Bazı esansiyel yağların Staphylococcus aureus ve Escherichıa coli üzerine antimikrobiyal etkisinin araştırılması
Investigation of the antimicrobial effect of some essential oils against Staphylococcus aureus and Escherichia coli
Yazar: DUYGU TURHAN
Danışman: YRD. DOÇ. DR. HATİCE FUNDA KARBANCIOĞLU GÜLER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2015
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Escherichia coli = Escherichia coli ; Gıda kontrolü = Food control ; Staphylococcus aureus = Staphylococcus aureus ; Uçucu yağlar = Essential oils

Özet:
Mikroorganizmalar gıdaların bozulmasına, ürün kalitesinin düşmesine ve hastalıklara neden olabilmektedir. Bu nedenle gıdaların korunmasında fiziksel ve kimyasal birçok yöntem kullanılmaktadır. Günümüzde tüketicilerde kimyasal koruyuculara karşı oluşan olumsuz düşünceler ve önyargılar, araştırmacıları doğal antimikrobiyal maddeleri araştırmaya yönlendirmiştir. Esansiyel yağların doğal olmaları ve toksisite göstermemeleri nedeniyle antimikrobiyal olarak kullanılabileceği yapılan bilimsel araştırmalarla kanıtlanmıştır.
Esansiyel yağlar farklı bileşenler içeren kompleks karışımlar olduklarından biyolojik etkileri yönünden farklılık göstermektedirler. Etki dereceleri içerdikleri etken maddenin özelliğine bağlı olarak değişiklik gösteren çok sayıda esansiyel yağın antimikrobiyal özelliğe sahip olduğu çeşitli araştırmalarca belirlenmiştir. Antimikrobiyal aktiviteleri, yapılarında bulunan fenolik (timol, karvakrol, öjenol vb.), terpenoid bileşenlerden, aldehitlerden ve organik asitlerden kaynaklanmaktadır. Bu bileşenlerin hücre membranının yapısını bozarak ve enzim sistemlerinin bozulmasına sebep olarak mikroorganizmaları öldürdüğü ifade edilmiştir. 
Esansiyel yağların uçucu ve sudaki çözünürlüklerinin az olması ile karmaşık yapıları antimikrobiyal aktivitelerini analizlemeyi ve elde edilen bulguları değerlendirmeyi güçleştirmektedir. Bu nedenle antimikrobiyal aktivitenin belirlenmesinde farklı yöntemler kullanılmakta ve aynı esansiyel yağ ile ilgili farklı bulgular elde edilebilmektedir.
Yapılan çalışmada ilk olarak dereotu, fesleğen, kakule, kekik, kimyon, mercanköşk, rezene ve zencefil esansiyel yağlarının E. coli ve S. aureus bakterilerine karşı antimikrobiyal aktivitesi agar kuyucuk difüzyon yöntemi kullanılarak etki çaplarının karşılaştırılmasıyla incelenmiştir. Esansiyel yağların antimikrobiyal aktiviteleri ölçülen etki çaplarının büyüklüğüne göre değerlendirilmiştir. Agar kuyucuk difüzyon yönteminde çalışılan konsantrasyonlarda E. coli'ye karşı fesleğen, zencefil ve rezene, S. aureus'a karşı ise zencefil ve rezene esansiyel yağları antimikrobiyal etkinlik göstermemiştir. Kekik ve mercanköşk diğer yağlara göre daha yüksek antimikrobiyal aktivite gösterirken, her iki mikroorganizmaya karşı en yüksek antimikrobiyal aktiviteyi mercanköşk esansiyel yağı göstermiştir. 2,5-5-10-15 µL esansiyel yağ miktarlarında kekik ve mercanköşk esansiyel yağları E. coli'nin gelişimini tamamen engellerken; 2,5 µL esansiyel yağ miktarında dereotu, kimyon ve kakule sırasıyla; 8 mm, 10 mm, 7 mm inhibisyon zonu oluşturmuştur. S. aureus'un gelişimini, kakule esansiyel yağı 15 µL'de, dereotu esansiyel yağı ise 15 µL ve 10 µL'de engellerken; kekik, kimyon ve mercanköşk esansiyel yağları 2,5 µL yağ miktarında dahi bakteri gelişimini tamamen inhibe etmiştir.
Çalışmanın ikinci kısmı için E. coli ve S. aureus bakterilerine karşı antimikrobiyal aktivite gösteren 4 esansiyel yağ (kekik, kimyon, kakule ve mercanköşk) seçilmiştir. Kekik, kimyon, kakule, mercanköşk esansiyel yağları varlığında E. coli ve S. aureus'un gelişimi inkübasyon süresince belirli aralıklarla (0; 2; 4; 6; 8; 10; 12; 18, 20; 22 ve 24 saat) izlenmiş; bakteri gelişimi, bakteri yoğunluğunun optik olarak ölçülmesiyle değerlendirilmiştir. Mikrodilüsyon yönteminin kullanıldığı çalışmada minimum inhibitör konsantrasyon (MİK) değeri görünür bulanıklığın olmadığı konsantrasyon olarak belirlenirken, mikroorganizmaların optik yoğunluğunun ölçülmesiyle de doğrulaması yapılmıştır. Bulanıklık tayini ve optik yoğunluk tayini ile E. coli ve S. aureus için belirlenen MİK değerleri kakule, kimyon ve mercanköşk esansiyel yağlarında farklılık göstermiştir. Bulanıklık tayiniyle belirlenen MİK değerleri E. coli için kimyon ve kakule esansiyel yağlarında 3,75 µL/mL, kekikte 0,93 µL/mL, mercanköşkte 0,46 µL/mL; S. aureus için ise kekik, kimyon, kakule ve mercanköşk esansiyel yağlarında sırasıyla 3,75 µL/mL, 7,5 µL/mL, 15 µL/mL, 0,93 µL/mL'dir. Optik yoğunluk ölçümüyle belirlenen MİK değerleri E. coli için kimyon ve kakule esansiyel yağlarında 7,5 µL/mL, mercanköşk esansiyel yağında 0,93 µL/mL iken; S. aureus için belirlenen MİK değerleri ise kimyon, kakule ve mercanköşk esansiyel yağlarında sırasıyla 15 µL/mL, 30 µL/mL, 1,87 µL/mL'dir. Bulanıklık tayiniyle belirlenen MİK değerleri, optik yoğunluğa göre belirlenen değerlerden daha yüksek konsantrasyonlarda tespit edilmiştir. Kekik esansiyel yağında ise her iki yöntemle de belirlenen MİK değeri aynıdır ve E. coli için 0,93 µL/mL, S. aureus için 3,75 µL/mL'dir.
MİK değerinin belirlendiği mikroplaklardan petri kutularına ekim yapılmış ve inkübasyon sonucunda minimum bakterisidal konsantrasyon (MBK) değerleri belirlenmiştir. MBK değerleri E. coli için kekik ve mercanköşk esansiyel yağlarında 0,93 µL/mL, kimyon ve kakule esansiyel yağlarında 7,5 µL/mL; S. aureus için ise kekik, kimyon, kakule ve mercanköşk esansiyel yağlarında sırasıyla 3,75 µL/mL, 15 µL/mL, 30 µL/mL, 1,87 µL/mL belirlenmiştir.
S. aureus'a karşı en düşük MİK ve MBK değeri mercanköşk esansiyel yağında belirlenirken, E. coli'ye karşı en düşük MİK ve MBK değeri mercanköşk ve kekik esansiyel yağlarında belirlenmiştir. Antimikrobiyal etkinliği en düşük olan esansiyel yağ E. coli için kakule ve kimyon, S. aureus için ise kakule esansiyel yağı olarak belirlenmiştir. Kakule, kimyon, kekik ve mercanköşk esansiyel yağlarının her birinde S. aureus için belirlenen MİK ve MBK değerleri, E. coli'ye göre daha yüksek olduğu için S. aureus'un E. coli'ye göre bu yağlara karşı daha dayanıklı olduğu tespit edilmiştir.

Abstract:
Preservation of food materials from spoilage, mainly by microbial activity, during production, storage and marketing is an important issue in the food industry. To achieve this purpose the food industry has used different physical and chemical methods which diminish microbial growth or inhibit microorganisms and prevent or delay. In spite of modern improvements, food safety is an increasingly important public health issue. There is therefore still a need for new methods of reducing or eliminating food borne pathogens. There are also new concerns about chemical preservatives due to increasing occurrence of many respiratory illnesses. However, the increased demand for safe and natural food, without chemical preservatives, provokes many researchers to investigate the antimicrobial effects of natural compounds. Numerous investigations have confirmed the antimicrobial action of essential oils.
Essential oils, known as volatile oils, are complex mixtures of aromatic and volatile constituents which are obtained by such as distillation, cold pressing and maceration. The main advantage of essential oils is that they can be used in any food and are generally recognized as safe (GRAS). Although, they are GRAS, the application of essential oils is limited because of organoleptic changes in food. However, the stereochemistry, lipophilicity and other factors affected the biological activity of these compounds which might be altered positively or negatively by slight modifications.
Essential oils can comprise more than sixty individual components. Major components can constitute up to 85% of the essential oils, whereas other components are present only as a trace. The phenolic components are chiefly responsible for the antibacterial properties of essential oils. There is some evidence that minor components have a critical part to play in antibacterial activity, possibly by producing a synergistic effect between other components.
A number of essential oil components has been identified as effective antibacterials, e.g. carvacrol, thymol, eugenol. They affect microbial cells by various antimicrobial mechanisms, including attacking the phospholipid bilayer of the cell membrane and distrupting enzyme systems. Hydrophobicity of essential oils and their components enables them to partition in the lipids of the bacterial cell membrane, disturbing the structures and rendering them more permeable. Leakage of ions and other cell contents can then occur. Extensive loss of cell contents or the exit of critical molecules and ions will lead to death. In a study, how mustard essential oil affected the cell membrane of Escherichia coli O157:H7 and Salmonella typhi was investigated. Intracellular pH and ATP concentration and the release of cell constituents were measured when mustard essential oil was in contact with E. coli and S. typhi at its minimal inhibitory concentration (MIC) and maximal tolerated concentration (MTC). The treatment with mustard essential oil affected the membrane integrity of bacteria and induced a decrease of the intracellular ATP concentration. Also, the extracellular ATP concentration increased and a reduction of the intracellular pH was observed in both bacteria. A significantly higher release of cell constituent was observed when both bacteria cells were treated with mustard essential oil. Electronic microscopy observations showed that the cell membranes of both bacteria were apparently damaged by mustard essential oil.
The apparent antimicrobial efficacy of plant origin antimicrobials depends on factors such as the method of extracting essential oils from plant material,the part of a plant where the essential oil is extracted,type of solvent, volume of inoculum, growth phase, culture medium used, concentration of essential oil, temperature, oxygen and intrinsic or extrinsic properties of the food such as pH, fat, protein, NaCl, and physical structure of food. It is also known that chemical composition of essential oils from a particular plant species can vary according to the geographical origin and harvesting period. In a study the effects of plants space and time of harvesting, plant height, plant diameter, yields of dry and fresh herbage, content (%) and yield of oil, thymol and carvacrol were measured. Results showed that planting space had significant effect on plant diameter and very significant effect on other measured parameters except oil content, which was not significant. Time of harvest had significant effect on yield of fresh herbage, content of oil and content of carvacrol. Its effect on other parameters was very significant except dry herbage and oil yield. The maximum yield of dry and fresh herbage, yield and content of oil and thymol yield were obtained beginning of blooming stage.
Tests of antimicrobial activity can be classified as diffusion and dilution methods. Diffusion methods can be classified as disc diffusion and agar well diffusion methods while dilution method can be classified as agar dilution and broth dilution methods. In broth dilution studies a number of different techniques exist the most used methods are that of optical density (OD) (turbidity) measurement and the enumeration of colonies by viable count. The obtained results are more sensitive than agar dilution method. The principles and practice of these test are explained in the literature but it appears that no standardised test has been developed for evaluating the antibacterial activity of essential oils against food-related microorganisms. Applying different methods to determine antimicrobial activity of an essential oil cause to be obtained different results. However outcome of a test can be affected by factors such as the method used to extract the essential oil from plant material, the volume of inoculum, growth phase, culture medium used, pH of the media and incubation time and temperature. Therefore comparison of published data is complicated.
Dilution methods are usually used to obtain minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). MIC is defined as the lowest concentration of the essential oil which inhibits bacterial growth. MBC is defined as concentration where 99.9% or more of the initial inoculum is killed.
The aim of this study was to determine the antimicrobial activity of some essential oils (dillweed, cardamon, basil, thyme, cumin, origanum, fennel and ginger essential oils) against Escherichia coli and Staphylococcus aureus and to determine the MIC and MBC values of the selected essential oils.
In this study, dillweed, cardamon, basil, thyme, cumin, origanum, fennel and ginger essential oils were studied for their antimicrobial activity against Escherichia coli and Staphylococcus aureus by agar well diffusion method. The results revealed that although some of the essential oils didn't show any antimicrobial activity against tested strains in the studied concentration range, most of them showed antimicrobial activity with varying levels. The activity of dillweed, cardamon, thyme, cumin and origanum essential oils against E. coli became apparent in the petri dishes with the emergence of a circular area of inhibition. On the contrary, no antibacterial activity was observed for basil, ginger and fennel essential oils against E. coli and for ginger and fennel essential oils against S. aureus. Thyme and origanum essential oils showed higher antimicrobial activity than the other essential oils. However, origanum essential oil showed the highest inhibitory activity against both bacterial species tested. The obtained inhibition diameters of essential oils against E. coli and S. aureus were in the range of 7-21 mm and 10-51 mm, respectively. E. coli and S. aureus had been completely inhibited by using 2.5 µL, 5 µL and 10 µL thyme and origanum essential oils.
Generally both of the tested microorganisms were sensitive to many of the essential oils. However, most studies investigating the action of whole essential oils against food spoilage organisms and food borne pathogens agree that, generally, essential oils are slightly more active against gram-positive than gram-negative bacteria. Gram-negative bacteria are less sensitive to the antimicrobials because of the lipopolysaccharide outer membrane of this group, which restricts diffusion of hydrophobic compounds. In this study the results show that gram-negative E. coli is less sensitive than gram-positive S. aureus, too. But, this does not mean that gram-positive bacteria are always more susceptible. 
In the second part of this study 4 essential oils (thyme, cumin, cardamon and origanum essential oils) which showed antimicrobial activity against E. coli and S. aureus were selected. Inhibitory effect of these selected essential oils were monitored during incubation period (0; 2; 4; 6; 8; 10; 12; 18, 20; 22 and 24 h) and the bacterial growth was determined by measuring optical density (OD) at 600 nm. Microdilution method was used to determine MIC. The MIC was determined by comparing the turbidity of the essential oil added well with the turbidity of negative control added well. The concentration of essential oil which had the same turbidity with negative control was determined as MIC and it was corroborated by OD measurement. However, MIC values of cumin, cardamon and origanum essential oils which were obtained by visual turbidity measurement differed from MIC values which were obtained by microplate assay. The obtained MIC values against E. coli and S. aureus of selected essential oils by turbidity measurement were in the range of 0.46-3.75 µL/mL and 0.93-15 µL/mL, respectively. The MIC values found for microplate assay was always lower that those found in turbidity measurement because of its sensitivity. In this method obtained MIC values of E. coli and S. aureus were in the range of 0.93-7.5 µL/mL, 0.93-30 µL/mL, respectively. 
To determine the MBC, broth was taken from each well, spread on Mueller Hinton Agar (MHA)  at which the microorganism did not show visible growth. MBC values of thyme and origanum essential oils against E. coli were 0.93 µL/mL and cumin and cardamon essential oils were 7.5 µL/mL, MBC values of thyme, cumin, cardamon and origanum were 3.75 µL/mL, 15 µL/mL, 30 µL/mL and 1.87 µL/mL, respectively. 
Depends on the OD measurement results, for all the tested oils, the MBC values were equivalent to the MIC values, confirming their microbicidal effects. The lowest MIC and MBC values were obtained from origanum essential oil against S. aureus. However, the lowest MIC and MBC values against E. coli were obtained from thyme and origanum essential oils. Cumin and cardamon showed the lowest antimicrobial effect against E. coli as cardamon had showed the lowest antimicrobial activity against S. aureus.