In ternationa l Scholars Journa ls African Journal of Agricultural Marketing ISSN 2375-1061 Vol. 8 (9), pp. 001-004, September, 2020. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Antimicrobial activity evaluation of the oleoresin oil of Pistacia vera L. Bachir Raho Ghalem* and Benali Mohamed Faculty of Biotoxicology Laboratory Science, Institute of Biology, Djillali Liabès University of Sidi Bel Abbès, Algeria. Accepted 05 May, 2020 The hydrodistilled essential oils from the exudates of Pistacia vera L. stems have been tested against three bacteria, Escherichia coli, Staphylococcus aureus and Proteus using three methods: agar disc diffusion method, determination of MIC (minimal inhibitory concentration) and in the liquid phase, by Maruzella method. The results obtained showed that E. coli was the greatest inhibitor of all the strains tested, and that Gram negative bacteria (E. coli and Proteus spp.) showed more inhibition than that observed on Gram positive bacteria (S. aureus) by the essential oil tested. Key words: Essential oils, oleoresin of Pistacia vera L., strains tested, antimicrobial activity determination. INTRODUCTION Among the aromatic plants belonging to the family of Anacardiaceae, the genus Pistacia is noteworthy for its numerous species and varieties of wild-growing plants. Many of these species are typical of the Mediterranean area. Pistacia has an economic value as it is the source of traditional medicinal agent "gum" mastic, an oleoresin exudates from the stem of this plant (Dogan et al., 2003). It is a traditional natural remedy used in very ancient civilizations in the Mediterranean like Greek and Egyptian (Pellecuer et al., 1980; Langenheim, 2003; Peachey, 1995). In Algeria, it is found in four species, namely Pistacia lentiscus, Pistacia terebinthus, Pistacia atlantica and Pistacia vera. According to ecology, the true pistachio (P. vera) is characterized by a large tolerance to climatic variations; it can grow under slices rainfall quite low and can cope in soils. In Algerian folk medicine, Pistacia has been used as an astringent, expectorant and cicatrisant agent (Benhammou et al., 2008). About the other genus of this plant, investigations have shown some pharmacological effects such as reducing blood pressure (Villar and Paya, 1987), anti- inflammatory *Corresponding author. E-mail:bachir_raho@yahoo.fr. Tel: 00213771063841. (Giner et al., 2001; Giner et al., 2000) and antimicrobial action (Ali-Shtayeh and Abu, 1999; Magiatis et al., 1999). The antiseptic activity of P. lentiscus essential oils and its resin on different microorganisms has been reported by several researchers (Tassou and Nychas, 1995; Iauk et al., 1996; Ali-Shtayeh and Abu, 1999; Marone et al., 2001; Benhammou et al., 2008; Douissa et al., 2005) but the antimicrobial effect of Pistacia vera extracts precisely its oleoresin oils have not been studied so far (Duru et al., 2003; Kordali et al., 2003; Özçelika et al., 2005) . In this study, we aimed to detect the inhibitory effect of the oils extracts from exudates of oleoresin from the P. vera stem on the growth of Escherichia coli, Staphylococcus aureus and Proteus tested by using three methods: agar disc diffusion method, determination of MIC (minimal inhibitory concentrations) and in the liquid phase, by Maruzella method. MATERIALS AND METHODS Plant material and extraction of the essential oil The essential oil of P. vera was extracted from the stem oleoresin by water distillation. The mastic gum was collected from the Technological Institute of fruit trees –T.I.F.T- of Tighennif (Wilaya of Mascara) situated in the northwest of Algeria, in the months of April, file:///C:\Users\user\Documents\REPUBLICATION\AGRICULTURAL%20SCIENCES\AppData\Local\Temp\www.internationalscholarsjournals.org Table 1. Antimicrobial activity evaluation of the essential oil resin of P. vera with agar disc diffusion method. Bacterial strain Resin concentration (µg/ml) Dilution 10 -1 10 -2 10 -3 10 -4 Standard 10 -1 10 9 8.5 7 7 E.coli 10-2 11 10.5 9 8.5 7 10 -3 11.5 10.9 9.5 9 6.5 10 -1 9 8.5 8 7 6.5 S.aureus 10 -2 10 9 8.5 7.5 7 10 -3 11 10.5 10 8.5 6.5 10 -1 9 8 8 7.5 7 Proteus spp. 10 -2 11 10.5 10.5 9 7.5 10 -3 11.5 11.5 11.5 10.5 7 May and June, which corresponds to the period of the oleoresin formation. Bacterial strains All bacterial strains (E. coli, Proteus spp and S. aureus) were provided by the Laboratory of Medical Analysis-–located in Dr. Yessaâd Khaled Hospital (YKH) of Mascara City, situated in the west of Algeria for patients suffering from certain infectious diseases. S. aureus was isolated from the pus of a patient, E. coli from blood specimens while Proteus was taken from a coproculture, and then confirmed by biochemical tests and morphological studies in Microbiology Laboratory of Biology Institute in the Mascara University (Euzéby, 1998; Marchal et al., 1982). Antimicrobial activity determination We used three methods to determine the antibacterial activity; agar disc diffusion method, determination of MIC (Minimal Inhibition Concentrations) and in the liquid phase, by Maruzella method. The agar disc diffusion method was employed to determine the anti- microbial activities of the essential oils in question. A suspension of each sample tested micro organism - diluted prior to 10 -1 , 10 -2 and 10 -3 - (1 ml of 10 8 cells/ml), was spread on the solid media plates. Filter paper discs (6 mm in diameter) were soaked in 13 µl of the resin oil and placed on the inoculated plates and, after drying for 15 min, were incubated at 37°C for 24 h .The diameters of the inhibition zones were measured in millimetres (Tepe et al., 2004). Minimal inhibitory concentration (MIC) was taken from the concentration of the lowest dosed test tube showing visually no growth. 10 µl from each visually no grown test tube was subcultured on Mueller- Hinton agar (Pauli and Kubeczka, 1996) . Each strain from the three S. aureus, Proteus spp and E. coli was diluted at 10 -1 , 10 -2 and 10 -3 (1 ml of 10 8 cells/ml); a standard was prepared with ethanol and oleoresin oil of P. vera. From each dilution a slick strain was spread over the surface of the Petri dish containing Mueller-Hinton agar medium liquid which was dried at 37°C for 15 min. Four discs were placed on agar containing the following quantities of the oleoresin oil dilution: 0.5, 1, 1.5, 2 and 2.5 l. Ethanol was added to the standard disc, placed in the Petri dish center, and was incubated toward the end at 37°C for 24 h. The principal technique of Maruzella method is to act in the liquid phase of increasing concentrations of oleoresin essential oil, after emulsifier addition (Singh et al., 2000; Larrondo et al., 1995). Serial dilutions (10 -1 , 10 - 2 and 10 -3 µml) were prepared from this essential oil solution. 1 ml of each dilution and 0.5 ml of tested culture strains were added to 8 ml of nutrient broth, maintained in a Bain Marie to 37°C under agitation for 24 h and then seeded by streaking the surface of agar medium and incubated at 37°C for 24 h. RESULTS AND DISCUSSION As indicated in Table 1, all strain bacteria were inhibited by essential oil resin of P. vera. A more significant inhibition was seen with a higher oleoresin oil con- centration. At low concentrations, a very limited inhibitory effect was observed on the growth of microorganisms in comparison with those of the standard. With increasing essential oil resin of P. vera concentration, an obvious inhibitory effect on growth of, E. coli, Proteus spp and S. aureus was significantly increased. The addition of mastic gum oil in broth culture inoculated with S. aureus, E. coli and Proteus spp inhi- bited the growth of these organisms .The rate of inhibition was greater, on Gram negative bacteria (E.coli , Proteus spp), than that observed on Gram positive bacteria (S. aureus). In most cases the size of inoculum and the con- centration of mastic gum oil affect the growth/survival of the organisms. These results are almost similar to those shown in other work on the antimicrobial activity of oil mastic gum of P. vera as well as those of similar species (Iauk et al., 1996; Koutsoudaki et al., 2005; Kamrani et al., 2007; Benhammou et al., 2008 and Özçelik et al., 2005). Minimal inhibitory concentration (MIC) values were defined as the lowest concentration of oils that completely inhibited microbial growth. The results were expressed in micrograms per millilitre. The results for the MIC are shown in Table 2. The MIC values regarding the antimicrobial activity of oil mastic gum of P. vera against Gram negative bacteria (E. coli and Proteus spp.) and S. aureus (Gram positive bacteria) were determined to be Table 2. MIC evaluation of the essential oil resin of P. atlantica. Bacterial strain Resin concentration (µg/ml) Dilution 0.5 1 1.5 2 2.5 Standard 10 -1 6.5 6.5 9 9.5 10 6 E.coli 10 -2 7 7 9.5 10 11 6.5 10 -3 6.5 8 10 10.5 11 7 10 -1 6 7 8 10 11 6 S. aureus 10 -2 6.5 7 9 10 11.5 6.5 10 -3 6.5 7.5 9.5 10.5 12 6.5 10 -1 6.5 8 9 10 10.5 6 Proteus spp. 10 -2 7 9 9.5 11 11 6 10 -3 7 8.5 10.5 11.5 12 6.5 Table 3. MIC evaluation essential oil resin of P. atlantica with the three bacterial strains. Bacterial strain Essential oil (µg/ml) Standard «0» 10 -1 ,10 -2 10 -3 , 10 -4 10 -5 E.coli + ++ + ++ +++ S. aureus +++ + ++ +++ Proteus spp +++ + +++ +++ ++: Comparable growth with that witness. +: Slow growth. 1.5 and 2.0 µg/ml, respectively. The results indicated that the oil mastic gum of P. vera showed antibacterial activity, according to Alma et al. (2004), and Özçelik et al. (2005), mainly against the Gram-negative bacteria (E. coli and Proteus spp). The oil mastic gum also exhibited an effect against the Gram-positive bacteria (S. aureus). However, this effect was less efficient than that presented against the Gram- negative bacteria, since a higher MIC value was obtained with the Gram-positive bacteria. Differences in MIC values of bacteria may be related to differential susceptibility of bacterial cell wall, which is the functional barrier to minor differences present in outer membrane in the cell wall composition (Zhao et al., 2001). Like previous tests, the application of the liquid phase method confirmed( by its results showed in Table 3) the important antibacterial activity of the oil mastic gum of P. vera on these three microbial strains, as it seems that Proteus spp is more sensitive than the other two. The change of inhibitory effect depends on the natural substance concentration. Conclusion The results of the antimicrobial activity tests indicate that essential oil of mastic gum P. Vera exhibited higher activity against the tested strains and confirm its traditional uses. 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