Laktik asit bakterileri tarafından üretilen antimikrobiyal maddelerin gıda patojeni olan mayalar üzerine etkisinin incelenmesi
Analysis of antimicrobial substances produced by lactic acid bacteria and determination of their activity on foodborn spoilage yeasts
Yazar: AHU ÜNER
Danışman: PROF. DR. DİLEK HEPERKAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Gıda Mühendisliği = Food Engineering ; Mikrobiyoloji = Microbiology ; Mühendislik Bilimleri = Engineering Sciences
Dizin: L-laktik asit = L-lactic acid ; Lactobacillus = Lactobacillus

Özet:
Biyokoruma, zararsız mikroorganizmaların veya metabolitlerinin, yapay kimyasal maddeler yerine kullanılmasıyla gıdalarda güvenliğin sağlanması ve bozulmanın önlenmesidir. Günümüzde tüketicilerin az işlem görmüş gıda ürünlerine talebinde artış olmuştur. Dolayısıyla gıda güvenliğini tehdit eden patojen mikroorganizmaların kontrolü için doğal bir koruma yöntemi olarak laktik asit bakterileri (LAB) ve bunlardan izole edilen antimikrobiyal maddelerin özelliklerine yönelik araştırmalar önem kazanmıştır.Mayalar gıda ürünlerinde sıklıkla bulunmaktadır. Genellikle düşük pH, yüksek şeker ve tuz konsantrasyonuna sahip gıdalarda mayalar bozulmaya sebep olabilmektedir. Debaryomyces, Candida ve Rhodotorula bozulmuş gıdalardan izole edilen maya türlerindendir. Ayrıca fırsatçı patojen özellik göstermektedirler.D- ve L- laktik asit izomerlerinin sentezi türe ve ortam koşullarına göre değişiklik göstermektedir. L izomeri insan metabolizmasında yer alıp, gıda uygulamalarında kullanılmaktadır. İnsan vücuduna zararlı etkileri olan D formunun ise gıdalarda kullanımı sınırlandırılmıştır. Peynir, şarap gibi bazı gıdalarda starter olmayan laktik asit bakterilerinin ürettiği D-laktik asit miktarının fazla olması istenmeyen doku değişikliklerine sebep olmaktadır. Bunun haricinde, D- ve L- laktik asitin mikroorganizmalar üzerine antimikrobiyal özellikleri de stereospesifiktir.Bu çalışmada, laktik asit bakterilerinden (Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus plantarum) elde edilen maddelerin, özellikle fermente et ürünlerinde kalite özelliklerini olumsuz yönde değiştiren ve insan sağlığına zararlı maya türleri (Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida zeylanoides, Debaryomyces hansenii ve Rhodotorula mucilaginosa) üzerine etkilerinin incelenmesi amaçlanmıştır. Laktik asit bakterileri tarafından sentezlenen antimaya özellikteki bileşiklerin belirlenmesi ve özelliklerinin incelenebilmesi için, öncelikle uygun bir ön tarama metoduna ihtiyaç bulunmaktadır. Çok sayıda örneği birarada test edebilecek, spektrofotometrik ölçüme dayanan mikrotitre plaka metodu bu amaçla kullanılmıştır. Antimaya özelliği en fazla olan suşun L. pentosus olduğu, antimaya aktivitenin organik asitlerden kaynaklandığı belirlenmiştir.Bu çalışmanın sonraki bölümünde, L. pentosus suşundan elde edilen supernatantın antimaya özellikleri araştırılmıştır. Bu anlamda, koloni sayım metodu ile spektroskopik ölçüme dayanan mikrotitre plaka metodu kullanılmış ve elde edilen sonuçlar birbirleriyle karşılaştırılmıştır. Çalışmada uygulanan spektrofotometrik ölçüm tekniği daha kolay, az zaman alan ve madde sarfiyatı daha az olan bir tekniktir. Ancak tek başına maya gelişimi üzerine inaktivasyon etkisini göstermede yanıltıcı sonuç vermiştir. Hücre sayımı tekniğinin daha doğru sonuç verdiği görülmüştür. Bu çalışma, antimaya özellikli maddelerin patojenler üzerine etkisinin gösterilmesi açısından özgün bir çalışma olup, bilimsel araştırmalara katkı sağlayacaktır.Bu çalışma ile, belirtilen maya türlerinin L. pentosus suşunun supernatantı varlığında gösterdiği hassasiyet de incelenmiştir. C. lusitaniae, C. zeylanoides ve R. mucilaginosa gelişimi tamamıyla inaktive edilmiştir. C. krusei, C. parapsilosis ve D. hansenii türlerinin dayanıklılık gösterip ortama adapte olduğu görülmüştür.Buna ek olarak, farklı LAB türlerinin ürettiği antimaya maddelerin içerdiği D- ve L-laktik asit izomerlerinin konsantrasyonları stereospesifik enzimatik metotla belirlenmiştir.

Abstract:
Biopreservation is ensuring food safety and prevention of food spoilage using harmless microorganisms or their antimetabolites instead of chemical additives. Nowadays, there has been an increase at demands of consumers for less processed food products. Therefore, to control pathogenic microorganisms which threaten food safety, researches for features of lactic acid bacteria (LAB) and antimicrobial substances isolated from them, as a natural preservation method, has become more important.LAB are ``generally recognized as safe?? (GRAS), because of their association with food fermentations and because they have long been used traditionally as food-grade microorganisms.LAB have inhibitory effect against other microorganisms as a result of the competition for nutrients and decrease in pH. They can produce antimicrobial compounds such as organic acids, hydrogen peroxide, carbon dioxide, bacteriocins and other proteinaceous compounds.Organic acids such as lactic and acetic acid, are fermentation end products of LAB and they decrease the pH in the environment. Carbohydrates are fermented either by homofermentative and heterofermentative species. Homofermentative LAB can produce only lactic acid and heterofermentative species can produce equimolar amounts of lactic acid, acetic acid/ethanol and carbon dioxid.Organic acids can reduce the pH and cause acidification in the cytosol, since undissociated forms of organic acids may penetrate through the plasma membrane. After the entrance, organic acid dissociate because of the increase of the pH in the cytosol. They have impact on the cellular activity of the sensitive organisms increasing the lag phase of their cell cycle.Lactobacillus is a genus of LAB. They are gram positive, facultative anaerobic or aerotolerant, homo/hetero-fermentative and rod-shaped bacteria. Lactobacillus species are used as starter cultures in fermented food products. These food products include yogurt, cheese, pickles, wine, beer and also animal feeds. Moreover, some lactobacillus strains have probiotic potential and positive health effects on human and animals.Yeasts often exist in food products and the level of yeast contamination is an important quality criteria in food safety. Generally, yeasts may cause spoilage of the foods that has low pH, high sugar and salt concentration such as fruit juices, wine, pickled mushrooms, fermented cacao, salads with dressing, jam and marmelades.The spoilage of meat and meat products is mainly associated with bacterial growth. In the recent years, modern food processing and storage techniques were developed and applied successfully against the bacteria responsible for food spoilage especially in meat products. As a result of this, yeast species have the opportunity to grow in these type of products where other microorganisms are not competitive. Debaryomyces, Candida and Rhodotorula are yeast strains that isolated from spoiled foods including fermented meat products. Also they have opportunistic pathogenic features.Synthesis of D- and L- lactic acid isomers shows differences according to type of bacteria and environmental conditions. L isomer takes part in human?s metabolism and is used in food applications. The usage of D form, which has harmful effects on human metabolism, in food is limited. Extra amount of D- lactic acid produced by non-starter lactic acid bacteria cause unwanted tissue changes on some kind of food like cheese and wine. Except that, antimicrobial properties of D- and L- lactic acid are also stereospecific. For example, D-lactic acid is more effective against Listeria monocytogenes, whereas L-lactic acid is more effective at killing Escherichia coli cells.In the study, it is aimed to research the effects of antimetabolites obtained from lactic acid bacteria (Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus plantarum), especially on yeast species (Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida zeylanoides, Debaryomyces hansenii ve Rhodotorula mucilaginosa) that affect the quality features of fermented meet products negatively. In the first place, an appropriate preliminary method is necessary to determine and examine features of antiyeast compounds that are synthesized by lactic acid bacteria. For that reason, microtiter plate method was used, which is based on spectrophotometric measurements and can test many samples at the same time.The activity of supernatants from five LAB strains were characterized in three modes. A small amount of cell free culture supernatant was filter sterilized by membrane filtration and stored at +4 ? C. This part contained organic acids, hydrogen peroxide and proteinaceous substances. The remaining supernatant was neutralized by NaOH to eliminate the antimicrobial effect of organic acids and stored at +4 ? C after filter sterilization in order to use in the second assay. As the third mode, catalase solution was added to the neutralized supernatant and incubated for 30 minutes to eliminate hydrogen peroxide and test the effect of bacteriocin-like substances on yeast strains. These three solutions were inoculated with yeast samples into the wells of the microtiter plate. Two types of control wells were prepared. The first one was inoculated without supernatant solutions and the second one was prepared without yeast inoculants. The microtiter plates were covered with sterile sealing films so as to minimize evaporation and prevent contamination. Antiyeast activity was detected using microplate reader at 600 nm at appropriate intervals. The results of microtiter plate method were confirmed by streaking samples from the wells on agar plate and observing the yeast cells on agar plates after 48 hours of incubation.It is determined that the effects of H2O2 and bacteriocin-like substances were not significant for all LAB strains. Since the yeasts produce the enzyme catalase, antimicrobial activity of hydrogen peroxide in the supernatant may be inactivated. Among the yeast strains L. pentosus has the highest antiyeast property and antiyeast activity is derived mainly from organic acids.In the next part of the study, antiyeast feature of supernatant obtained from the strain L. pentosus was investigated. In this sense, colony counting method and microtiter plate method that based on spectrophotometric measurements were used and the obtained results were compared to each other.Spectrophotometric method is rather easier, takes less time and has less material consumption. However, it gave misleading results about showing inhibition effects on yeast growth, because the amount of the cells in the sample solution should be higher than a limit concentration. It was seen that colony counting method gives more accurate results although it is labor-intensive, takes much more time and needs more material consumption. Inactivation curve determination is important to provide information about antimicrobial effect on increased lag phase, reduced growth rate during log phase, reduced stationary phase level and lethality on the yeast strains. In a food product total inhibition of microorganisms is not always needed. For example, increased lag phase may be enough to maintain the food safety. The effect of LAB supernatants on growth curves of yeasts was determined by colony counting and spectrophotometric turbidity methods. This study is unique because of showing effects of substances has antimicrobial properties on yeast strains and will contribute to the future studies.With this study, susceptibility of mentioned yeast species in the presence of cell free L. pentosus? supernatant was investigated. Growth of C. lusitaniae, C. zeylanoides and R. mucilaginosa was completely inhibited. It was seen that C. krusei, C. parapsilosis and D. hansenii were more resistant and adaptive to environment. Comparing the results of the current study to the studies in the literature, C. krusei was determined as a resistant yeast to many antimicrobial drugs. C. parapsilosis, D. hansenii and C. lusitaniae are less resistant to the chemicals than C. krusei. In general, C. zeylanoides is susceptible to the antimicrobial drugs. Our findings are consistent with the literature. The cell membrane of C. krusei has a different mechanism to control intracellular acidity that makes the yeast resistant to extracellular low pH conditions.In addition, the concentration of D- and L-lactic acid isomers contained in antiyeast supernatants produced by the LAB species were determined using rapid and simple stereospecific enzymatic method based on the oxidation of D/L-lactate to pyruvate by NAD+ in the presence of the enzymes D/L-lactate dehydrogenase. In the second reaction, pyruvate is catalyzed by alanine aminotransferase. These two coupled reactions lead the way to the formation of NADH molecule which is measured by a spectrophotometer at 340 nm.The results show that the supernatant of L. pentosus has the highest lactic acid content, whereas L. plantarum has the lowest. The amount of L-lactic acid in the supernatant of L. brevis 2 is very similar to that of L. pentosus which shows that D-lactic acid causes the antiyeast activity of L. pentosus supernatant. The pH value of the supernatants of L. pentosus, L. plantarum was measured as 3,77 and 5,48, respectively. On the other hand, the pH levels of L. brevis strains were very close to each other (between 4,26 and 4,31). The total lactic acid content of L. pentosus supernatant was calculated as 11,88 g/l. The concentration of lactic acid produced by L. plantarum was nearly zero. Moreover, L. brevis strains? supernatants included lactic acid between 6,23 and 7,41 g/l. According to the results, pH values of the cell free supernatants, the total lactic acid content of the supernatants and antiyeast activities of them are consistent.