Nar kabuğundan enzim ve ultrason destekli ekstraksiyon yöntemi ile fenolik madde ve pektin elde edilmesi
Enzyme and ultrasound-assisted extraction of phenolic compounds and pectin from pomegranate peel
Yazar: ZEYNEP MERVE KAHVECİ
Danışman: DOÇ. DR. DERYA KAHVECİ KARINCAOĞLU
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı / Gıda Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2022
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Bitki fenolikleri = Plant phenolics ; Fenolik maddeler = Phenolic matters ; Nar = Pomegranate ; Pektin = Pectin ; Ultrason = Ultrasound ; Özütleme = Extraction

Özet:
Günümüzde, gıda kaynaklarının azalması, nüfusun hızla artışı gibi sebeplere bağlı olarak gıda atıklarının azaltılması ve atıklardan katma değeri yüksek bileşenlerin geri kazanımı önem kazanmaktadır. Geri kazanım alanında metotların geliştirilmesi ile birlikte, sürdürülebilir ve çevre dostu üretim kavramları da gündeme gelmeye başlamıştır. Çevre dostu metotlar arasında yer alan enzim ve ultrason destekli ekstraksiyon; çözgen kullanımını azaltarak, daha az enerji sarfiyatı sağlayarak ve verimi artırarak fayda sağlamayı amaçlamaktadır. Meyve sebzeler gıda atıklarının büyük bir çoğunluğunu oluşturduğu için geri kazanım konusunda pek çok çalışmaya konu olmaktadırlar. Nar kabuğun, meyvenin yaklaşık yarısını oluşturması ve değerli bileşenler bakımından zengin olması ile bu çalışmada tercih edilmiş; endüstriyel nar suyu üretiminden arta kalan nar kabuğundan fenolik madde ve pektin ekstraksiyonu için enzim ve ultrason destekli ekstraksiyon yöntemleri kullanılarak optimum koşullar belirlenmiştir. 
Öncelikle, enzimin hücre duvarını parçalaması özelliğinden faydalanılarak nar kabuğundan fenolik madde ekstraksiyonu için optimum enzim ön işlem süresi 60 dk, sonrasında etanol ile ekstraksiyon süresi ise 30 dk olarak belirlenmiştir. Çeşitli katı:sıvı oranı ve enzim konsantrasyonu seviyelerinde yapılan esktraksiyon sonucunda,optimum katı sıvı oranı 1:40, enzim konsantrasyonu %6 (%v/m) olarak belirlenmiştir ve bu koşullarda 329,5±37,8 mg GAE/g fenolik madde konsantrasyonu elde edilmiştir. Belirlenen bu optimum koşullarda elde edilen fenolik maddelerin profili HPLC ile çıkarılmış, baskın olarak punikalajin, gallik asit, ellagik asit ve (-)-apigallokateşin içerdiği tespit edilmiş ve antioksidan aktivite CUPRAC ve DPPH metotları ile tayin edilerek sırasıyla 3108,5±303 mg TE/g ve 1586±15,9 mg TE/g olarak tespit edilmiştir. Elde edilen fenolik madde verimi ve antioksidan aktivitesinin enzim kullanılmayan klasik yönteme kıyasla daha yüksek olduğu tespit edilmiştir. 
Projenin amacı çevre dostu bir metotla birden çok fraksiyonda geri kazanımı sağlamak olduğu için, fenolik madde ekstraksiyonu gerçekleştirildikten sonra kalan nar kabuğundan pektin ekstraksiyonu gerçekleştirilmiştir. Ultrason kullanımının kavitasyon etkisi ile hücre duvarını parçalayarak verimi artırdığı tespit edilmiş ve yapılan optimizasyon çalışmasında 1:20 katı sıvı oranı ve 45 dk süre ile gerçekleştirilen ultrason ön işlem sonucunda 3,7±0,002% ile maksimum verim elde edilmiştir. Elde edilen pektinin karakterizasyonu için galakturonik asit oranı ve esterleşme derecesi analiz edilmiştir. %18,2±1,4 galakturonik asit oranı ve %74±0,07 esterleşme derecesi elde edilmiş; yüksek metoksipektin olduğu ve şeker varlığında jelleşebileceği tespit edilmiştir. Bu kriterlere bakıldığında, geleneksel metot ile direk olarak nar kabuğundan elde edilen pektine kıyasla önemli bir farklılık olmadığı görülmüştür.  
Çalışma sonucunda, enzim ve ultrason ile ön işlem uygulamasının fenolik madde ve pektin verimini artırabileceği tespit edilmiştir.

Abstract:
Food waste refers to decrease in the quality and amount of food after reaching final consumer while food loss refers to decrease in the amount of food during production, transportation and storage. Nowadays, accesabilty to safe food becomes more important and depending on the reasons such as the decrease in food resources and the rapid increase in the population, taking precaution, reducing food waste and recycling high value-added components from waste become an significant issue. 
In order to take precaution, there are different initiatives in different countries to increase consciousness about the reasons of food waste, possible solutions and advices. Since waste of food also refers to the waste of sources like electric and water which is used during the production of wasted food, it is also important to decrease food waste to preserve sources. However, reuse of waste is still necessary for the remaining waste. There are different methods for the recycling of the food waste and continue to be developed with the increasing importance of this issue. With the development of methods in the field of recycling, the concepts of sustainable and environmentally friendly production began to become more important. 
Enzyme and ultrasound assisted extraction, which are among the environmentally friendly methods; aim to provide benefits by reducing solvent usage, providing less energy consumption and increasing efficiency. Food wastes contain different type of valuable substances according to their types. Pectin and phenolic compounds are two important compounds that can be extracted from food waste by this environmentally friendly methods. Commonly used methods to extract pectin and phenolic compounds are conventional extraction, supercritic fluid extraction, high pressure extraction, ultrasound assisted extraction, microwave assisted extraction and enzyme assisted extraction.
Since fruits and vegetables constitute the majority of food waste, they are the subject of many studies on recycling. Citrus peel, apple peel, carrot residue and grape peel are common examples that are used in many studies. Pomegranate peel was preferred in this study because it constitutes about half of the fruit and is rich in valuable components like pectin and phenolic compounds; optimum conditions were determined by using enzyme and ultrasound assisted extraction methods for phenolic substance and pectin extraction from pomegranate peel remaining from industrial pomegranate juice production.
In this study, it is planned to obtain higher efficiency than the conventionel method in the extraction of phenolic substance and pectin from the pomegranate peel, using enzymes and ultrasound assisted method and an improvement is expected in the quality parameters measured by the antioxidant activity of the obtained phenolic substance and the degree of esterification of pectin and galacturonic acid concentrations. There are studies in literature that are about extraction of phenolic compounds and pectin from pomegranate peel but there is no study about extraction of both compounds at once.
First of all, enzyme pretreatment was applied to pomegranate peel for 60 minutes at 50°C. Celluclast enzyme which contains cellulase was used to degrade cellulosic cell wall structure. Then, the mixture was waited in boiled water for 10 minutes to deactivate enzyme. After it was cooled down to room temperature, equal volume of ethanol with water was added. It was mixed for 30 minutes. During enzyme pretreatmend and extraction with ethanol, 1 ml sample is collected after 10, 20, 30 and 60 minutes. The same procedure was applied when ultrasound pretreatment was applied by itself and with enzymatic pretreatment. 
According to phenolic substance analysis results of these samples, decision to apply enzyme or ultrasound pretreatment was made and optimum time for enzymatic pretreatment and extraction with ethanol was determined. Maximum phenolic substance was obtained with use of ethanol pretreatment and ultrasound pretreatment. Since the difference between results for enzymatic pretreatment and ultrasound pretreatment was very low and they were both higher then the combination of two pretreatments, in order to reduce energy consumption, it was decided to apply only enzymatic pretreatment. By this decision, advantage of the enzyme's ability to break down the cell wall was taken. Moreover, lower yield or no detectable increase was observed when pretreated with ultrasound because increase of temperature and pressure around cavity, which was formed as a result of ultrasound caused decrease in phenolic compounds. The optimum enzyme pretreatment time for phenolic substance extraction from pomegranate peel was determined as 60 minutes because phenolic substance concentration increased with time and the extraction time with ethanol was determined as 30 minutes. Although there was a little decrease in phenolic substance contentration, 30 minutes was chosen because decrease was very low and it was seen in the literature that generally, phenolic substance concentration increases with increasing time. To determine optimum extraction conditions, 1:10, 1:25, 1:40 solid-liquid ratios and 2, 6, 10% enzyme concentrations were used. As a result of the extraction at various solid:liquid ratio and enzyme concentration levels, the optimum solid-liquid ratio was determined as 1:40, enzyme concentration was determined as 6% (v/w). It was observed that phenolic substance concentration increases with increasing solid-liquid ratio. It was also seen that increase in the concentration of enzyme affected results to a certain level due to limited substrate. Under determined optimum conditions 329.5±37.8 mg GAE/g phenolic compound concentration has been obtained as the maximum. It was seen that phenolic compound concentration was higher when enzyme was used compared to unused.
Antioxidant activity of extract at optimum conditions was determined with CUPRAC and DPPH methods. Antioxidant activity was determined as  3108.5±303 mg TE/ g by CUPRAC method and 1586±15.9 mg TE/g by DPPH method. When enzyme was no used, 1327,4±58,5 and 951,8±69,9 TE/g  antioxidan activity were obtained relatively. It was determined that the obtained antioxidant activity were higher than the classical method without using enzymes because cell wall was broken down by enzyme application and antioxidant activity increased with increase in components indicating antioxidant activity.
The profile of the phenolic compounds obtained under these optimum conditions was determined by HPLC, it was determined that the extract contain predominantly punicalagin, gallic acid, ellagic acid and (-)-apigallocatechin. Punicalagin had the highest concentration among the others. A greater variety of phenolic substances were obtained by enzyme assisted extraction, and the same ones were detected in higher concentrations compared to conventional method. 
Since the aim of the project was to recover multiple fractions with an environmentally friendly method, pectin was extracted from the remaining pomegranate peel after the phenolic substance extraction was carried out. Pectin was extracted from the remaining peel in 1% citric acid solution at 80°C for 60 minutes. Then the mixture was cooled down to room temperature and then equal volume of ethanol with water was added. Pectin was precipitated at 4°C for 60 minutes. The same procedure was applied after ultrasound pretreatment at 80°C for 60 minutes . 3±0,006% pectin yield was obtained with ultrasound pretreatment while 2±0,006% pectin yield was obtained with convetional method. It was decided to apply ultrasound pretreatment due to its cavitation effect which was resulted in break down of cell wall and release of pectin molecules esily. 
In the optimization study, 1:10, 1:20, 1:30 solid-liquid ratio and 15, 30, 45 minutes ultrasound pretreatment times were applied. Not too long durations were chosen because long time application of ultrasound may results in hydrolysis of pection molecules and decrease in the pectin yield. The maximum pectin efficiency of 3.7±0.002% was obtained as a result of the ultrasound pretreatment carried out with 1:20 solid-liquid ratio for 45 minutes. Increase of solid-liquid ratio to a certain level and increase in ultrasound pretreatrment time affected pectin yield in a positive direction. However, when pectin was extracted directly from pomegranate peel %13 pectin yield was obtained. Precipitated pectin was collected during phenolic substance extraction but total amount of yield showed that a part of pectin was lost during phenolic substance extraction.
For the characterization of the obtained pectin, galacturonic acid ratio and degree of esterification were analyzed. These criteria are important to determine gel properties of pectin. Galacturonic acid concentration of 18.2±2% and an esterification degree of 74±0.07% were obtained. It has been determined that it is high methoxypectin, can gel at low pH and  in the presence of sugar due to its degree of esterification. When these criteria were examined, it was seen that there was no significant difference compared to the pectin obtained directly from the pomegranate peel with the traditional method. Galacturonic acid contentration of obtained pectin is lower than the galacturonic acid concentration of pectin obtained with citric acid solution. 
As a result of this study, it was determined that pretreatment with proper enzyme may increase the yield of phenolic compound concentration and therefore antioxidant activity. However, pectin yield was less then expected due to loss during phenolic compound extraction. Due to decrease in the yield of pectin, galacturonic acid concentration was also decreased. Nevertheless, degree of esterification was found very similar to pectin obtained directly from pomegranate peel. 
All in all, high sensitivity of the obtained compounds makes it difficult to implement this type of process on a large scale production. However, due to increasing demand, sustainability and environmental concerns, recycling with environmentally friendly methods will become more important. Based on the findings obtained in this study, studies can be carried out to obtain valuable compounds with higher yield or better quality characteristics.