Kuzu ayaklarından bazı ekstrasellüler matriks bileşenlerinin (Kolajen ve GAG) ultrason destekli ekstraksiyonu ve karakterizasyonu
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Dosyalar
Tarih
2019
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında, ekstrasellüler matriks bileşenlerinden kolajen ve glukozaminoglikanların (GAG) ultrason destekli esktraksiyonu ve saflaştırılması hedeflenmiştir. Çalışma kapsamında, kuzu ayakları, hammadde kaynağı olarak kullanılmış ve en yüksek düzeyde verim eldesi için uygun ekstraksiyon koşulları belirlenmiştir. Farklı sıc aklık ve sürelerde uygulanan ultrason işleminin kolajen ve GAG yapısı üzerine etkileri incelenmiştir. Çalışmanın ilk aşamasında, kolajen eldesi için uygun ekstraksiyon parametreleri belirlenmiştir. Ekstraksiyon işleminde enzimatik hidroliz yöntemi seçilmiş ve pepsin enzimi kullanılmıştır. Ön işlemleri tamamlanan kuzu ayakları, ekstraksiyon işlemi için % 0.1 (w/v) pepsin içeren 1:20 (w/v) oranda %5'lik laktik asit çözeltisi içinde 6 saat bekletilmiştir. Ardından 3 farklı sıcaklık (20± 2°C, 25± 2°C ve 30± 2°C) ve sürede (20, 40, 60 dk.) ultrason destekli ekstraksiyon gerçekleştirilmiştir. Ekstraksiyon işleminin ardından örnekler saflaştırma işlemi için diyaliz edilerek -56°C'de 32 sa boyunca dondurarak kurutulmuştur. En yüksek kolajen konsantrasyonuna 25°C-60 dk parametresinde (5061,75±171,41 μg/μl) ulaşılmıştır. SDS-PAGE elektroforez ve FTIR analizi sonuçları, farklı sıcaklık ve sürelerde uygulanan ultrason işleminin, kolajen yapısına zarar vermediğini ve tüm kolajenlerin yapısal bütünlüğünü koruduğunu göstermiştir. SEM analizi sonucunda, tüm kolajen örneklerinin lifli ve gözenekli yapıya sahip olduğu gözlenmiştir. viii Kolajen örneklerinin b* ve L* değerleri arasındaki fark istatistiksel olarak anlamlı bulunmuştur. Örneklerin a* değerleri arasındaki fark ise sadece dört grup için anlamlı bulunmuştur (p<0.05). Çalışmanın ikinci aşamasında, GAG eldesi için uygun ekstraksiyon parametreleri belirlenmiştir. Ekstraksiyon işleminde papain enzimi ve sodyum asetat çözeltisi kullanılmıştır. Katı:çözgen oranı 1:20 (w/v), enzim miktarı (5 mg papain / 1 gr kuzu ayağı), sıcaklık (60°C) ve ekstraksiyon süresi 20 saat olarak belirlenmiştir. 20 saat ekstraksiyon işleminin ardından ultrason uygulamasının etkilerinin incelenmesi amacıyla, 3 farklı sıcaklık (60± 2°C, 65± 2°C ve 70± 2°C) ve sürede (20, 40, 60 dk.) ultrason destekli ekstraksiyon gerçekleştirilmiştir. Ekstraksiyon işleminin ardından örnekler saflaştırma işlemi için diyaliz edilerek -56°C'de 24 sa boyunca dondurarak kurutulmuştur. En yüksek kondroitin sülfat konsantrasyonuna 60°C-20 dk parametresinde (471,38±49,96 mg/L) ulaşılmıştır. FTIR analizi sonucunda, farklı ekstraksiyon koşulları altında elde edilen GAG örnekleri ile standarda ait spektrumlarının benzerlik gösterdiği görülmüştür. SEM analizi sonuçları, GAG örneklerindeki kümeleşmiş yapının ultrason süresi ve sıcaklığına bağlı olarak değiştiğini göstermiştir. Genel olarak farklı sıcaklık ve sürelerde uygulana ultrason işleminin GAG örneklerinin renk değerleri üzerine anlamlı bir etkisi olmadığı görülmüştür.
In this thesis, ultrasound-assisted extraction and purification of collagen and glucosaminoglycans (GAG), one of the extracellular matrix components, was aimed. Within the scope of the study, lamb legs were used as raw material source and suitable extraction conditions were determined for obtaining the highest yield The effects of ultrasound treatment on collagen and GAG structure were investigated at different temperatures and durations. In the first stage of the study, suitable extraction parameters for the production of collagen were determined. Enzymatic hydrolysis method was selected for extraction and pepsin enzyme was used. The lambs which were pre-treated were left for 6 hours in a 1:20 (w / v) 5% lactic acid solution containing 0.1% (w / v) pepsin for extraction. Then, ultrasound-assisted extraction was performed at 3 different temperatures (20± 2°C, 25± 2°C and 30± 2°C) and time (20, 40, 60 min). After extraction, the samples were dialyzed for purification and freeze-dried at -56 ° C for 32 hours. The highest collagen parameter was reached at 25°C-60 min (5061,75±171,41 μg/μl). The results of SDS-PAGE electrophoresis and FTIR analysis showed that ultrasound applied at different temperatures and times did not damage the collagen structure and retain the structural integrity of all collagen. As a result of SEM analysis, it was observed that all collagen samples have fibrous and porous structure. The difference between b * and L * values of collagen samples was statistically significant x (p<0.05). The difference between the a * values of the samples was significant only for the four groups. In the second stage of the study, suitable extraction parameters for GAG were determined. Papain enzyme and sodium acetate solution were used in the extraction process. The solid to solvent ratio was 1:20 (w / v), the amount of enzyme (5 mg papain / 1 g lamb's foot), temperature (60°C) and extraction time was determined as 20 hours. After 20 hours of extraction, ultrasound assisted extraction was performed in 3 different temperatures (60± 2°C, 65± 2°C and 70± 2°C) and time (20, 40, 60 min) to examine the effects of ultrasound application. After extraction, the samples were dialyzed for purification and freeze-dried at -56°C for 24 hours. The highest chondroitin sulfate concentration was reached at 60°C-20 min (471,38±49,96 mg/L). As a result of FTIR analysis, it was seen that GAG samples obtained under different extraction conditions were similar to the spectra of the standard. The results of SEM analysis showed that the clustered structure of GAG samples changed depending on the ultrasound time and temperature.
In this thesis, ultrasound-assisted extraction and purification of collagen and glucosaminoglycans (GAG), one of the extracellular matrix components, was aimed. Within the scope of the study, lamb legs were used as raw material source and suitable extraction conditions were determined for obtaining the highest yield The effects of ultrasound treatment on collagen and GAG structure were investigated at different temperatures and durations. In the first stage of the study, suitable extraction parameters for the production of collagen were determined. Enzymatic hydrolysis method was selected for extraction and pepsin enzyme was used. The lambs which were pre-treated were left for 6 hours in a 1:20 (w / v) 5% lactic acid solution containing 0.1% (w / v) pepsin for extraction. Then, ultrasound-assisted extraction was performed at 3 different temperatures (20± 2°C, 25± 2°C and 30± 2°C) and time (20, 40, 60 min). After extraction, the samples were dialyzed for purification and freeze-dried at -56 ° C for 32 hours. The highest collagen parameter was reached at 25°C-60 min (5061,75±171,41 μg/μl). The results of SDS-PAGE electrophoresis and FTIR analysis showed that ultrasound applied at different temperatures and times did not damage the collagen structure and retain the structural integrity of all collagen. As a result of SEM analysis, it was observed that all collagen samples have fibrous and porous structure. The difference between b * and L * values of collagen samples was statistically significant x (p<0.05). The difference between the a * values of the samples was significant only for the four groups. In the second stage of the study, suitable extraction parameters for GAG were determined. Papain enzyme and sodium acetate solution were used in the extraction process. The solid to solvent ratio was 1:20 (w / v), the amount of enzyme (5 mg papain / 1 g lamb's foot), temperature (60°C) and extraction time was determined as 20 hours. After 20 hours of extraction, ultrasound assisted extraction was performed in 3 different temperatures (60± 2°C, 65± 2°C and 70± 2°C) and time (20, 40, 60 min) to examine the effects of ultrasound application. After extraction, the samples were dialyzed for purification and freeze-dried at -56°C for 24 hours. The highest chondroitin sulfate concentration was reached at 60°C-20 min (471,38±49,96 mg/L). As a result of FTIR analysis, it was seen that GAG samples obtained under different extraction conditions were similar to the spectra of the standard. The results of SEM analysis showed that the clustered structure of GAG samples changed depending on the ultrasound time and temperature.
Açıklama
Anahtar Kelimeler
Ekstrasellüler Matriks, Kolajen, Glukozaminoglikan, Ultrason, Ekstraksiyon, Extracellular Matrix, Collagen, Glucosaminoglycan, Ultrasound, Extraction
