Gıda işleme sürecinde ortaya çıkan domates çekirdeği atığından protein, protein hidrolizatı ve peptitlerin eldesi, karakterizasyonu ve biyoaktivitelerinin belirlenmesi
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Mevcut çalışmada, gıda atıklarının yol açtığı çevresel zararlar ve bu zararın
yanı sıra ortaya çıkan ciddi ekonomik kayıplar göz önüne alınarak, domates işleme
sanayisinde önemli bir atık olan domates çekirdeğine odaklanılmış ve domates
çekirdeğinin gıda kaynaklı protein ve protein hidrolizatı/biyoaktif peptit eldesi için
sahip olduğu potansiyel araştırılmıştır.
Bu hedef doğrultusunda öncelikle domates çekirdeğinden protein eldesi için
ultrasonikasyon destekli ekstraksiyon yöntemi kullanılmış ve ekstraksiyon işlemi
Cevap Yüzey Yöntemi ile optimize edilmiştir.
İkinci aşamada, domates çekirdek proteininin enzimatik hidrolizi için enzim
seçiminde in silico yaklaşımdan faydalanılmış ve in silico analiz sonucu seçilen
pepsin enzimi ile hidroliz işlemi gerçekleştirilmiştir.
Üçüncü aşamada, elde edilen protein ve protein hidrolizatının, fizikokimyasal
özellikleri, antioksidan, antihipertansif ve antidiyabetik özellikleri araştırılmıştır.
Domates çekirdek proteininin antioksidan ve ACE inhibitör aktivitesine sahip
olduğu fakat DPP-IV inhibitör aktivitesinin bulunmadığı belirlenirken, protein
hidrolizatında bu üç biyoakitivite de tespit edilmiş ve hidroliz işleminin başlangıç
proteinine göre antioksidan, ACE inhibitör ve DPP-IV inhibitör aktiviteyi arttırdığı
belirlenmiştir. Ayrıca, hidroliz öncesi uygulanan ön işlemlerin (ısısal ve
ultrasonikasyon) elde edilen hidrolizatın yapısına ve biyoaktivitesine olan etkisi bu aşamada ayrı bir alt hedef olarak araştırılmıştır. Uygulanan ön işlemlerin hidrolizat
yapısını değiştirdiği ve hidrolizatın sahip olduğu antioksidan, ACE inhibitör ve
DPP-IV inhibitör aktivitesi üzerinde negatif bir etkisi olduğu belirlenmiştir.
Dördüncü aşamada, elde edilen protein hidrolizatının saflaştırılması ve
fraksiyonlarına ayrılması amacıyla sırasıyla ultrafiltrasyon ve jel filtrasyon
kromatografisi teknikleri arka arkaya uygulanmış ve ayrılan hidrolizat
fraksiyonlarının moleküler ağırlık dağılımları (SDS-PAGE), antioksidan, ACE
inhibitör ve DPP-IV inhibitör aktiviteleri belirlenmiştir. Ultrafiltrasyon sonucu
protein hidrolizatının 10 kDa’dan küçük moleküler büyüklüğüne sahip fazının (<10
kDa) daha yüksek biyoaktiviteye sahip olduğu tespit edilmiş ve bu faz jel filtrasyon
kromatografisi ile 15 fraksiyona ayrılmıştır.
Beşinci aşamada, jel filtrasyon kromatografisi ile elde edilen 15 peptit
fraksiyonu içinden yüksek antioksidan, ACE inhibitör ve DPP-IV inhibitör
aktivitesi gösteren fraksiyon, içerdiği peptitlerin amino asit dizilimlerinin
belirlenmesi amacıyla LC-QTOF-MS ile analiz edilmiştir. Analiz sonucunda
potansiyel antioksidan, ACE inhibitör ve DPP-IV inhibitör aktivitesine sahip
olabilecek 24 peptit belirlenmiştir.
Çalışmanın sonucunda, meyve sebze işleme sektöründe önemli bir gıda atığı
olan domates çekirdeğinin, bitkisel bir protein ve biyoaktif peptit kaynağı olarak
kullanılabileceği ve günümüzde önemi giderek artan bitkisel protein kaynakları için
bir alternatif oluşturabileceği kanaatine varılmıştır.
In the present study, considering the environmental damage caused by food waste and the serious economic losses that arise as well as this damage, we focused on the tomato seed, which is an important waste in the tomato processing industry and the potential of tomato seed to obtain food-derived protein and protein hydrolyzate/bioactive peptide was investigated. In line with this goal, firstly, ultrasonication assisted extraction method was used to obtain protein from tomato seed and the extraction process was optimized by Response Surface Method. In the second step, the in silico approach was used in the selection of enzymes for the enzymatic hydrolysis of tomato seed protein and the hydrolysis process was performed with the pepsin enzyme selected as a result of in silico analysis. In the third stage, the physicochemical properties, antioxidant, antihypertensive and antidiabetic properties of the obtained protein and protein hydrolyzate were investigated. While it was determined that tomato seed protein has antioxidant and ACE inhibitory activity but not DPP-IV inhibitory activity, these three bioactivities were determined in protein hydrolyzate and it was determined that the hydrolysis process increased antioxidant, ACE inhibitor and DPP-IV inhibitory activity compared to the parent protein. In addition, the effect of pre-treatments (thermal and ultrasonication) applied before hydrolysis on the structure and bioactivity of the obtained hydrolyzate was investigated as a separate sub-target at this stage. It was determined that the pre-treatments applied changed the hydrolyzate structure and had a negative effect on the antioxidant, ACE inhibitor and DPP-IV inhibitory activity of the hydrolyzate. In the fourth step, ultrafiltration and gel filtration chromatography techniques were applied consecutively in order to purify the obtained protein hydrolyzate and separate it into its fractions, and the molecular weight distributions (SDS-PAGE), antioxidant, ACE inhibitor and DPP-IV inhibitory activities of the separated hydrolyzate fractions were determined. As a result of ultrafiltration, it was determined that the phase of the protein hydrolyzate with a molecular size less than 10 kDa (<10 kDa) had higher bioactivity and this phase was separated into 15 fractions by gel filtration chromatography. In the fifth step, the fraction content showing high antioxidant, ACE inhibitor and DPP-IV inhibitory activity among the 15 peptide fractions obtained by gel filtration chromatography was analyzed by LC-QTOF-MS to determine the amino acid sequences of the peptides. As a result of the analysis, 24 peptides that may have potential antioxidant, ACE inhibitor and DPP-IV inhibitory activity were determined. As a result of the study, it was concluded that tomato seed, which is an important food waste in the fruit and vegetable processing sector, can be used as a vegetable protein and bioactive peptide source and can be an alternative for vegetable protein sources, which are increasingly important today.
In the present study, considering the environmental damage caused by food waste and the serious economic losses that arise as well as this damage, we focused on the tomato seed, which is an important waste in the tomato processing industry and the potential of tomato seed to obtain food-derived protein and protein hydrolyzate/bioactive peptide was investigated. In line with this goal, firstly, ultrasonication assisted extraction method was used to obtain protein from tomato seed and the extraction process was optimized by Response Surface Method. In the second step, the in silico approach was used in the selection of enzymes for the enzymatic hydrolysis of tomato seed protein and the hydrolysis process was performed with the pepsin enzyme selected as a result of in silico analysis. In the third stage, the physicochemical properties, antioxidant, antihypertensive and antidiabetic properties of the obtained protein and protein hydrolyzate were investigated. While it was determined that tomato seed protein has antioxidant and ACE inhibitory activity but not DPP-IV inhibitory activity, these three bioactivities were determined in protein hydrolyzate and it was determined that the hydrolysis process increased antioxidant, ACE inhibitor and DPP-IV inhibitory activity compared to the parent protein. In addition, the effect of pre-treatments (thermal and ultrasonication) applied before hydrolysis on the structure and bioactivity of the obtained hydrolyzate was investigated as a separate sub-target at this stage. It was determined that the pre-treatments applied changed the hydrolyzate structure and had a negative effect on the antioxidant, ACE inhibitor and DPP-IV inhibitory activity of the hydrolyzate. In the fourth step, ultrafiltration and gel filtration chromatography techniques were applied consecutively in order to purify the obtained protein hydrolyzate and separate it into its fractions, and the molecular weight distributions (SDS-PAGE), antioxidant, ACE inhibitor and DPP-IV inhibitory activities of the separated hydrolyzate fractions were determined. As a result of ultrafiltration, it was determined that the phase of the protein hydrolyzate with a molecular size less than 10 kDa (<10 kDa) had higher bioactivity and this phase was separated into 15 fractions by gel filtration chromatography. In the fifth step, the fraction content showing high antioxidant, ACE inhibitor and DPP-IV inhibitory activity among the 15 peptide fractions obtained by gel filtration chromatography was analyzed by LC-QTOF-MS to determine the amino acid sequences of the peptides. As a result of the analysis, 24 peptides that may have potential antioxidant, ACE inhibitor and DPP-IV inhibitory activity were determined. As a result of the study, it was concluded that tomato seed, which is an important food waste in the fruit and vegetable processing sector, can be used as a vegetable protein and bioactive peptide source and can be an alternative for vegetable protein sources, which are increasingly important today.
Açıklama
Anahtar Kelimeler
Atık Değerlendirme, Domates Çekirdek Proteini, Biyoaktif Peptit, Ultrasonikasyon Destekli Ekstraksiyon, Antioksidan Aktivite, ACE İnhibitör Aktivite, DPP-IV İnhibitör Aktivite, Waste Utilization, Tomato Seed Protein, Bioactive Peptide, Ultrasonication Assisted Extraction, Antioxidant Activity, ACE Inhibitor Activity, DPP-IV Inhibitory Activity
