Streptococcus zooepidemicus ile hyaluronik asit üretiminde optimum koşulların belirlenmesi
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Dosyalar
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Bursa Teknik Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Hyaluronik asit (HA) geleneksel olarak kimyasal yollarla horoz ibiği, göbek kordonu gibi hayvansal kaynaklardan saflaştırılmaktayken, bu yöntemlerin kaynakların sınırlı olması, viral enfeksiyon riski taşıması ve HA'nın yıkıcı enzimlerle kontaminasyona maruz kalması gibi sorunlara yol açtığı bilinmektedir. Bu nedenlerle son yıllarda mikrobiyal fermantasyon yoluyla HA üretimi tercih edilmekte ve protokol geliştirme çalışmaları devam etmektedir. Bu çalışma kapsamında HA üretiminde genel üretici olduğu bilinen Streptococcus zooepidemicus ile çalışmalar gerçekleştirilmiştir. S. zooepidemicus ile HA üretimi üzerinde etkisi olduğu düşünülen pH ve sıcaklık gibi kültür şartları eski protokoller yerine tam kontrollü (oksijen, sıcaklık, pH ve karıştırma) paralel fermentör sistemi kullanılarak farklı değerler uygulanarak denenmiştir. Beş saatlik fermantasyon süresi sonucunda oluşan HA miktarları karbazol yöntemiyle belirlenerek farklı değerlerde uygulanan kültür şartları arasında karşılaştırmalar yapılmıştır. Fermantasyonla HA üretimine pH etkisi incelendiğinde HA üretiminin pH 6,5 ve pH 7 değerlerinde pH 7,5 ve pH 8'e göre yüksek olduğu görülmüştür. HA üretimine pH etkisi incelendiğinde pH 6,5, pH 7, pH 7,5 ve pH 8 değerleri arasında HA üretimleri açısından istatistiksel olarak herhangi bir farkın olmadığı görülmüştür (Duncan Test, P>0,05). HA üretimine sıcaklık etkisi incelendiğinde en yüksek HA üretimi 35°C ve 37°C'de elde edilmesine rağmen kullanılan farklı sıcaklık değerleri arasında istatistiki bir fark bulunamamıştır (Duncan Test, P>0,05). Farklı sıcaklık değerlerinin denendiği gruplar parametrik olmayan test ile de karşılaştırılmıştır, ancak gruplar arasında önemli bir fark tespit edilememiştir (Kruskal Wallis Testi, P>0,05). HA üretiminde en az varyasyon 32°C ve 35°C sıcaklıklarında gözlemlenmiştir. En yüksek HA üretimi 37°C sıcaklıkta ve pH 6,5'te olmuştur. Fermantasyon boyunca her saatte numune alınıp bakteri sayıları izlenerek farklı pH ve sıcaklık değerlerinde bakteri sayısı değişimi karşılaştırılmıştır. Her bir deney grubunda fermantasyon ortamındaki bakteri sayısı arttıkça oluşan HA miktarının da arttığı belirlenmiştir. Deney grupları incelendiğinde bakteri sayısı artışının en yüksek 37°C sıcaklıkta ve pH 7'de olduğu görülmüştür. Ön fermantasyon aşamasının incelendiği bu çalışmada farklı pH ve sıcaklık değerlerinde HA miktarındaki eğilim belirlenmiştir.
While hyaluronic acid (HA) is traditionally purified from animal sources such as cockbones, umbilical cord by chemical means, these methods are known to cause problems such as limited resources, risk of viral infection, and contamination of HA with destructive enzymes. For these reasons, HA production is preferred by microbial fermentation and protocol development studies are still going on. Within the scope of this study, studies were carried out with Streptococcus zooepidemicus, which is known to be general producer in HA production. The culture conditions such as pH and temperature, which are thought to have an effect on the production of HA with S. zooepidemicus, have been tested by applying different values using a fully controlled (oxygen, temperature, pH and mixing) parallel fermenter system instead of the old protocols. The amount of HA formed as a result of the five hour fermentation period was determined by the carbazole method and the different culture conditions were compared. When the pH effect of HA production by fermentation was examined, HA production was found to be higher than pH 7,5 and pH 8 in pH 6,5 and pH 7. When pH effect was examined for HA production, it was seen that there was no statistically any difference between the pH 6,5, pH 7, pH 7,5 and pH 8 in terms of HA production (Duncan Test, P>0,05). When the temperature effect of HA production was examined, although the highest HA production was obtained at 35°C and 37°C, no statistical difference was found amongst the different temperature values used (Duncan Test, P>0,05). Groups with different temperature values were also compared with the nonparametric test, but there was no significant difference between the groups (Kruskal Wallis Test, P> 005). The lowest variation in HA production was observed at 32°C and 35°C. The highest HA production was at 37°C and pH 6,5. Bacterial count changes in different pH and temperature values were compared by taking samples at every hour during fermentation. In each experimental group, it was determined that as the number of bacteria in fermentation environment increases, the amount of formed HA increased. When the experimental groups were examined, it was observed that the number of bacteria increased at the highest 37°C and pH 7. In this study where pre-fermentation stage was examined, the trend of HA in different pH and temperature values were determined.
While hyaluronic acid (HA) is traditionally purified from animal sources such as cockbones, umbilical cord by chemical means, these methods are known to cause problems such as limited resources, risk of viral infection, and contamination of HA with destructive enzymes. For these reasons, HA production is preferred by microbial fermentation and protocol development studies are still going on. Within the scope of this study, studies were carried out with Streptococcus zooepidemicus, which is known to be general producer in HA production. The culture conditions such as pH and temperature, which are thought to have an effect on the production of HA with S. zooepidemicus, have been tested by applying different values using a fully controlled (oxygen, temperature, pH and mixing) parallel fermenter system instead of the old protocols. The amount of HA formed as a result of the five hour fermentation period was determined by the carbazole method and the different culture conditions were compared. When the pH effect of HA production by fermentation was examined, HA production was found to be higher than pH 7,5 and pH 8 in pH 6,5 and pH 7. When pH effect was examined for HA production, it was seen that there was no statistically any difference between the pH 6,5, pH 7, pH 7,5 and pH 8 in terms of HA production (Duncan Test, P>0,05). When the temperature effect of HA production was examined, although the highest HA production was obtained at 35°C and 37°C, no statistical difference was found amongst the different temperature values used (Duncan Test, P>0,05). Groups with different temperature values were also compared with the nonparametric test, but there was no significant difference between the groups (Kruskal Wallis Test, P> 005). The lowest variation in HA production was observed at 32°C and 35°C. The highest HA production was at 37°C and pH 6,5. Bacterial count changes in different pH and temperature values were compared by taking samples at every hour during fermentation. In each experimental group, it was determined that as the number of bacteria in fermentation environment increases, the amount of formed HA increased. When the experimental groups were examined, it was observed that the number of bacteria increased at the highest 37°C and pH 7. In this study where pre-fermentation stage was examined, the trend of HA in different pH and temperature values were determined.
Açıklama
Anahtar Kelimeler
Biyomühendislik, Bioengineering, Biyoteknoloji, Biotechnology
