As customer awareness grows, there is an increasing consumer demand for minimally processed food products containing reduced amounts of additives. In order to preserve the safety and stability of foodstuffs, the use of preservatives is necessary in most cases, but their amounts can be reduced or they can be replaced by using alternative preservatives. There are numerous research findings in the scientific literature that support the antimicrobial effect, oxidative stability and scavenger abilities of black cumin seed oil. These properties, combined with its beneficial physiological effects, make it particularly suitable for use as a natural preservative. The goal of this work was to investigate the antimicrobial effect of black cumin seed oil in the case of Staphylococcus aureus, a pathogen relevant from a food safety point of view. In order to be able to map the mode of action more accurately, we worked with erythromycin, and to test the suitability of the oil in a combined preservation process, it was combined with organic acids

Frece Jadranka

Mikulka Petra

Mohácsiné Farkas Csilla

Čanak Iva

English

University of Szeged

1642 Journal of Food Investigation – Vol. 63, 2017 No. 31 National Food Chain Safety Office, Directorate for Food Safety Risk Assessment2 University of Zagreb, Faculty of Food Technology and Biotechnology3 Szent István University, Department of Microbiology and BiotechnologyInvestigation of the antimicrobial effect of black cumin seed oil using Staphylococcus aureus strainsPetra Mikulka1, Iva Čanak2, Jadranka Frece2, Csilla Mohácsiné Farkas3Received: 2017. June – Accepted: 2017. August1. SummaryAs customer awareness grows, there is an increasing consumer demand for minimally processed food products containing reduced amounts of additives. In order to pre-serve the safety and stability of foodstuffs, the use of preservatives is necessary in most cases, but their amounts can be reduced or they can be replaced by using alter-native preservatives. There are numerous research findings in the scientific literature that support the antimicrobial effect, oxidative stability and scavenger abilities of black cumin seed oil. These properties, combined with its beneficial physiological effects, make it particularly suitable for use as a natural preservative.The goal of this work was to investigate the antimicrobial effect of black cumin seed oil in the case of Staphylococcus aureus, a pathogen relevant from a food safety point of view. In order to be able to map the mode of action more accurately, we worked with erythromycin, and to test the suitability of the oil in a combined preservation process, it was combined with organic acids.2. IntroductionFood-borne illnesses affect about 3 million people annually in Hungary [10], and so efforts to minimize food safety risks are of utmost importance [15], [25].The use of preservatives can offer a partial solution, however, there has been an increasing demand on the parts of consumers to reduce the amounts of ad-ditives in foodstuffs, because harmful health effects, sometimes legitimately, are attributed to them [9]. Using alternative substances with antimicrobial ef-fects the amounts of preservatives added to food-stuffs can be reduced, without any additional adverse health effects.The extract of the seeds of black cumin (Nigella sa-tiva), a plant native to Southern Europe, North Africa and Southwest Asia, inhibits several isolates of bac-terial origin, especially Gram-positive cocci, with a wide working spectrum [1], [4]. Its positive effect on the human body – mainly in the case of digestive, gynecological and respiratory diseases – has been proven by phytochemical studies [3], [14], [31]. Its blood glucose stabilizing, cholesterol-lowering, gas-tric mucosal regenerative and parasiticidal effects are also supported by numerous studies [1], [2], [7], [8], [21], [28].Figure 1 illustrates the main active components of black cumin seed oil, as determined by HPLC analy-sis: thymoquinone (TQ), dithymoquinone (DTQ), thy-mol (THY) and thymohydroquinone (THQ) [23], of which TQ exhibits the highest biological activity [14]. Based on preliminary research, TQ could be one of the new antibacterial and multiresistance-modifying agents [16]. If its Efflux pump inhibiting effects can be confirmed by further tests, it can be helpful in the fight against the rapidly spreading multidrug resist-ance (MDR), posing an international problem [22], [24], [26], [30].INVESTIGATION OF THE ANTIMICROBIAL EFFECT OF BLACK CUMIN SEED OILSCIENCEKeywords: black cumin seed oil, thymoquinone, alternative preservation, multidrug resistance,  Staphylococcus aureus1643Journal of Food Investigation – Vol. 63, 2017 No. 33. Materials and methods3.1. Materials usedIn our experiments, 100% pure black cumin seed oil was used, containing 9.6 mg TQ per ml, according to information from the manufacturer (Amazing Herbs Rt.). Extraction of the oil was carried out by cold pressing, therefore, it contained significant amounts of lipophilic components (e.g., antioxidants, TQ de-rivatives) [19].We worked with 10 Staphylococcus aureus strains, obtained from the National Collection of Agricultural and Industrial Microorganisms of Szent István Uni-versity. Our choice was based on literature data, ac-cording to which Staphylococcus aureus appeared to be the most sensitive the effects of black cumin seed oil [16], [27]. Code numbers of the strains and infor-mation on their sources are summarized in Table 1.Experiments were carried out using nutrient agar: 28 g of powdered medium (composition: 1 g of beef ex-tract, 5 g of peptone, 5 g of NaCl, 2 g of yeast extract, 15 g agar-agar) was dissolved in 1000 ml of distilled water, and then sterilized at 121 °C for 15 minutes.3.2.  Applied methodFor the qualitative analysis of natural antimicrobial substances, the agar diffusion method is considered to be the most efficient procedure [32], therefore, in the case of black cumin seed oil, the agar well dif-fusion method was used. A microbe suspension of 107 TKE/ml concentration was used, 1 cm3 of which was pipetted onto a sterilized Petri dish. Plate pour-ing was performed with 15 cm3 of 45 °C temperature medium. Into the wells of 10 mm diameter, drilled into the solidified agar plates, 100 µl of the compo-nent to be analyzed was pipetted. Samples were in-cubated for 24 hours at 37 °C, and at the end of the incubation period, diameters of the inhibition zones were determined.4. Results4.1.  Minimum inhibitory concentration (MIC)The minimum inhibitory concentration (MIC) value of black cumin seed oil for the Staphylococcus aureus strains was 50 mg/ml, equivalent to a TQ content of 0.48 µg/ml. Diameters of the inhibition zones are shown in Table 2.  Most resistant was the strain iso-lated from mammary gland inflammation (2033), while the most sensitive proved to be the strain used in the FDA’s antibiotic sensitivity test (1755). It is important to note that the required microbe inhibition concen-tration in foods might be different from the 5% de-termined by the laboratory experiment, because the food matrix can influence the bacteriostatic or bacte-ricidal effects through several interactions [13].4.2.  Combination with erythromycinThe combination with erythromycin, aimed at a more accurate understanding of the antimicrobial mode of action of black cumin seed oil, was preceded by the determination of the MIC value of the antibiotic. In the case of 8 of the 10 strains, values ranged between 3.5 and 7.0 µg/ml. The strain from human clinical sam-ples, with a code number of 2174, and the strain iso-lated from pus, with a code number of 1186, proved to be resistant, therefore, in the case of these strains, the concentration of 7.0 µg/ml was used. The MIC values of erythromycin for the Staphylococcus aureus strains and the diameters of the inhibition zones that developed are summarized in Table 3.Results of the combination confirmed the additive ef-fect found in earlier studies [16], in which tetracycline was used, but the mode of action of the two antibiot-ics are similar: peptide chain elongation is inhibited by tetracycline in the 30S, and by erythromycin in the 50S ribosomal subunit [5], [20]. Table 4. illustrates that black cumin seed oil and erythromycin ampli-fied each other’s effects for most of the strains. In the case of strain no. 2174, resistant to erythromy-cin, there was a 100% increase in the inhibition zone, while in the case of the also resistant strain no. 1186, the increase was 50%. The efficacy of the combina-tion is also supported by the nearly 50% increase in the inhibition zone of strain no. 2033, least sensitive to the effect of black cumin seed oil.4.3. Combination with acetic acid and citric acidApplication of combinations with food acids was jus-tified by the fact that the minimum inhibitory concen-tration of the oil is relatively high: 5%. At this concen-tration, it would probably reduce the enjoyment value of the foods (it has a characteristic peppery, slightly hot flavor) [29], however, in a combined preserva-tion process, together with another inhibitory factor, it could be effective at lower concentrations [17]. Results in Table 5 clearly show that, in the 5 to 15 mg/ml concentration range, growth of the ten strains was inhibited to nearly the same extent by the food acids. As a result of the combinations, there was no significant increase in the inhibition zone in the case of most strains. For the acetic acid combinations, the diameters of the inhibition zones increased by 5 and 6 mm in the case of two strains (2399, 1314), while in the case of the citric acid combinations, a 5 mm increase in the inhibition zone was only observed for strain no. 2033, isolated from sheep mammary gland inflammation. The results obtained do not let us assume that the combinations could be effective.5. ConclusionsBased on our experiments, combined preservation procedures are recommended as the main applica-tion possibility for black cumin seed oil, for exam-ple, together with substances of biological origin that INVESTIGATION OF THE ANTIMICROBIAL EFFECT OF BLACK CUMIN SEED OILSCIENCE1644are effective in low concentrations, such as nisin or pimaricin. In case of effective combinations, it could even be used to reduce the energy demand of gen-tle preservation procedures, such as high hydrostatic pressure treatment or modern irradiation methods.Based on the results of studies aimed at understand-ing the mode of action, erythromycin resistant strains can be sensitized in the presence of black cumin seed oil. It is likely that, in the case of strains no. 2174 and 1186, due to the presence of TQ, the Efflux pump was unable to pump out erythromycin from the cell plasma, and so their resistance to erythromy-cin was eliminated. The increase in the sensitivity of the other strains can also be explained by the Efflux pump inhibiting effect of TQ: thanks to the lipophilic properties of the oil, it can penetrate the cell mem-brane itself, and can inhibit cell functions (e.g., ATP synthesis) [11], [12], [18]. In the combinations, eryth-romycin also entered the cells, blocking the growth of the Staphylococcus aureus strains by inhibiting pro-tein synthesis. In this case, functioning of the cells was inhibited by the oil not only because of its hydro-phobic properties, but also by helping erythromycin’s entry into the cytoplasm, and so cells were subjected to a double stress effect. If further experiments suc-ceed in supporting the Efflux pump inhibiting effect of black cumin seed oil, or the TQ extracted from it, it could provide real help in the fight against rapidly spreading multidrug resistant strains.Combinations with food acids were not sufficiently effective, which could be explained by the fact that the antimicrobial effect of the acids is based mainly on the inhibition of membrane transport processes through decreasing the pH and eliminating the pro-ton gradient [6], and the Efflux pump inhibiting effect of TQ could not be exerted.6. AcknowledgementWe would like to thank the staff of the Department of Microbiology and Biotechnology of Szent István Uni-versity and of the Faculty of Food Technology and Bi-otechnology of the University of Zagreb, for contrib-uting their expertise to carrying out our experiments. 7. References[1] Ali, B. H., Blunden, G. (2003): Pharmacologi-cal and Toxicological Properties of Nigella sativa. Phytotherapy Research. 17. p. 299-305[2] Bhatt, I., Fazal, U. R., Khan, M. A., Khan, S. M. (2009): Effect of prophetic medicine Ka-lonji (N. sativa) on lipid profile of human be-ings: An In Vivo Approach. World Applied Science Journal. 6. (8) p. 1053-1057[3] Boskabady, M. H., Shirmohammadi, B. (2002): Effect of Nigella sativa on Isolated Gu  inea Pig Trachea. Arch Iranian Med. 5. (2) p. 103-107[4] Chaieb, K., Kouidhi, B., Jrah, H., Mahdouani, K.,  Bakhrouf, A. (2011): Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial bio-film formation. BMC Complementary and Alter-native Medicine. 11:29[5] Chopra, I., Roberts, M. (2001): Tetracycline An-tibiotics: Mode of Action, Applications, Molecu-lar Biology, and Epidemiology of Bacterial Re-sistance. Microbiology and Molecular Biology Rewievs. 65.2. p. 232-260[6] Deák, T., Kiskó, G., Maráz, A., Mohácsiné, F. Cs. (2006): Élelmiszer-mikrobiológia. Mezőgazda Kiadó. Kémiai tartósítási módszerek.[7] Demir, H., Kanter, M., Coskun, O., Uz ,Y. H., Koc, A., Yildiz, A. (2006): Effect of black cumin (Nigella sativa) on heart rate, some hematologi-cal values and pancreatic beta-cell damage in cadmium-treated rats. Biological Trace Ele-ment Research. 110. (2) p. 151-162[8] El Shenawy, N. S., Soliman, M. F., Reyad, S. I. (2008): The effect of antioxidant properties of aqueous garlic extract and Nigella sativa as anti-schistosomiasis agents  in  mice. Journal of the São Paulo Institute of Tropical Medicine. 50. (1) p. 29-36[9] European Food Information Council (2004): Tartósítószerek az élelmiszerek hosszabb és biztonságosabb eltarthatóságáért. Food To-day. [10] Élelmiszerlánc-biztonsági Stratégia 2013-2022 (2013): Földművelésügyi Minisztérium. Nemzeti Élelmiszerlánc-biztonsági Hivatal.  http://elelmiszerlanc.kormany.hu/download/4/39/70000/%C3%89LBS%204_1_20130711.pdf  Acquired: 20. 02. 2017[11] Friedly, E. C., Crandall, P. G., Ricke, S. C., Ro-man M., O’Bryan, C., Chalova, V. I. (2009): In vitro antilisterial effects of citrus oil fractions in combination with organic acids. Journal of Food Science. 74. p. 67-72[12] Guinoiseau, E., Luciani A., Rossi, P. G., Quili-chini, Y., Ternengo, S., Bradesi, P. (2010): Cel-lular effects induced by Inula graveolens and Santolina corsica essential oils in Staphylococ-cus aureus. European Juornal of Clinical Micro-biology and Infectious Diseases. 29. p. 873-879[13] Hyldgaard, M., Mygind, T., Meyer, R. L. (2012): Essential Oils in Food Preservation: Mode of Action, Synergies, and Interactions with Food Matrix Components. Frontiers in Microbiology. 3:12[14] Ismail M. Y. M. (2009): Therapeutic Role of Pro-phetic Medicine Habbat El Baraka (Nigella sa-tiva L.). World Applied Sciences Journal. 7. (9) p. 1203-1208Journal of Food Investigation – Vol. 63, 2017 No. 3SCIENCEINVESTIGATION OF THE ANTIMICROBIAL EFFECT OF BLACK CUMIN SEED OIL[15] Kasza Gy., Szeitzné Sz. M., Mészáros L., Oravecz, M., Zoltai, A., Vásárhelyi, A., Cseh, J., Hidi, E., Horváth, Zs., Süth, M., Laczay, P. (2011): Élelmiszer eredetű megbetegedések Magyarországon, EU-tagságunk tükrében. Magyar állatorvosok lapja, 133. 6. p. 368-375[16] Kouidhi, B., Zmantar, T., Jrah, H., Souiden, Y., Chaieb, K., Mahdouani, K., Bakhrouf, A. (2011): Antibacterial and resistance-modify-ing activities of thymoquinone against oral pathogens. Annals of Clinical Microbiology and Antimicrobials. 10:29[17] Leistner, L. (2000): Basic aspects of food preservation by hurdle technology. Interna-tional Journal of Food Microbiology. 55, p. 181–186[18] Li, M., Muthaiyan, A., O’Bryan, C. A., Gustaf-son, J. E., Li, Y., Crandall, P. G. (2011): Use of natural antimicrobials from a food safety perspective for control of Staphylococcus aureus. Current Pharmaceutical Biotechnol-ogy. 12. p. 1240-1254[19] Lutterodt, H., Luther, M., Slavin, M., Yin, J., Parry, J., Gao, J., Yu, L. (2010): Fatty acid profile, thymoquinon content, oxidative sta-bility, and antioxidant properties of cold-pressed black cumin seed oils. LWT - Food Science and Technology. 43. p. 1409-1413[20] Mazzei, T., Mini, E., Novelli, A., Periti, P. (1993): Chemistry and mode of action of macrolides. Journal of Antimicrobial Chemo-therapy. 31. p. 1-9[21] Meddah, B., Ducroc, R., El Abbes Faouzi, M., Eto, B., Mahraoui, L., Benhaddou-Andalous-si, A., Martineau, L. C., Cherrah, Y., Haddad, P. S. (2009): Nigella sativa inhibits intestinal glucose absorption and improves glucose tolerance in rats. Journal of Ethnopharmacol-ogy. 121. (3) p. 419-424[22] Monecke, S., Coombs, G., Shore, A. C. (2011): A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus. Plos One Tenth An-niversary[23] Omar, A., Ghoshe, S., Abdulghani, A., Hou-di, A., Crookscor, P. A. (1999): High perfor-mance liquid chromatographic analysis of the pharmalogycally active quinones and related compounds int he oil of the black seed (Ni-gella sativa L.). Journal of Pharmaceutical and Biomedical Analysis. 19. (5) p. 757-762[24] Pál, T. (2012): Az orvosi mikrobiológiai tankönyve. Medicina Könyvkiadó. p. 425-524[25] Pesavento, G., Calonico, C., Bilia, A. R., Bar-nabei, M., Calesini, F., Addona, R., Mencarelli, L., Carmagnini, L., DiMartino, M.C., Lo Nos-tro, A. (2015): Antibacterial activity of Oregano, Rosmarinus and Thymus essential oils against Staphylococcus aureus and Listeria monocy-togenes in beef meatballs. Food Control. 54. p. 188-199[26] Piddock, L. J. (2006): Clinically relevant chro-mosomally encoded multidrug resistance ef-flux pumps in bacteria. Clinical Microbiologi-cal Reviews. 19. (2) p. 382-402[27] Rafati, S., Niakan, M., Naseri, M. (2014): Anti-microbial effect of Nigella sativa seed extract against staphylococcal skin Infection. Medi-cal Journal of the Islamic Rebublic of Iran. 28:42[28] Salem, M. L. (2005): Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. International Immunopharmacology. 5. (13-14) p. 1749-1770[29] Sharma, N. K., Ahirwar, D., Jhade, D., Gupta, S. (2009): Medicinal and Phamacological Po-tential of Nigella sativa: A Review. Ethnobot-anical Leaflets. 13. p. 946-955[30] Szeitzné, Sz., M., Krisztalovics, K., Sréterné, L., Zs., Fehér, Á., Cseh, J. (2008): Magyaror-szág mikrobiológiai élelmiszerbiztonsági he-lyzete. Élelmiszervizsgálati Közlemények. 54. p. 36-37[31] Toma, C., Simu, G. M., Hanganu, D., Olah, N., Vata, F. M. G., Hammami, C., Hammami, M. (2010): Chemical Composition of the tunisian Nigella sativa. Note 1. Profile on Essential oil. Farmacia. 58. p. 454-458[32] Valgas, C., Souza, S., Smânia, E. F. A., Smânia Jr., A. (2007): Screening methods to determine antibacterial activity of natural products. Brazilian Journal of Microbiology. 38. p. 369-3801645Journal of Food Investigation – Vol. 63, 2017 No. 3SCIENCEINVESTIGATION OF THE ANTIMICROBIAL EFFECT OF BLACK CUMIN SEED OIL

Investigation of the antimicrobial effect of black cumin seed oil using staphylococcus aureus strains

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Investigation of the antimicrobial effect of black cumin seed oil using staphylococcus aureus strains

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