Isolation and Molecular Characterization of Lactic Acid Bacteria From Cheese By ˙isem BULUT A Dissertation Submitted to the Graduate School in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Depertmant: Biotechnology and Bioengineering Major: Biotechnology İzmir Institute of Technology Izmir, Turkey September, 2003 ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor Assistant Prof. Ali Fazıl Yenid(cid:252)nya for his endless encouragement, support and patience during this reseach. I also wish to express my thanks to my co-advisors Prof. Dr. Şebnem Harsa and Assoc. Prof. Hatice G(cid:252)neş for their all kind of support and help. I would also like to thank Prof. Dr. Sevda Kılı(cid:231) from Department of Dairy Technology at Ege University for her guidance and the valuable information she provided. I want to add thanks to Prof. Dr. Mehmet Karapınar and (cid:214)mre Sıkılı from Food engineering Department at Ege University for their support. I want to express my thankfulness to my friends; Burcu Okuklu, H. Sevgi ˙oban, Elif Yavuz, G(cid:252)ney Akbalık, Seda Elmacı, ˙elenk ˙ınar and Ay(cid:231)a ˙akın. Additionaly, special thanks for Mert Sudağıdan due to sharing all kinds of experience with me. Also, I would like to thank G(cid:246)n(cid:252)l Yenid(cid:252)nya for special help and kindness. I would like to thank managers and employers of Ministry of Agriculture in Nevşehir and Avanos; Mustafa Titrek, Ali ˙opur and the Mayor of the Avanos; M. Seyhan Duru for their kindness and supports on collecting of cheese samples. Finally, I offer sincere thanks to my family members for their support and love. ii ABSTRACT Specially selected starter cultures are required for the industrial production of cheese. These starter cultures are mainly composed of lactic acid bacteria (LAB). Starter LAB have many functions in cheese production. They produce lactic acid during the fermentation process and provide formation of the curd. Futhermore, they show proteolytic activity and also they play a role in the production of aroma compounds and antimicrobial substances. In order to prevent loss of LAB biodiversity and loss of traditional cheese diversity, it is important to identify novel LAB from traditional cheese. The aim of this project was to isolate and identify natural LAB flora involved in traditional (cid:147)˙(cid:246)mlek Peyniri(cid:148) fermentation. In order to achive this goal, LAB were isolated and characterized by using phenotypic ( cell morphology, Gram staining, physiological and biochemical tests ) and genotypic methods (PCR- Restriction Fragment Length Polymorphism). Moreover, technological characterization was performed by monitoring the acid production profiles of the isolates. At the end of the study, a total of 113 coccal and 21 mesophilic lactobacilli were obtained and maintained for future use. It was found that cocci shaped isolates included 54 lactococci and 59 enterecocci. Further identification at the species level indicated that all of the lactococci isolates were L. lactis ssp. lactis. Thirty of the enterecocci were E. faecium, 8 of them were E. faecalis , 3 of them were E. avium, 2 of them were E. durans and 16 of them were other Enterococcus ssp. Lactobacilli isolates were identified as Lb. paracasei ssp. paracasei (3 isolate), Lb. casei ( 3 isolate ) and other Lactobacillus spp ( 15 isolate) . PCR-RFLP method which is based on the amplification of 16S rRNA- ITS genes and restriction digestion with HaeIII and TaqI endonucleases was found to be useful for further identification. Finally, acid production profiles of isolates indicated that 35 of the isolates could lower the pH of UHT skim milk below 5.3 for 6 h incubation at 30 (cid:176)C and these isolates were therefore the best starter candidates for industrial applications. iii (cid:214)Z End(cid:252)striyel peynir (cid:252)retimi (cid:246)zel olarak se(cid:231)ilmiş starter k(cid:252)lt(cid:252)rleri gerektirmektedir. Bu starter k(cid:252)lt(cid:252)rler başlıca laktik asit bakterilerinden (LAB) oluşmaktadır. Starter LAB peynir (cid:252)retiminde pek (cid:231)ok fonksiyona sahiptir. Fermentasyon prosesi boyunca laktik asit (cid:252)retirler ve pıhtının oluşmasının sağlamaktadırlar. Ayrıca, proteolitik aktivite g(cid:246)stermekte, aroma bileşikleri ve de antimikrobiyal madde (cid:252)retiminde rol oynamaktadırlar. LAB bio(cid:231)eşitliliğinin ve de geleneksel peynir (cid:231)eşitliliğinin kaybolmasını (cid:246)nlemek i(cid:231)in, geleneksel peynirlerden yeni laktik asit bakterilerinin tanımlanması (cid:246)nemlidir. Bu projenin amacı, geleneksel (cid:147)˙(cid:246)mlek Peyniri(cid:148) fermentasyonuna dahil laktik asit bakterilerinin izolasyonu ve tanımlanmasıdır. Bu amacı ger(cid:231)ekleştirmek (cid:252)zere, laktik asit bakterileri izole edilmiştir ve fenotipik ( h(cid:252)cre morfolojisi, Gram boyama, fizyolojik ve biyokimyasal testler) ve de genotipik ((cid:147)PCR- Restriction Fragment Length Polymorphism(cid:148)) metodlar kullanılarak karakterize edilmiştir. Ayrıca , izolatların laktik asit (cid:252)retme profilleri monitor edilerek teknolojik karakterizasyon ger(cid:231)ekleştirilmiştir. ˙alışmanın sonunda, toplam 113 kok ve 21 mezofil laktobasil elde edilmiştir. Kok şeklindeki izolatların 54 adedi laktokok ve 59 adedi enterokok olarak bulunmuştur.T(cid:252)r d(cid:252)zeyindeki ileri tanımlama g(cid:246)stermiştir ki; t(cid:252)m laktokok izolatlar L. lactis ssp. lactis(cid:146)dir ve enterekokların 30(cid:146)u E. faecium, 8(cid:146)i E. faecalis, 3(cid:146) (cid:252) E. avium, 2(cid:146) si E. durans ve 16 (cid:145)sı Enterococcus ssp. olarak tanımlanmıştır. Laktobasil izolatları Lb. paracasei ssp paracasei (3 izolat) , Lb. casei (3 izolat) ve Lactobacillus ssp. (15 izolat) olarak tanımlanmıştır. 16S rRNA-ITS genlerinin amplifikasyonuna ve Taq I ve Hae III endon(cid:252)kleazları ile restriksiyonuna dayalı PCR- RFLP ((cid:147)PCR- Restriction Fragment Length Polymorphism(cid:148)) metodu, ileri tanımlama i(cid:231)in faydalı bulunmuştur. Son olarak izolatların asit (cid:252)retme profilleri g(cid:246)stermiştir ki; 35 izolat, UHT yağsız s(cid:252)t(cid:252)n pH sını 30 (cid:176)C de 6 saat inkubasyon sonunda 5.3 (cid:252)n altına d(cid:252)ş(cid:252)rebilmiştir ve bu izolatlar end(cid:252)striyel uygulama i(cid:231)in en iyi starter adayları olmuştur. iv TABLE OF CONTENTS LIST OF FIGURES....................................................................................................viii LIST OF TABLES........................................................................................................ix CHAPTER 1. INTRODUCTION..................................................................................1 CHAPTER 2. STARTER LACTIC ACID BACTERIA.............................................5 2.1. Main Groups Of Lactic Starters İn Cheese İndustry.....................................7 2.1.1. Mesophilic Starter Cultures.............................................................7 2.1.2. Thermophilic Starter Cultures.........................................................9 2.1.3. Artisanal or (cid:147)Natural(cid:148) Starter Cultures.........................................10 2.2. Starter Functions..........................................................................................11 2.2.1. Acid Production............................................................................11 2.2.2. Proteolytic Activity.......................................................................12 2.2.3. Flavor Formation...........................................................................12 2.2.4. Exopolysacchride Production........................................................13 2.2.5. Antimicrobial Property..................................................................13 2.3. Commercial Production of Dairy Starter Cultures.......................................14 2.4. Genetically Modified Lactic Acid Bacteria and Culture Improvement.......15 CHAPTER 3. IDENTIFICATION METHODS FOR DAIRY BACTERIA...........17 3.1. Phenotypic Methods.....................................................................................17 3.1.1.Morphological Methods.................................................................17 3.1.2. Physiological and Biochemical Methods......................................17 3.2.Genotypic Methods......................................................................................19 3.2.1. Randomly Amplified Poylmorphic DNA.....................................21 3.2.2. PCR Ribotyping............................................................................21 3.2.3. PCR-FRLP....................................................................................21 3.2.4. Rep-PCR.......................................................................................21 3.2.5. Pulsed Field Gel Electrophoresis..................................................22 CHAPTER 4. MATERIALS AND METHODS.........................................................24 4.1. Materials.......................................................................................................24 4.1.1. Chemicals......................................................................................24 4.1.2. Samples.........................................................................................24 4.1.3. Reference strains...........................................................................26 v 4.2. Methods........................................................................................................26 4.2.1. Isolation of Lactic Acid Bacteria..................................................26 4.2.1.1. Culture Media and Growth Conditions..........................26 4.2.2. Phenotypic identification..............................................................27 4.2.2.1.Selection of the Isolates According to Their Fermentative Properties...............................................................27 4.2.2.2. Morphological Examination...........................................28 4.2.2.2.1. Simple Staining...............................................28 4.2.2.2.2. Colony Morphology........................................28 4.2.2.2.3. Gram Staining.................................................28 4.2.2.3. Catalase Test...................................................................29 4.2.2.4. Long Term Preservation of the Isolates .........................29 4.2.2.5. Physiological and Biochemical Identification................30 4.2.2.5.1. Identification of Cocci.....................................30 4.2.2.5.1.1. Gas Production from Glucose...........30 4.2.2.5.1.2. Growth at Different Temperatures....30 4.2.2.5.1.3. Growth at Different NaCl Concentrations.....................................................31 4.2.2.5.1.4. Arginine Hydrolysis and Gas Production From Citrate......................................31 4.2.2.5.1.5. Carbohydrate Fermentations...........31 4.2.2.5.2. Identification of Lactobacilli...........................34 4.2.3. Genotypic Identification by PCR-RFLP.......................................34 4.2.3.1. Genomic DNA Isolation.................................................34 4.2.3.2. Amplification of 16S rDNA and ITS(Internally Transcribed Spacer) Region by PCR Reaction...........................35 4.2.3.3. Separation of amplified Fragments................................36 4.2.3.4. Purification of PCR Products.........................................36 4.2.3.5. Restriction Fragment Length Polymorphism (RFLP)...37 4.2.3.6. Purification of Digested DNA Fragments......................37 4.2.3.7. Electrophoresis of Restriction Fragments......................37 4.2.3.8. Interpretation of Results.................................................38 4.2.4. Technological Characterization of Isolates...................................38 4.2.4.1. Acidifying Activity of Isolates.......................................38 vi 4.2.4.1.1. Monitoring of pH............................................38 4.2.4.1.2. Monitoring Lactic Acid Production................39 4.2.4.1.2.1. Standardization of 0.1 N NaOH and determination of Factor Value......................39 4.2.4.1.3. Evaluation of Results.......................................40 CHAPTER 5. RESULTS AND DISCUSSION..........................................................41 5.1. Isolation of Lactic Acid Bacteria.................................................................41 5.2. Phenotypic Identification.............................................................................42 5.2.1. Examination of Homofermentative Properties..............................42 5.2.2. Morphological Examination..........................................................42 5.2.3. Subculturing of Isolates.................................................................42 5.2.4. Gram Staining and Catalase test ...................................................42 5.2.5. Physiological and Biochemical Tests............................................44 5.2.5.1. Physiological and Biochemical Differentiation of Cocci shaped Isolates..................................................................44 5.2.5.2. Identification of Bacilli Shaped Isolates........................57 5.3. Genotypic Identification..............................................................................61 5.3.1. Amplification of 16 S rRNA and ITS region................................61 5.3.2. Digestion of Amplified 16 S rRNA and ITS region.....................62 5.3.2.1. Hae III digestion.............................................................63 5.3.2.2. Taq I digestion...............................................................68 5.4. Acid Production...........................................................................................69 CHAPTER 6. CONCLUSION AND FUTURE PERSPECTIVE............................71 REFERENCES..............................................................................................................72 APPENDIX A................................................................................................................78 APPENDIX B................................................................................................................81 APPENDIX C................................................................................................................83 APPENDIX D ...............................................................................................................87 APPENDIX E ...............................................................................................................88 APPENDIX F................................................................................................................90 APPENDIX G................................................................................................................91 APPENDIX H................................................................................................................92 APPENDIX I...............................................................................................................102 vii LIST OF FIGURES Figure1.1. Flow sheet of (cid:147)˙(cid:246)mlek peyniri(cid:148) making process.......................................2 Figure 1.2. Flowsheet of (cid:147)Beyaz peynir(cid:148) manufacturing...............................................3 Figure 2.1. Glucose utilization metabolic pathways of LAB.........................................6 Figure 3.1. Polymease Chain Reaction-Restriction Fragment Length Polymorphism............................................................................................23 Figure 5.1. Gram Staining and typical colony morphology of LAB............................43 Figure 5.2. Representative 16 S and ITS amplification products of isolates...............61 Figure 5.3. Hae III digests of 16S-ITS rRNA genes of representative isolates and reference strains.........................................................................................62 Figure 5.4. TaqI digests of 16S-ITS rRNA genes of representative isolates and reference strains.........................................................................................64 Figure 5.5. Dendogram of Hae III digest of representative isolates and reference strains.........................................................................................................65 Figure 5.6. Dendogram of Taq I digest of representative isolates and reference strains ........................................................................................................66 Figure 5.7. pH changes in UHT skim milk broths during incubation for 3 h, 6 h, 9 h ,24 h at 30 (cid:176)C..........................................................................................70 Figure 5.8. Lactic acid production in UHT skim milk broths during incubation for 3 h, 6 h, 9 h, 24 h at 30 (cid:176)C.........................................................................70 viii LIST OF TABLES Table 2.1. Examples of starters used for specific type of cheeses.............................8 Table 2.2. Starter bacteria used in Turkish (cid:147)beyaz peynir(cid:148).....................................11 Table 2.3. Classes of bacteriocins Produced by LAB..............................................14 Table 3.1. Differential characteristics of the cocci shaped lab found in starter cultares....................................................................................................18 Table 3.2. Procedural steps of main genotypic methods.........................................20 Table 4.1. Sample types and location......................................................................25 Table4.2. Differential characteristic of cocci shaped LAB.....................................33 Table 5.1. Logarithmic microbial count (log cfu/g) and standard deviations..........41 Table 5.2. Enterecoccal isolates which produced carbon dioxide from citrate.......46 Table 5.3. Biochemical results of cocci shaped isolates..........................................47 Table 5.4. Physiological and biochemical test results of cocci shaped LAB..........48 Table 5.5. Biochemical identification results of lactobacilli...................................58 Table 5.6. Physiological and biochemical test results of lactobaccili......................59 Table 5.7. Fragment sizes of Hae III digests of the isolates and reference strains..67 Table 5.8. Fragment sizes of Taq I digests of the isolates and reference strains.....67 Table 5.9. Acidification characteristics of the isolates...........................................69 Table H1.1. pH Changes in UHT skim milk during incubation at 30 (cid:176)C for Lactococcus genus..................................................................................92 Table H1.2. pH Changes in UHT skim milk during incubation at 30 (cid:176)C for Enterococcus genus................................................................................94 Table H1.3. pH Changes in UHT skim milk during incubation at 30 (cid:176)C for Lactobacillus genus................................................................................96 Table H2.1. Lactic acid production in UHT skim milk during incubation at 30 (cid:176)C for Lactococcus genus............................................................................97 Table H 2.2. Lactic acid production in UHT skim milk during incubation at 30 (cid:176)C for Enterococcus genus..........................................................................99 Table H 2.3. Lactic acid production in UHT skim milk during incubation at 30 (cid:176) C for Lactobacillus genus.....................................................................101 Table I.1 Reference strains subjected to physiological and biochemical tests.....102 ix CHAPTER 1 INTRODUCTION The production of cheese from milk is a very ancient process. Cheese manufacturing started about 8 000 years ago in the (cid:147)Fertile Crescent(cid:148) between Tigris and Euphrates rivers (Hayaloğlu et al., 2002). Production of cheese is essentially achieved by bringing four ingredients together: milk, rennet, microorganisms, and salt. The process includes the following steps: gel formation, acid production, whey expulsion, salt addition, and finally ripening period. The main biochemical changes that occur in cheese manufacture is the production of lactic acid from lactose. This is achieved by different species of lactic acid bacteria (LAB). The responsible flora that form acid development during cheese production are starter cultures that cause decrease in the pH, formation of curd, expulsion of whey (Beresford et al., 2001). Since the early 1900s there has been a remarkable increase in the industrial production of cheese. Because, hygiene is the most important criterion in the large scale production (M(cid:228)yra-M(cid:228)kinen et al.,1998). For this reason, pasteurized milk is used. Therefore, natural LAB flora contained in the milk is lost. Consequently, in order to make cheese from pasteurized milk, an external LAB source is needed. This source includes predefined strains of LAB and are called starter strains. In Turkey there has been no starter strain developed as yet that could represent our native LAB flora. This prompted us to collect LAB from the regions where traditional cheese making is still dominating. Traditionally, raw milk of cow, goat and sheep are fermented by the help of naturally occurring indigenous LAB. Beside the technological parameters like curd handling and cooking temperature, the quality of cheese is mainly dependent on the microbial associations within the respective region. Therefore, the LAB flora of traditional cheese making can be taken as the basis of starter strains with unique characteristics. In order to prevent the loss of microbial diversity and loss of wide range of cheese variety, it is very important task to build up LAB collections. (cid:147)˙(cid:246)mlek Peyniri(cid:148) is one type of traditional cheese that is very common in central Anatolia. It has been produced from raw cow or sheep milk for many millennia. Although production recipes change from one village to another, and even among