Fermentasyon yoluyla fungal rennin üretimi
Küçük Resim Yok
Tarih
1991
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
ÖZET Bu çalışmada, yüzey fermentasyonu yolu ile buğday kepeği üzerinde büyütülen Mucor pusillus CBS 183.67 straininin rennin üretme yeteneği araştırılmıştır. Bu araştırma, fermentasyon öncesi işlemler, fermentasyon ve fermentasyon sonrası işlemler olmak üzere üç ayrı temel safhada gerçekleştirilmiştir. Fermentasyon öncesi işlemler aşaması; kültürün aktif hale getirilmesi ve inokulumun hazırlanmasını içermektedir. Organizmanın aktif hale gelmesi için, liyofilize kültür 1-2 mi. malt-pepton solüsyonu içinde açılmış ve YPSSA ortamında 37°C'de 5 gün süre ile büyütülmüştür. Daha sonra, bu kültür aynı ortamı içeren yatık ağarlara transfer edilmiş ve 37°C'de 5 gün inkübe edilerek sporlanması sağlanmıştır. Sporlar sodyum lauril sülfat ile yıkanarak spor süspansiyonu hazırlanmış ve spor süspansiyonu lxl(P adet spor/ml. olacak şekilde ayarlanmıştır. Fermentasyon için temel substrat olan kepek ortamı, karbon kaynağı olarak buğday unu ve azot kaynağı olarak skim milk powder içermektedir. Bu ortam 500 ml.'lik erlenlere koyulmuş ve pH'ı 1 olan 15 mi. asidik mineral tuz solüsyonu ile karıştırılmıştır. Fermentasyon değişik süre ve sıcaklıklarda denenmiş ve en iyi aktivite (600 SU/ml. ) 37°C'de 72 saat sonunda alınmıştır. Bununla birlikte, en yüksek enzim aktiviteleri; organik azot kaynakları varlığındaalınmıştır. Fermentasyon sonrası işlemler aşaması; renninin ekstrakte edilmesi ve enzimatik aktivitenin ölçülmesini kapsamaktadır. Fermentasyon sonunda, kepek ortamı 200 mi. çeşme suyu ile karıştırılarak yıkanmış ve rennin enzimini içeren solüsyon filtrasyon ile hücre ve misellerden ayrılmıştır. Elde edilen enzim filtratının pH'ı, renninin stabil olduğ pH olan 4.5' a ayarlanmıştır. Enzim bu pH'da, +4°C'de buzdolabında ve renkli şişeler içinde saklanmış ve yine bu pH'da aktivite kontrolleri yapılmıştır. Pıhtılaşacak sütün sıcaklığı, pH'ı ve CaCİ2 miktarının, renninin pıhtılaştırma aktivitesini etkilediği gözlenmiş ve en ıyı sonuçlar; pH 4-5.5'de, 55-60C'de ve 5x10 J M. CaCİ2.varlığında alınmıştır. Fakat en iyi pıhtı yapısı 30-40°C arasında oluşmuş; bu nedenle de süt 35°C'ye kadar ısıtılmıştır. Ayrıca hem pH 4.2-5.4 arasında protein yapısında olmayan diğer azotlu bileşikler de çökeceğinden ve hem de rennin K-kazeinin fenilalanin-metiyonin bandını pH 6-7 arasında kopardığından, sütün pH'ı 6.0' ya ayarlanmıştır. Berridge'nin (1952) belirttiği yönteme göre hazırlanan süt örneklerine enzim solüsyonu ilave edilmiş ve renninin sütü pıhtılaştırma süresi 40 sn. olarak bulunmuştur. Pıhtılaştırma aktivitesi ise, Soxhlet formülü kullanılarak 600 SU/ml. olarak hesaplanmıştır
89 7. SUMMARY In this study, the ability of rennin production of Mucor puslllus CBS 183. 67 strain which has grown on wheat bran using surface culture fermentation was examined. This investigation was conducted in three main stages as the processes previous of fermentation, fermentation period and the post fermentation processes. The processes previous of fermentation stage includes the activation of culture and the preparation of inoculum. In order to activate the organism, the Iyofilize culture was opened in 1-2 ml. of malt-peptone solution and it was grown in YPSSA medium at 37°C for 5 days period. Later on, this culture was transferred to slant agars containing same medium and incubated at 37°C for 5 days period for sporulation. The spore suspantion was prepared washing by sodium lauril sulfate and it was prepared as 1x10^ spores /ml. in number. The wheat bran medium which has wheat flour as carbon source and skim milk powder as nitrogen source was used for fermantation as basic substrate. This substrate was transferred to 500 ml. erlenmayar flasks and 15 ml. acidic mineral salt solution having pH 1 was added to this. Fermentation was experimented at various periods and temperatures and the best activity (600 SU/ml) was obtained at 37°C at the end of 72 hours. As a matter of fact, the90 highest enzyme activities were obtained by organic nitrogen sources. The post fermentation processes stage includes the exraction of rennin and the calculation of the enzymatic activity. At the end of the fermentation, wheat bran medium was washed by mixing with 200 ml. of top water and the solution containing rennin was purified from cells and mycelia by using filtration. The pH of the enzyme filtrate was adjusted to 4.5 since rennin is stabil in this pH. This enzyme was stored at about this pH in the colored bottles at +4°C in the refrigerator and the activity controls were also made at about this pH. The temperature, pH and amount of CaCİ2 of milk which will be clotted, have affected clotting activity of this enzyme and the best results were obtained at pH 4.5-5, 55-60°C and 5xl0~3 M. CaCİ2. But, the best clot type was formed at 30-40°C, for this reason the milk was heated until 35°C. In addition, since the other nitrogen compounds which are not protein in structure precipitate at pH 4-5.5 and rennin split phenilalanin-methionin bond of K-casein at pH 6-7, the pH of milk was adjusted to 6.0. To the milk samples prepared according to Berridge Method (1952), the enzyme solution was added and the milk clotting period of rennin was found as 40 second. The milk clotting activity was also calculated as600 SU/ml. by using Soxhlet formula.
89 7. SUMMARY In this study, the ability of rennin production of Mucor puslllus CBS 183. 67 strain which has grown on wheat bran using surface culture fermentation was examined. This investigation was conducted in three main stages as the processes previous of fermentation, fermentation period and the post fermentation processes. The processes previous of fermentation stage includes the activation of culture and the preparation of inoculum. In order to activate the organism, the Iyofilize culture was opened in 1-2 ml. of malt-peptone solution and it was grown in YPSSA medium at 37°C for 5 days period. Later on, this culture was transferred to slant agars containing same medium and incubated at 37°C for 5 days period for sporulation. The spore suspantion was prepared washing by sodium lauril sulfate and it was prepared as 1x10^ spores /ml. in number. The wheat bran medium which has wheat flour as carbon source and skim milk powder as nitrogen source was used for fermantation as basic substrate. This substrate was transferred to 500 ml. erlenmayar flasks and 15 ml. acidic mineral salt solution having pH 1 was added to this. Fermentation was experimented at various periods and temperatures and the best activity (600 SU/ml) was obtained at 37°C at the end of 72 hours. As a matter of fact, the90 highest enzyme activities were obtained by organic nitrogen sources. The post fermentation processes stage includes the exraction of rennin and the calculation of the enzymatic activity. At the end of the fermentation, wheat bran medium was washed by mixing with 200 ml. of top water and the solution containing rennin was purified from cells and mycelia by using filtration. The pH of the enzyme filtrate was adjusted to 4.5 since rennin is stabil in this pH. This enzyme was stored at about this pH in the colored bottles at +4°C in the refrigerator and the activity controls were also made at about this pH. The temperature, pH and amount of CaCİ2 of milk which will be clotted, have affected clotting activity of this enzyme and the best results were obtained at pH 4.5-5, 55-60°C and 5xl0~3 M. CaCİ2. But, the best clot type was formed at 30-40°C, for this reason the milk was heated until 35°C. In addition, since the other nitrogen compounds which are not protein in structure precipitate at pH 4-5.5 and rennin split phenilalanin-methionin bond of K-casein at pH 6-7, the pH of milk was adjusted to 6.0. To the milk samples prepared according to Berridge Method (1952), the enzyme solution was added and the milk clotting period of rennin was found as 40 second. The milk clotting activity was also calculated as600 SU/ml. by using Soxhlet formula.
Açıklama
Bu tezin, veri tabanı üzerinden yayınlanma izni bulunmamaktadır. Yayınlanma izni olmayan tezlerin basılı kopyalarına Üniversite kütüphaneniz aracılığıyla (TÜBESS üzerinden) erişebilirsiniz.
Anahtar Kelimeler
Biyoloji, Biology, Fermentasyon, Fermentation, Renin, Renin
