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Fby-11-3515 Yüksek Lisans Te T.C. ERCİYES ÜNİVERSİTESİ BİLİMSEL ARAŞTIRMA PROJELERİ KOORDİNASYON BİRİMİ FERMENTE SUCUKTAN İZOLE EDİLEN BAZI LAKTİK ASİT BAKTERİLERİNİN STARTER KÜLTÜR OLARAK KULLANIM POTANSİYELİNİN BELİRLENMESİ Proje No: FBY-11-3515 YÜKSEK LİSANS TEZ PROJESİ SONUÇ RAPORU Proje Yürütücüsü Prof. Dr. Hasan YETİM Gıda Mühendisliği Bölümü Araştırmacı Yük. Lis.Öğr. Yasemin ÇELEBİ SEZER 10/2012 KAYSERİ FERMENTE SUCUKTAN İZOLE EDİLEN BAZI LAKTİK ASİT BAKTERİLERİNİN STARTER KÜLTÜR OLARAK KULLANIM POTANSİYELİNİN BELİRLENMESİ Yasemin ÇELEBİ SEZER Erciyes Üniversitesi, Fen Bilimleri Enstitüsü Yüksek Lisans Tezi, Ağustos 2012 Danışman: Prof. Dr. Hasan YETİM ÖZET Geleneksel fermente sucuklarda üretim süresince gelişen mikroflora ile ürünün kalitesi, teknolojik özellikleri ve güvenilirliği arasında kuvvetli bir ilişki bulunmaktadır. Bu nedenle geleneksel Türk tipi sucuklarda spontan olarak gelişen, sucuk kalitesi üzerinde olumlu etkileri bulunan doğal floranın starter kültür olarak kullanılma potansiyelinin belirlenmesi gerekmektedir. İşte bu çalışmada da geleneksel yöntemlerle üretilen sucuklardan izole edilen bazı bakteri kültürlerinin starter kültür olarak kullanılma potansiyeli araştırılmıştır. Araştırmada, starter kültürsüz hazırlanan kontrol grubuna ilave olarak laktik asit bakterilerinin (Lactobacillus sakei, Lb. curvatus, Lb. plantarum) ve Staphlococcus cinsi bakterilerin (S. xylosus ve S. carnosus) 9 farklı kombinasyonu oluşturularak toplam 10 farklı grup sucuk üretilmiştir. Fermantasyon süresince (0., 4., 8 ve 12. günler) sucuk örneklerinde nem, pH, su aktivitesi analizleri ile laktik asit bakteri sayımları yapılmıştır. Ayrıca sucuğun bakteriyel florasınındaki değişim fermentasyon süresince (0., 4., 8. ve 12. günler) PCR-DGGE yöntemi ile izlenmiştir. PCR-DGGE analizi sonuçlarına göre tüm sucuk örneklerinde, fermantasyonun 4. gününden itibaren şiddetli bir Lb. sakei bandı gözlenmiştir. Kontrol grubu örneklerde Brochothrix thermosphacta, Leuconostoc gasicomitatum, Carnobacterium divergens ve Lactococcus piscium bakterileri fermentasyonun ilk günlerinden itibaren gözlenmiştir. Lb. plantarum, Lb. sakei ve Lb. curvatus bakterilerine ait bantlar sucuk örneklerinde ayırt edilebilmelerine rağmen, S. carnosus ve S. xylosus bakterilerine ait bantlar ayırt edilememiştir. Ayrıca, fermantasyon sonrası, sucuk örneklerinde bazı fiziksel, kimyasal, mikrobiyolojik ve duyusal analizler ile uçucu bileşiklerin analizi yapılmıştır. Üretilen sucuklarda olgunlaşmanın son gününde pH değerleri; 4.96-5.45, aw değerleri; 0.89-0.92, kuru madde değerleri; %58.08-59.48 ve titrasyon asitliği değerleri; %1.25-1.80 aralıklarında bulunurken, ortalama LAB sayısının 8.65 log kob/g, TMAB sayısının 8.83 log kob/g, Enterobacteriaceae sayısının 3.70 log kob/g ve maya-küf sayısının ise 4.5 log kob/g civarında olduğu tespit edilmiştir. Duyusal açıdan en çok Lb. curvatus+Lb. sakei bakteri kültürlerini içeren (CS) sucuk grubu örnekleri beğenilmiştir. Üretilen tüm sucuklarda uçucu bileşik fraksiyonun önemli bir kısmını, baharat kaynaklı terpenlerin oluşturduğu tespit edilmiştir. Elde edilen bulgulara göre, ilave edilen bakteri kültürlerinin sucukların kalite ve teknolojik özellikleri üzerine istatiksel olarak önemli bir etkisi gözlenememiş konuyla ilgili daha çok sayıda ve daha detaylı araştırmaların yapılması gerektiği sonucuna varılmıştır. Anahtar Kelimeler: Türk tipi sucuk, yerel flora, doğal starter kültür, moleküler teknikler DETERMINATION OF STARTER CULTURE POTENTIAL OF SOME LACTIC ACID BACTERIA ISOLATED FROM TURKISH SUCUK Yasemin ÇELEBİ SEZER Erciyes University, Graduate School of Natural and Applied Sciences M. Sc. Thesis, August 2012 Supervisor: Prof. Dr. Hasan YETİM ABSTRACT In general, there is a strong relationship between the growing microflora during the Turkish sucuk production and the quality, technological characteristics and safety of the product. Hence, the determination of potantial use of natural flora as a starter culture is important since they grow spontaneously and have positive effects on quality of the sucuks. In this research, the potantial use of bacterial cultures used as starter culture and obtained from the Turkish sucuks produced by traditional methods was investigated. In research, ten different groups of sucuk were produced, thereby creating nine different combination of lactic acid bacterias (Lactobacillus sakei, Lb. curvatus, Lb. plantarum) and Staphlococcus type bacteria (S. xylosus ve S. carnosus) beside a control group produced without starter culture. Sample groups were fermented in a special cabin (15- 24 ºC, %75-95 relative humidity) for 12 days. Percent (%) moisture, pH, water activity and lactic acid bacteria count determined in the sucuk samples during the fermentation (0., 4., 8. and 12. days). PCR-DGGE analysis was also performed in order to determine the bacterial flora of sucuk during the fermentation. After the fermentation, some physical, chemical, microbiological, sensory and volatile compound analysis were conducted in sucuk samples. At the last day of fermentation in sucuk samples, pH values were between 4.96-5.45, water activity values were between 0.89-0.92, dry matter values were between 58.08-59.48%, titration acidity values between 1.25-1.80% and the number of average LAB was 8.65 log cfu/g, TMAB was 8.83 log cfu/g, number of Enterobacteriaceae was 3.70 log cfu/g and number of yeast and mold was 4.5 log cfu/g. From the sensory points of view, the most favoured sample group amoung the sucuks was Lb. curvatus+Lb. sakei bacterial cultures (CS) containing samples. In all samples, the terpenes originated from the spices were the important portion of volatile compound fraction. In terms of PCR-DGGE analysis results, in all sucuk samples, several Lb. sakei band was observed. At the beginning of fermentation in control group, Leuconostoc mesenteroides, Carnobacterium divergens, Brochothrix thermosphacta, Leuconostoc gasicomitatum bacteria were observed. Although Lb. plantarum, Lb. sakei and Lb. curvatus bacteria bands were distinguished in the all sucuk samples, S. carnosus and S. xylosus bacteria bands could not. In terms of the data obtained, added bacterial culture in the production of fermented sucuk did not have statiscally significant effect on the quality and technological characteristics of the sucuk samples. In conclusion, it might be suggested that more detailed research should be conducted on the quality of sucuk produced with added starter culture or isolated bacterial cultures. Keywords: Turkish sucuk, house flora, indigenous starters, molecular techniques KISALTMA ve SİMGELER Sembol Anlamı LAB Lactic acid Bacteria; Laktik Asit Bakterisi TMAB Toplam Mezofilik Aerobik Bakteri MRS De Man Rogosa and Sharpe Agar PCA Plate Count Agar VRB Violet Red Bile Agar DRBC Dichloran Rose Bengal Chloramphenicol Agar Kob Koloni Oluşturan Birim KM Kuru madde aw Water activity; Su Aktivitesi TBA Tiyobarbütirik Asit ABS Absorbans SFS Serum Fizyolojik Su A Adenin T Timin G Guanin C Sitozin g Gram V Volt vb. ve benzeri MS Mass spectrometry; Kütle spektrometrisi SPME Solid Phase Microexraction; Katı faz mikroekstraksiyon μmol Mikromol TABLOLAR LİSTESİ Tablo 2.1. Sucuk üretiminde kullanılan bakteri kültürlerinin kombinasyonları ......................... 20 Tablo 2.2. Fermente sucukların olgunlaştırma koşulları ............................................................. 21 Tablo 2.3. V1 ve V3 bölgesinin amplifikasyonu için kullanılan primerlerin baz dizilimi ......... 26 Tablo 3. 1. Fermentasyon süresince sucuk örneklerine ait LAB sayısının değişimi (log kob/g) 34 Tablo 3. 2. Sucuk örneklerine ait mikrobiyolojik sayım sonuçları (log kob/g) .......................... 34 Tablo 3. 3. Sucuk örneklerinin fermentasyon süresince fizikokimyasal analiz sonuçları .......... 40 Tablo 3. 4. Sucuk örneklerine ait fermentasyon sonundaki fizikokimyasal analiz sonuçları ..... 43 Tablo 3. 5. Sucuk örneklerine ait renk ölçüm değerleri .............................................................. 43 Tablo 3. 6. Çiğ Sucuk Örneklerine Ait Duyusal Analiz Sonuçları ............................................. 47 Tablo 3. 7. Pişmiş Sucuk Örneklerine Ait Duyusal Analiz Sonuçları ........................................ 47 Tablo 3. 8. Kontrol grubu ile bakteri kültürü ilave edilen sucuklarda olgunlaşma süresince belirlenen uçucu bileşiklerin alanlarına ait ortalamalar .......................................... 55 ŞEKİLLER LİSTESİ Şekil 2. 1. Araştırmada hazırlanan sucuk üretim aşamaları ............................................ 22 Şekil 3.1. Sucuk örneklerinin LAB sayılarının fermentasyon süresince değişimi ................. 32 Şekil 3. 2. Sucuk Örneklerinin Fermentasyon Süresince pH Değerlerinin Değişimi ............ 36 Şekil 3.3. Sucuk öneklerinin fermentasyon süresince su aktivitesi değerlerinin değişimi..... 37 Şekil 3.4.Sucuk örneklerinin fermentasyon süresince kuru madde değerlerinin değişimi .... 39 Şekil 3.5. Laboratuarda üretilen çiğ ve pişirilmiş sucuk örneklerinin fermentasyon sonunda kesit yüzey rengi değerlerinin örnekler arasındaki değişimi ............................. 45 Şekil 3.6. Laboratuarda üretilen çiğ ve pişirilmiş sucuk örneklerinin fermentasyon sonunda dış yüzey rengi değerlerinin örnekler arasındaki değişimi ................................ 46 Şekil 3.7. Laboratuarda üretilen çiğ sucuk örneklerinin fermentasyon sonunda kesit yüzey görünüş değerlerinin örnekler arasındaki değişimi ........................................... 48 Şekil 3.8. Laboratuarda üretilen çiğ sucuk örneklerinin fermentasyon sonunda kabuk oluşum değerlerinin örnekler
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