Bazı organik bileşiklerin tayini için mikrobiyal esaslı biyosensör geliştirilmesi
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Dosyalar
Tarih
2009
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada fenolik bileşiklerin tayin edilmesine yönelik probiyotik bakteri esaslı bir biyosensör geliştirilmesi amaçlandı ve Lactobacillus acidophilus, Lactobacillus delbrueckii bulgaricus, Streptococcus thermophilus probiyotik bakterilerinin değişik formlarının biyosensör geliştirmede doğal enzim kaynağı olarak kullanılabilirlikleri incelendi. Uygun bulunan probiyotik bakteri formu ile hazırlanan mikrobiyal esaslı biyosensörün optimizasyon ve karakterizasyon çalışmaları gerçekleştirildi. Bu amaçla çalışmalarda substrat olarak kullanılacak fenolik bileşikler belirlendi. Optimizasyon çalışmalarında hazırlanan biyosensörün biyosensör cevapları üzerine pH, tampon konsantrasyonu ve sıcaklığın etkisi incelendi. Ayrıca biyosensörün biyoaktif tabaka bileşenlerini oluşturan probiyotik bakteri, jelatin ve glutaraldehid miktarının biyosensör cevapları üzerine etkisi incelendi. Karakterizasyon çalışmalarında belirlenen optimum koşullarda hazırlanan biyosensörün fenolik bileşiklerin analizi için standart kateşole göre tayin sınırları belirlendi. Ayrıca analiz sonuçlarının tekrarlanabilirliği, operasyonel kararlığı ve depo kararlılığı incelendi. Belirlenen optimum koşullarda hazırlanan biyosensörün çeşitli örneklerdeki fenolik bileşiklerin belirlenmesinde uygulanabilirliği incelendi. Uygun probiyotik bakterinin belirlenmesinde; Lactobacilluslar için uygun olan MRS Broth besi ortamında substrata adapte edilmiş; karışık haldeki Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus themophilus; saflaştırılmış Lactobacillus acidophilus, saflaştırılmış Lactobacillus bulgaricus ve liyofilize formda karışık haldeki Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus themophilus bakterileri kullanılarak biyosensörler hazırlandı. En iyi biyosensör cevabı L. acidophilus, L. bulgaricus ve S. thermophilus'u içeren liyofilize form olarak belirlendi. Optimizasyon ve karakterizasyon çalışmalarında; standart olarak kullanılacak fenolik bileşiğin belirlenmesi için orsinol, rezorsin, p-kresol, laktik asit, L-dopa, fenol, kateşol, gallik asit ve pirogallolün hazırlanan biyosensörle verdiği biyosensör cevapları incelendi ve en uygun cevap kateşol ile elde edildi. Optimizasyon çalışmalarında; hazırlanan biyosensörün optimum pH'ı 8.0, en uygun tampon sistemi 50 mM fosfat tamponu, optimum sıcaklık 37,5 ºC olarak belirlendi. Biyoaktif tabaka bileşenlerinin optimizasyon çalışmalarında; optimum bakteri miktarı, optimum jelatin miktarı ve optimum glutaraldehit yüzdesi sırasıyla 5 mg, 5 mg ve % 0,625 olarak belirlendi. Karakterizasyon çalışmalarında; belirlenen optimum koşullarda hazırlanan probiyotik esaslı biyosensörün fenolik bileşiklerin tayininde standart olarak kullanılan kateşol için tayin sınırları 0,5 ? 5 mM aralığı olarak belirlendi. Operasyonel karalılığın ve tekrarlanabilirliğin belirlenmesinde; 1 mM standart kateşol için arka arkaya ölçümler alındı. Xort=1,022 mM, standart sapma (S.D.) ± 0,045, varyasyon katsayısı (C.V.) % 4,39 olarak belirlendi. Bu durum hazırlanan biyosensörün, fenolik bileşik tayini için uygun ve kararlı bir sistem olduğunu gösterdi. Geliştirilen biyosensörün depo kararlılığının belirlenmesinde; 22 gün boyunca belirli periyotlarla ölçümler alındı. İlk 10 gün, aktivite korundu. Sonra 18. güne kadar biyosensörün başlangıç aktivitesinin % 20'sini kaybettiği gözlendi. 19 günden sonra, biyosensör hızlı bir şekilde aktivitesini kaybetmeye başladı. Geliştirilen biyosensör kullanılarak yapılan ölçümlere ait standart sapma ve varyasyon katsayıları dikkate alındığında; fenolik bileşik analizi yapılan bütün örneklerde uygulanabileceği görüldü. Anahtar kelimeler: Mikrobiyal biyosensör, fenolik bileşik, probiyotik, Lactobacillus acidophilus
In this study, for the determination of phenolic compounds, a biosensor development based on probiotic bacterium was purposed and for developing a biosensor, suitability of different forms of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus probiotic bacterium were investigated. The biosensor was prepared by using probiotic bacterium form which was found suitable, and the optimization and characterization conditions of this biosensor were carried out. For this purpose, the phenolic compounds which was used as a substrate in assays was determined. In the optimization studies of the biosensor, the effects of pH, concentration of buffer and temperature on the biosensor response were investigated. Furthermore, for the determination of the effects of bioactive layer materials such as the amount of probiotic bacterium and gelatin, percentage of glutaraldehyde on the biosensor response were investigated. In the characterization studies of the biosensor, determination of phenolic compounds, a biosensor was prepared at the determined optimum working conditions and linear measurement range was determined according to catechol. Besides, repeatability of biosensor responses, operational and storage stability of the biosensor were investigated. For determination of phenolic compounds in various samples, suitability of the biosensor which was prepared in determined optimum conditions, were investigated. For the determination of the suitable probiotic bacterium, mixed form of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus which was adapted to substrat in Lactobacilli MRS Broth, pure form of Lactobacillus acidophilus and Lactobacillus bulgaricus which were isolated from mixed form in Lactobacilli MRS Broth, liyophilized mixed form of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus were used and using these bacteriums, biosensors were prepared. The best biosensor response was taken with liyophilized mixed form of L. acidophilus, L. bulgaricus and S. thermophilus. In the optimization and characterization studies, for the determination of a phenolic compounds which was used as a substrate in assays, biosensor responses which were taken with some phenolic compounds such as orsinol, rezorsin, p-cresol, lactic acid, L-dopa, phenol, catechol, gallic acid and pyrogallol were investigated and the most suitable response was obtained with catechol. In the optimization studies, pH 8,0 was selected as optimum pH value for the biosensor and the most suitable buffer system was determined as 50 mM phosphate buffer and optimum temperature was determined as 37,5 ºC. In the optimization studies of bioactive layer, optimum bacterium amount, optimum gelatin amount and optimum percentage of glutaraldehyde were determined as 5 mg, 5 mg and % 0,625, respectively. In the characterization studies, for determination of phenolic compounds, a biosensor was prepared at the determined optimum working conditions and linear measurement range was determined on catechol concentration between 0,5 and 5 mM. For determination of operational stability and repeatability; measurements were done for 1 mM concentration of catechol. Xort=1,022 mM, S.D. ± 0,045, C.V. 4,39 % were determined. This case proved that this biosensor system was suitable and stable for determination of phenolic compounds. For determination of storage stability of developed biosensor, the experiments were carried out periodically during 22 days. The first 10 days, the activity was protected constantly and then up to 18 days it was observed that the biosensor lost 20 % of its initial activity. After 19 days, the biosensor lost its activity rapidly. When the standard devination and the coefficient of variation of the measurements which were done by using developed biosensor were paid attention; suitability of the biosensor were seen for all samples which phenolic compounds analysis were done in. Keywords: Microbial biosensor, phenolic compound, probiotic, Lactobacillus acidophilus
In this study, for the determination of phenolic compounds, a biosensor development based on probiotic bacterium was purposed and for developing a biosensor, suitability of different forms of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus probiotic bacterium were investigated. The biosensor was prepared by using probiotic bacterium form which was found suitable, and the optimization and characterization conditions of this biosensor were carried out. For this purpose, the phenolic compounds which was used as a substrate in assays was determined. In the optimization studies of the biosensor, the effects of pH, concentration of buffer and temperature on the biosensor response were investigated. Furthermore, for the determination of the effects of bioactive layer materials such as the amount of probiotic bacterium and gelatin, percentage of glutaraldehyde on the biosensor response were investigated. In the characterization studies of the biosensor, determination of phenolic compounds, a biosensor was prepared at the determined optimum working conditions and linear measurement range was determined according to catechol. Besides, repeatability of biosensor responses, operational and storage stability of the biosensor were investigated. For determination of phenolic compounds in various samples, suitability of the biosensor which was prepared in determined optimum conditions, were investigated. For the determination of the suitable probiotic bacterium, mixed form of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus which was adapted to substrat in Lactobacilli MRS Broth, pure form of Lactobacillus acidophilus and Lactobacillus bulgaricus which were isolated from mixed form in Lactobacilli MRS Broth, liyophilized mixed form of Lactobacillus acidophilus, Lactobacillus bulgaricus, Streptococcus thermophilus were used and using these bacteriums, biosensors were prepared. The best biosensor response was taken with liyophilized mixed form of L. acidophilus, L. bulgaricus and S. thermophilus. In the optimization and characterization studies, for the determination of a phenolic compounds which was used as a substrate in assays, biosensor responses which were taken with some phenolic compounds such as orsinol, rezorsin, p-cresol, lactic acid, L-dopa, phenol, catechol, gallic acid and pyrogallol were investigated and the most suitable response was obtained with catechol. In the optimization studies, pH 8,0 was selected as optimum pH value for the biosensor and the most suitable buffer system was determined as 50 mM phosphate buffer and optimum temperature was determined as 37,5 ºC. In the optimization studies of bioactive layer, optimum bacterium amount, optimum gelatin amount and optimum percentage of glutaraldehyde were determined as 5 mg, 5 mg and % 0,625, respectively. In the characterization studies, for determination of phenolic compounds, a biosensor was prepared at the determined optimum working conditions and linear measurement range was determined on catechol concentration between 0,5 and 5 mM. For determination of operational stability and repeatability; measurements were done for 1 mM concentration of catechol. Xort=1,022 mM, S.D. ± 0,045, C.V. 4,39 % were determined. This case proved that this biosensor system was suitable and stable for determination of phenolic compounds. For determination of storage stability of developed biosensor, the experiments were carried out periodically during 22 days. The first 10 days, the activity was protected constantly and then up to 18 days it was observed that the biosensor lost 20 % of its initial activity. After 19 days, the biosensor lost its activity rapidly. When the standard devination and the coefficient of variation of the measurements which were done by using developed biosensor were paid attention; suitability of the biosensor were seen for all samples which phenolic compounds analysis were done in. Keywords: Microbial biosensor, phenolic compound, probiotic, Lactobacillus acidophilus
Açıklama
Yüksek Lisans
Anahtar Kelimeler
Biyokimya, Biochemistry
