Bazı laktik asit bakteri suşlarının Staphylococcus aureus ve Salmonella typhimurium'a karşı antimikrobiyal özelliklerinin araştırılması
Antimicrobial effect of some lactic acid bacteria  
strains on Staphylococcus aureus and Salmonella typhimurium 
pathogen bacteria
Yazar: ECE BEKPINAR
Danışman: PROF. DR. DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Mikrobiyoloji = Microbiology

Özet:
Günümüzde gıdaların muhafazasında çeşitli metotlar ve teknolojiler kullanılmaktadır. Ancak kimyasal katkı maddelerinin sağlığa zararlı bulunması, uygulanan işlemlerin de gıdanın tadında, yapısında, dokusunda hasara neden olması ve besin değerlerinde kayıplara neden olması gibi nedenlerden ötürü doğal koruma yöntemlerine yönelimler her geçen gün artmaktadır. Laktik asit bakterileri gibi biyokontrol ajanları bu anlamda doğal koruma metotlarına örnek olarak verilebilir. Laktik asit bakterilerinin metabolitleri arasında yer alan organik asitler, hidrojen peroksit, bakteriyosinler?vb. maddeler, gıdalarda insan sağlığı açısından tehlike yaratan biyolojik tehlikeler olan patojen mikroorganizmalara karşı inhibe edici etki göstermektedir.
Staphylococcus aureus ve Salmonella typhimurium gıdalarda sıkça rastlanan patojen bakterilerdir ve sırasıyla gastroenteritis ve salmonellosis hastalıklarına yol açmaktadırlar. Özellikle Salmonella türleri kaynaklı salgınlar çok sayıda hayat kaybına neden olmaktadır. Staphylococcus aureus ve Salmonella typhimurium açısından riskli başlıca gıdalar tavuk, yumurta, et ürünleri, süt ve süt ürünleridir.
Çalışmada, tulum peynirinden izole edilmiş 47 laktik asit bakterisi izolatının Staphylococcus aureus ve Salmonella typhimurium patojen bakterileri üzerine etkileri ELISA microplate metodu ile araştırılmıştır. Bu bakterilerden elde edilen supernatantlar toplam supernatant etkisi, nötralize supernatant etkisi ve nötr, katalaz eklenmiş supernatant etkisi olarak incelenmiş ve bu şekilde organik asitler, hidrojen peroksit ve diğer antimikrobiyal maddeler olarak laktik asit bakteri metabolitleri üç ayrı grup olarak incelenmiştir. Staphylococcus aureus üzerindeki antimikrobiyal etki Baird-Parker katı besi yerine yapılan ekimlerle doğrulanmıştır. Salmonella typhimurium bulguları ise Xylose Lysine Deoxycholate katı besiyerine yapılan ekimlerle doğrulanmıştır.
Çalışmadan elde edilen bulgular, Staphylococcus aureus patojeni inhibisyonunda çalışılan üç grup supernatantın da, yani organik asitlerin, hidrojen peroksitin ve diğer antimikrobiyal metabolitlerin etkili olduğunu göstermiştir. Çalışmanın Salmonella typhimurium ile gerçekleştirilen kısmında ise organik asitlerin inhibisyonda etkili olduğunu, ancak hidrojen peroksit ve diğer antimikrobiyal maddelerin Salmonella typhimurium inhibisyonu üzerine hiçbir etki göstermediği tespit edilmiştir. Lactobacillus brevis türünün Salmonella typhimurium patojeni üzerindeki antimikrobiyal etkisi ilk defa bu çalışmada incelenmiş ve bu tür için de çalışılan diğer laktik asit bakteri türlerine benzer olarak, metabolitleri arasında yalnızca organik asitlerin Salmonella typhimurium inhibisyonunda etkili olduğu görülmüştür.

Abstract:
Food products are consumed by everyone including sensitive division of people like babies, children, elders. That?s why food safety is very important and food products are supposed to be prepared regarding every possible hazard and risk which may affect human health. Various methods and technologies are being used in food preservation nowadays. However, reasons like chemical additives being considered az hazards against human health, processing food cause damage in texture, nutrition and sensory attributes of the product, interest in natural preservation methods keep increasing. Heat treatments like pasteurization and sterilization cause nutritional loss like denaturation of protein. Consumers prefer natural and safe food with no chemical additive and loss in nutrition. In this means, use of lactic acid happens to be a natual and preferable preservation method. Lactic acid is an ancient food preservation method whic has been improved with time. This study examines the antibacterial effect of some lactic acid bacteria strains isolated from tulum cheese against the growth of Staphylococcus aureus and Salmonella typhimurium pathogen bacteria.
Lactic acid bacteria generates an example for natural preservation methods. Lactic acid bacteria metabolites like organic acids, hydrogen peroxide, bacteriocins, carbohydrate?etc. show inhibitory effect against pathogen microorgnisms. Organic acids generate a low pH medium and therefore decrease the pathogen microorganism viability. Hydrogen peroxide is decomposed by catalase enzyme, lactic acid bacterium is catalase (-) so they don?t decompose hydrogen peroxide. However Staphylococcus aureus and Salmonella typhimurium are catalase (+) pathogens and decompose hydrogen peroxide, released free oxygen causes a strong oxidative reaction in pathogen cells. Carbohydrate effects aerob strains of pathogens by creating anerob medium and another effect mechanism of carbohydrate is affecting the pathogen cells by causing functional disorders on the cell membrane.
Staphylococcus aureus and Salmonella typhimurium are common food pathogen bacteria. Staphylococcus aureus can cause gastroenteritis which affects stomach and intestines. Gastroenteritis symptoms are nausea, vomiting, stomach aches and in some cases high fever. Staphylocoocus aureus affects human health in two ways. This pathogen can affect directly with it?s presence in food products or the enterotoxins produced by Staphylococcus aureus can affect health. Salmonella typhimurium can cause salmonellosis which is seriously fatal. Symptoms for salmonellasis are like gastroenteritis nausea, vomiting, stomach aches ans high fever. Especially Salmonella sourced outbreaks can cause high death ratios. Foods that are risky in means of Staphylococcus aureus and Salmonella typhimurium are mainly poultry, eggs, meat and dairy products. Especially Salmonella typhimurium pathogen bacteria is a serious and important risk in poultry and egg products.
In the study, effect of 47 lactic acid bacteria strains isolated from tulum cheese are used. Variaty of these lactic acid bacteria is given as 16 strains of  Lactobacillus brevis 1, 10 strains of Lactobacillus brevis 3, 13 strains of Lactobacillus plantarum 1, 4 strains of Lactobacillus paracasei subsp. paracasei, 2 stains of Lactobacillus pentosus, 1 strain of Lactobacillus brevis 2 and 1 strain of Lactococcus lactis subsp. lactis. The antibacterial effect of the lactic acid bacteria metabolites on Staphylococcus aureus and Salmonella typhimurium pathogen bacteria are observed via ELISA microplate method. Supernatants obtained from lactic acid bacteria strains by centrifuging the 24-48 hour incubated lactic acid bacteria strains. Supernatants are studied for three effect mechanisms. Firstly the total antibacterial effect is observed by using the unprocessed supernatant. The effect of each lactic acid bacteria?s supernatant are observed within 6hours, 24hours and 48 hours period. To observe the antibacterial effect of organic acids only, neutralized supernatants are used. Initial oH values of unprocessed supernatants vary between 3.77 and 5,55. Neutral supernatants are prepared by adding Sodium Hydroxide to the supernatant until the pH value reaches 6. By neutralizing supernatants the effect of organic acids are excluded. To see only the effect of hydrogen peroxide in the neutralized supernatant, catalyse enzyme is used. Catalase enzyme decomposes hydrogen peroxide. This way we can exlude the effect of hydrogen peroxide besides organic acids and can observe the antibacterial effect of hydrogen peroxide by looking at the difference. For preparation of the ELISA microplates, 24 hour incubated pathogen bacteria is inoculated in three cells and non pathogen broth is inoculated in other three cells to compare and extract the absorbance value of the supernatant?s own color and turbidity. Control groups are prepared with using MRS broth without lactic acid bacteria metabolites in it to see the normal growth of the pathogen bacteria. Likewise pathogen inoculated three cells and non-pathogen three cells are prepared. Antimicrobial effect on Staphylococcus aureus is verified with Baird-Parker Agar inoculations and for Salmonella typhimuriumSalmonella typhimurium, this verification is done with Xylose Lysine Deoxycholate Agar.
Results of the study are obtained from the observation of the absorbance values of each supernatant with pathogen bacteria. Microplates are incubated at 37?C and absorbance values are measured at the 6th, 24th and 48th hour of the incubation. This measurement is done for unprocessed supernatant containing cells as well a microplates prepared with neutralized supernatant and catalase added neutral supernatant. Absorbance values of the pathogen added cells are compared with non-pathogen cells and numerical difference between these absorbance values is evaluated as pathohen activity. To see the inhibitory effect of lactic acid bacteria metabolites on activity of Staphylococcus aureus and Salmonella typhimurium pathogens these absorbance values are compared with the control group which does not contain any antibacterial materials and enable normal pathogen growth. 
ELISA results showing the antibacterial effect of unprocessed supernatants on Staphylococcus aureus indicated that all 47 strains of lactic acid bacteria inhibited the growth of Staphylococcus aureus pathogen bacteria. Neutral supernatants are found to be effective in inhibition as well, however the absorbance values are close to the control group values which means neutral supernatants are much less effective and the main metabolite that stops Staphylococcus aureus growth is organic acids. To see the part of hydrogen peroxide among antibacterial metabolites catalase added neutral supernatants are used and we found out that the aborbance values are even closer to the control group values than the neutral supernatants. This shows that hydrogen peroxide is also has antibacterial effect against Staphylococcus aureus pathogen bacteria. Catalase added supernatant absorbance values are also found to be below control group values which indicates that lactic acid bacteria metabolites other than organic acids and hydrogen peroxide have antibacterial effect against Staphylococcus aureus pathogen bacteria. However the biggest antibacterial effect is found to be coming from organic acids.
ELISA results obtained related to Salmonella typhimurium are evaluated likewise. Unprocessed supernatants are found to be very effective in inhibiting the growth of Salmonella typhimurium pathogen bacteria, the absorbance values are found to be a lot below control grouğ absorbance values. However neutralized supernatants and catalase added neutral supernatants did not show any inhibitory effect against Salmonella typhimurium. These results show that among lactic acid bacteria metabolites, only organic acids are effective in inbiting the growth. Other matabolites like hydrogen peroxide, diacetyl, carbon dioxide and bacteriocins had no antibacterial effect on Salmonella typhimurium growth. Considering the results obtained with both pathogens, it is seen that organic acids are the most antibacterial agents among lactic acid bacteria metabolites against pathogens. Salmonella typhimurium is found to be more resistant to antibacterial agents than Staphylococcus aureus. 
Antibacterial effect is also verified with inoculation on petri dishes. Antimicrobial effect on Staphylococcus aureus is verified with Baird-Parker Agar inoculations and for Salmonella typhimuriumSalmonella typhimurium, this verification is done with Xylose Lysine Deoxycholate Agar. Supernatant containing pathogen bacteria broth in inoculated on petri dishes on incubation periods 24 hours and 48 hours. After incubating the petri dishes for 24 hours at 37?C the growth level on petri dishes is compared with ELISA absorbance values and the results are found to be paralel.
Studies related to determining lactic acid bacteria?s antibacterial effect on pathogens usually use well diffusion method, inoculationg the antibacterial agent to the petri dish where pathogenic growth took place and watching the inhibition zones on the petri dish afterwards in certain periods. ELISA method for determining the effect of antimicrobial agents like organic acids on pathogen microorganisms is newer and fewer among these type of experiments. 
Additionally, this study is first to examine the antimicrobial effect of Lactobacillus brevis on Salmonella typhimurium and only organic acids of Lactobacillus brevis are found to be inhibitory against Salmonella typhimurium.