Laktik asit bakterilerinin Enterobacter sakazakii üzerine antibakteriyal etkisinin incelenmesi
Antibacterial effect of some lactic acid bacteria on 
Enterobacter sakazakii
Yazar: SİNEM DOĞAL
Danışman: PROF. DR. ZEYNEP DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2015
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Antibakteriyel aktivite = Antibacterial activity ; Enterobacter sakazakii = Enterobacter sakazakii ; Laktik asit bakterileri = Lactic acid bacteria

Özet:
Enterobacter sakazakii, Enterobacteriaceae familyasına ait Gram negatif, fakültatif anaerob, çubuk şeklinde, spor oluşturmayan, peritrik flagellalarıyla hareketli bir bakteridir. Literatürde bakteri tanımlanırken hem Cronobacter spp. hem de Enterobacter sakazakii ismi kullanılmaktadır. E. sakazakii' nin bebeklerde enfeksiyona neden olabilecek ve bebek mamalarında bulunabilecek mikroorganizmalar arasında en fazla riske sahip olduğu belirlenmiştir. E. sakazakii, özellikle 1 yaşın altındaki bebekler ve bağışıklık sistemi baskılanmış kişilerde hastalığa neden olabilmektedir. Prematüre yeni doğanlar ve bebeklerde menenjit (beyin zarının iltihabı) ve ince ve kalın bağırsak iltihabına sebep olmaktadır. Ölüm oranı yüksek olan E. sakazakii sağ kalanlarda devam eden nörolojik hastalıklar sebebiyle önemli bir patojendir. Literatürde yapılan çalışmalarda E. sakazakii' nin asidik pH değerlerinde önemli ölçüde dirençli olduğu belirlenmiştir. Aynı zamanda, E. sakazakii' nin düşük su aktivitesine de dirençli olduğu belirlenmiştir. E.sakazakii; lateks, silikon, plastik, polikarbon, paslanmaz çelik gibi yüzeylere yapışmakta ve biyofilm oluşturmaktadır.
Laktik asit bakterileri gram pozitif, spor oluşturmayan, katalaz negatif, oksidaz negatif, anaerobik ve aerotolerant özellikte olan, genellikle hareketsiz, aside toleranslı, kuvvetli fermentatif özellikte olan çubuk ve koklardır. Şeker fermantasyonu sırasında başlıca son ürün olarak laktik asit üreterek ortam asitliğini düşürmektedir. Karbonhidratları metabolize ederken takip ettikleri yola göre homofermantatif ve heterofermantatif özellik göstermektedir. Homofermantatif laktik asit bakterileri, son ürün olarak laktik asit oluştururken; heterofermantatifler ise laktik asit yanında etanol, CO2, asetik asit ve formik asit de oluştururlar. 
Laktik asit bakterilerinin fermentasyon sonucu ürettiği antimikrobiyal maddelerin gıda koruması için kullanılması çok eski çağlardan beri uygulanan bir yöntemdir. Fermantasyon sonucu kullanılabilir karbonhidrat miktarı azalır ve antimikrobiyal etkiye sahip küçük molekül ağırlığında organik moleküller oluşur. Laktik asit bakterilerinin ürettiği antimikrobiyal maddeler; düşük molekül ağırlığına sahip bileşenler (H2O2, CO2 ve diasetil gibi) ve bakteriyosin gibi yüksek molekül ağırlığına sahip bileşenler olarak sınıflandırılmaktadır. Üretilen bu antimikrobiyal maddeler gıdalardaki patojen ve bozulma yapan bakterilerin inhibe edilmesini sağlamaktadır. 
Günümüzde tüketicilerin daha az işlem uygulanmış gıdaları tercih etmeleri nedeniyle doğal koruma yöntemleri ile ilgili çalışmalar artmıştır. Laktik asit bakterileri ile raf ömrü uzatılırken, işleme maliyetleri de düşürülmekte ve patojen mikroorganizmaların kontrolüne olanak sağlanmaktadır. Bu nedenle besin öğelerinde kayba neden olan ve gıdanın aroma, tat ve bazı tekstürel özelliklerinin değişmesine neden olan doğal olmayan gıda koruma işlemlerine alternatif olarak laktik asit bakterileri tarafından üretilen bu antimikrobiyal maddeler üzerine yapılan çalışmalar önem kazanmaktadır. 
Çalışma kapsamında 10 farklı laktik asit bakterisi supernatantının 4 farklı E. sakazakii suşu üzerine etkisi ELISA cihazı ve ekim yöntemi ile incelenmiştir. Supernatantların patojen bakteri üzerine etkisi; toplam etki, nötr etki ve protein etki olmak üzere üç farklı şekilde incelenmiştir. Laktik asit bakteri supernatantlarının Enterobacter sakazakii patojen bakterisi üzerine toplam etkisi incelendiğinde; çoğu izolata ait supernatantın inhibe edici etkisi olduğu görülürken bazılarının etkisinin suşa göre değiştiği, bazılarının ise etkisiz olduğu görülmüştür. Laktik asit bakteri supernatantlarının E. sakazakii patojen bakterisi üzerine nötr ve protein etkisi incelendiğinde ise tüm suşların inhibisyonda etkisiz olduğu gözlemlenmiştir. Bu durum, kullanılan laktik asit bakterilerine ait antimikrobiyal metabolitlerden sadece organik asitlerin E. sakazakii patojen bakterisi üzerinde etkili olduğunu göstermiştir.

Abstract:
Enterobacter sakazakii, which belongs to the family Enterobacteriaceae, is a Gram-negative, facultative anaerobic, rod-shaped, spore-forming bacteria with a moving peritrik flagella. In literature, Enterobacter sakazakii is also named as Cronobacter sakazakii. Enterobacter sakazakii is a bacteria which causes chronic or long-lasting, life-threatening diseases and is classified as extremely dangerous. Although it may cause diseases all-aged people, it is more effective on humans who have a weak immune system. Especially, babies under 1 year-old are more affected compared to other age groups. E. sakazakii causes enteritis and meningitis on babies and also causes high rates of death at the same time. This organism has been isolated from a wide range of foods including cheese, meat, vegetables, grains, herbs and spices, but its major habitat is still unknown. 
Studies in the literature determined that, E. sakazakii is resistant to low water activity and acidic pH values. This pathogen bacteria also adhere to surfaces such as stainless steel, latex, silicone, plastic, polycarbonate and form biofilms. Neonatal infections have been associated with E. sakazakii colonization of food preparation equipment such as brushes, blenders and spoons. E. sakazakii produces a capsule with a heteropolysaccharide structure. The capsule can be involved in the organism's ability to survive the long IFM shelf-life. It may also enable the organism to attach to surfaces and form a biofilm that is more resistant to cleaning and disinfectant agents. E. sakazakii is not resistant to pasteurization process of powdered infant formula production but it can be contaminated while adding micronutrient and reconstitution after pasteurization. It is an obligation to make E.sakazakii analysis at infant formula milk and follow-up formulas in Turkish Food Codex.
Lactic acid bacteria (LAB) are gram-positive, nonsporing, nonrespiring cocci or rods, which produce lactic acid as the major end product during the fermentation of carbohydrates. Generally, all lactic acid bacteria can grow in the medium where is any oxygen but the bacteria can grow in the presence of oxygen as aerotolerant anaerobes, unlike most anaerobes. 
As a major end product, lactic acid, decreases acidity of the environment during sugar fermentation. They show homofermentative and heterofermantative property during metabolizing carbohydrates. Homofermentative lactic acid bacteria, produce lactic acid as the major end product. Beside lactic acid, ethanol, CO2, acetic acid and formic acid are produced by heterofermantative lactic acid bacteria. The obligate homofermenters are Lb. acidophilus, Lb. delbrückii and Lb. helveticus. The facultative heterofermenters are Lb. plantarum, Lb. casei, and Lb. sake. Lastly, the obligate heterofermenters are Lb. brevis, Lb. fermentum and Lb. kefir.

LAB have a GRAS (Generally Regarded As Safe) status. LAB are generally associated with habitats rich in nutrients, such as various food products (milk, meat, sausages, beverages, winemaking, curing fish, pickling of vegetables), but some of them are also members of the normal flora of the mouth and intestine. 
Lactic acid fermentation is one of the oldest forms of preparation and preservation of foods. LAB are essential to the fabrication of fermented products such as cheese, yogurt, fermented milk and butter. Lactic acid also gives fermented milks a slightly tart taste. Likewise, they are also responsible for formation and maturation of cheeses and improvement of nutritional and technological value of food products. LAB also contribute to the development of the organoleptic characteristics (flavour, aroma, texture) of the final products. Their ability to promote food preservation is linked to the fact that they cause a decrease in pH. They produce lactic acid and also other antimicrobial agents. All of these factors inhibit undesirable microorganisms (spoilage or pathogenic microorganisms). In addition, the bacteria can be used as starter cultures, as additives and as helping metarials that are produced by them for fermentation or other process.
Antimicrobial substances, produced by lactic acid bacteria during fermentation, have been used for food preservation since ancient times. As a result of this fermentation process, the amount of available carbohydrate reduces and small molecular weight organic molecules with antimicrobial activity are produced. Antimicrobial substances produced by lactic acid bacteria classified as low molecular weight components (H2O2, such as CO2 and diacetyl) and high molecular weight components such as bacteriocins. It is very clear that the primary antimicrobial effect exerted by LAB is the production of organic acids and reduction of pH. Levels and types of organic acids produced during the fermentation process depend on LAB species or strains, culture composition and growth conditions. The antimicrobial effect of organic acids lies in the reduction of pH, as well as the undissociated form of the molecules. All of these antimicrobial agents produced by lactic acid bacteria, provide inhibition of pathogenic bacteria that cause spoilage in foods.
Today, studies about the natural protection methods are increased due to the consumer choice of untreated foods. The shelf life is extended by lactic acid bacteria. Processing costs are decreased and also this technology allows the control of pathogenic microorganisms. Other food preservation methods can cause loss of nutrients, aroma, taste and can change some textural characteristics of foods. Therefore, antimicrobial substances produced by lactic acid bacteria is an alternative method for preservation of foods.
In this study, effect of 10 different lactic acid bacteria strains isolated from tulum cheese and boza are used. These lactic acid bacteria are Streptococcus macedonicus, Weissella confusa, Pediococcus parvulus, Lactococcus garvieae, Lactobacillus paracasei, Leuconostoc citreum, Lactobacillus coryniformis, Lactococcus lactis, Lactobacillus brevis and Lactobacillus plantarum. The antibacterial effect of metabolites produced by lactic acid bacteria on 4 different E. sakazakii strains are observed via ELISA microplate and cultivation 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 0hours, 6hours, 10hours, 24hours and 48 hours period. To observe the antibacterial effect of organic acids only, neutralized supernatants are used. Neutral supernatants are prepared by adding Sodium Hydroxide to the supernatant until the pH value reaches 6,5. By neutralizing supernatants the effect of organic acids are excluded. Finally the effect of proteinaceous compounds of LAB strains were determined. These compounds of LAB strains were obtained by pH-mediated cell adsorption-desorption method. 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 by using MRS broth without lactic acid bacteria metabolites to see the normal growth of the pathogen bacteria. Likewise three pathogen inoculated cells and three non-pathogen cells are prepared. In cultivation method, Triptic Soy Agar is used.
ELISA microplates are incubated at 37˚C and absorbance values are measured at the 0th, 6th, 10th, 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 proteinaceous compounds. Absorbance values of the pathogen added cells are compared with non-pathogen cells and numerical difference between these absorbance values is evaluated as pathogen activity. To see the inhibitory effect of lactic acid bacteria metabolites on activity of E. sakazakii strains, these absorbance values are compared with the control group.
ELISA and cultivation method results showing the antibacterial effect of unprocessed supernatants on E. sakazakii indicated that, most of the lactic acid bacteria inhibited the growth of E. sakazakii. This shows that all of the metabolites; organic acids, hydrogen peroxide and bacteriocins inhibit the growth of E. sakazakii. The supernatants of Streptococcus macedonicus (A15), Weissella confusa (C19), Lactobacillus paracasei (D41), Leuconostoc citreum (A31) and Lactococcus lactis (F39) are effective to the all of the E. sakazakii strains (ATCC 51329, 800, 37-10, 22). Some of them have bacteriostatic effect and two of them have not inhibitory effect on E. sakazakii. The effect of the supernatants Pediococcus parvulus (E42), Lactobacillus coryniformis (C55) and Lactococcus garvieae (E32) changes with the E. sakazakii strains.  The effect of the supernatants Lactobacillus brevis (25A) and Lactobacillus plantarum (13.1) are ineffective to all of the E. sakazakii strains (ATCC 51329, 800, 37-10, 22). Neutral supernatants and proteinaceous compounds are found to be ineffective in inhibiting all strains of E. sakazakii.