oil was dissolved in 50 mL petroleum ether using a 250‐mL extraction funnel, and then extracted three times with 30 mL of a mixture consisting of methanol : water (60:40, v/v). The lower layers containing phenolic compounds were removed. The petroleum ether was evaporated in a rotary evaporator (Büchi, Flawil, Switzerland) at 40C to obtain phenolic compounds‐free oil. Samples of phenolic compounds‐free oil were kept at −18C for analysis.

The Fatty Acid Composition Determination.  The fatty acid composition of the oil was determined following the International Standards Organization (ISO) draft standard ([14]). In brief, one drop of the oil was dissolved in 1 mL of n‐heptane, 50 mL 2 M sodium methanoate in methanol was added, and the closed tube was agitated vigorously for 1 min. After addition of 100 mL of water, the tube was centrifuged at 4,500 g for 10 min at room temperature and the lower aqueous phase was removed. After that, 50 mL 1 M HCl was added to the heptane phase; the two phases were shortly mixed and the lower aqueous phase was drained. The top n‐heptane phase was transferred into a vial and 0.15 µL of it was injected into a Hewlett Packard 6,890 gas chromatograph (Agilent, Waldbronn, Germany) with a capillary column, CP‐Sil 88 (Varian GmbH, Darmstadt, Germany).The temperature program was from 155C, heated to 220C (1.5C/min), 10 min isotherm; injector 250C, detector 250C; carrier gas 1.07 mL/min hydrogen; split ratio 1:50.

Tocopherol Determinations.  For determination of tocopherols, a solution of 250 mg oil in 25 mL n‐heptane was directly used for the high‐performance liquid chromatography (HPLC) analysis. The HPLC analysis was conducted using a Merck‐Hitachi low‐pressure gradient system, fitted with an L‐6000 pump, a Merck‐Hitachi F‐1000 Fluorescence Spectrophotometer (detector wavelengths for excitation 295 nm, for emission 330 nm) and a D‐2500 integration system. The samples (20 mL) were injected by a Merck 655‐A40 Autosampler onto a Diol phase HPLC column (25 cm × 4.6 mm ID; Merck, Darmstadt, Germany) using a flow rate of 1.3 mL/min. The mobile phase used was n‐heptane/tert‐butyl methyl ether (99 + 1, v/v) ([3]). All determinations were carried out in triplicate.

Bacterial Strains and Culture Conditions.  The bacterial strains used as test organisms were the standard organisms E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and B. subtilis BGA DSM 618. Local isolates were B. cereus MK 131 (isolated from food) and S. paratyphi and E. faecalis (clinical isolates). All bacterial strains were cultured aerobically at 37C in nutrient broth and agar medium (contained in gram/litre: beef extract 3; peptone 5; sodium chloride 8 and for solid medium, agar 15). Before the susceptibility test, cultures from solid mediums were sub‐cultured in liquid media, incubated for 18 h and used as the source of inoculums for each experiment.

Antimicrobial Activity Determination.  The antibacterial activity of the extracts was determined using the standard well diffusion method according to [6]). Briefly, 100 µL of oil was applied to the well (10 mm in diameter) in nutrient agar plates cultured completely with standardized concentration of 106 colony forming unit (cfu)/mL. For each test organism, the inoculum concentration was standardized by measuring the turbidity of overnight nutrient broth culture using a spectrophotometer (wavelength 450 nm).

Thirty‐microgram standard antibiotic discs (6 mm in diameter each) of Chloramphenicol, Novobiocin, Penicillin, Streptomycin, Tetracycline and Vancomycin were used as controls. The concentrations (106 cfu/mL) of test organisms were used for inoculating nutrient agar plates for antibiotic sensitivity discs. All tests were carried out in quadruplicate.

Some natural products have been part of food in the same time that they exhibit antimicrobial activity and extend the shelf life of food. The same situation is shown by melon bug oil.

The major fatty acids of the crude oil, as determined by gas chromatography, were palmitic, stearic, oleic and linoleic acids. The oil of melon bugs showed a low amount of saturated fatty acids (37.4%) and a high content of unsaturated fatty acids (61.5%), mainly oleic (46.63%). This high content of oleic acid contributes in the antibacterial properties of this oil against gram‐positive bacteria ([29]). Melon bugs had low amounts of tocopherols (0.3 mg/100 g) in comparison to 64.7 and 97.6 mg/100 g in sesame and sunflower oils, respectively. Thus, low tochopherol levels cannot contribute in the stability of this oil against oxidation – but oleic acid, which has high antioxidant properties – can give the oil long shelf life and a very low rate of rancidity ([22]; [21]).

The crude oil of A. viduatus showed relatively high antibacterial activity (Table 1) against pathogenic bacteria S. aureus and B. cereus. The activity also targeted non‐pathogenic strains B. subtilis BGA. Many S. aureus strains are well known for their high antibiotic resistance against different antibacterial agents ([27]). Against B. cereus, 100 µL crude oil showed equal activity in its inhibition zone diameter to 30 mg of Chloramphenicol (18 mm). Also, it produced an inhibition diameter similar to that produced by Vancomycin (19 mm). The crude oil showed moderate activity against the gram‐positive organism E. faecalis.

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ANTIMICROBIAL ACTIVITY OF A. VIDUATUS OIL

1 R, resistance; NT, not tested; CR, crude oil; SA, salicic column purified oil; PH, phenolic compounds‐free oil, diameter of well = 10 mm; CHL, Chloramphenicol; NOV, Novobiocin; PEN, Penicillin; STR, Streptomycin; TET, Tetracycline; VAN, Vancomycin.

S. aureus ATCC 25923 was not affected by phenolic compounds‐free oil, which means that the phenolic compounds in melon bug oil is the active antimicrobial compounds against S. aureus. The phenolic compounds‐free oil showed relatively high antimicrobial activity against B. cereus MK131, B. subtilis BGA DSM 618 and S. paratyphi. The phenolic compounds‐free oil activity against S. enterica serovar Paratyphi is the only activity against gram‐negative bacteria detected in this study. No other significant antimicrobial properties of melon bug oil were recognized against gram‐negative bacteria tested.

The standard antibiotic discs were used as controls for sensitivity and resistance of the test organisms against common antibiotics and also to compare them with melon bug oil antimicrobial activity. The resistance showed by P. aeruginosa against these antibiotics is not surprising because it is known that this organism is difficult to control with antibiotics or disinfectants ([19]). The clinical isolates S. enterica serovar Paratyphi and E. faecalis showed clear resistance against some of the antibiotics tested.

The antimicrobial activity of the oil was lost when the oil was applied to the silicic column; we believe that this may indicate that the effective materials are pigments, tocopherols or phospholipids, which were lost as a result of this binding to the column. Previous studies ([2]; [24]; [17]) indicate the possibility of using a silicic acid column as chromatographic techniques to provide an effective means of purifying and stripping vegetable oils of their minor components. [17]) reported that no peroxides, hexanal, pigments, tocopherols and phospholipids were detected in borage and evening primrose oils, which were purified using a silicic acid column. The phenolic compounds‐free extract of the oil preserve its antibacterial activity against most of the organisms studied.

[15]) stated that unsaturated fatty acids with long chain and more double bonds are active against microorganisms, and melon bug oil contains a high amount of unsaturated fatty acids. Although phenolic compounds are known antimicrobials ([32]), the extraction of the phenolic compounds from the oil did not clearly affect its antibacterial activity except for S. aureus.

Numerous herbs, spices and plants have been reported to be potential sources of antimicrobial agents but not many animal origins have been studied with respect to levels and range of activity ([13]). In particular, some insects of limited distribution, such as those restricted to particular regions or countries, are poorly studied. The current work clearly demonstrates that simple extraction and purification methods of stable melon bug oil has activity against a range of food‐related bacterial species. This oil in its crude and purified form, therefore, has potential to extend the shelf life or improve the safety of foods. The levels and range of activity, however, also indicate that application of the melon bug oil will strongly depend on the specific bacterial problem to be addressed.

The old traditions of using such oil as part of the human diet in some places combined with its antimicrobial activity indicate its possible use for food preservation. More studies are needed to identify exactly the active antimicrobial compounds and to explore its uses as a food preservative.