Zeytinyağı koku giderme atığından modifiye edilmiş soxhlet ekstraksiyon yöntemi ile skualenin geri kazanılması ve tepki yüzey metodolojisi ile optimizasyonu
Recovery of squalene by modified soxhlet extraction from olive oil deodorizer distillate and optimization with response surface methodology
Yazar: BUKET KOÇAK
Danışman: PROF. DR. MELEK TÜTER ; PROF. DR. SİBEL TİTİZ SARGUT
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Mühendisliği Ana Bilim Dalı / Kimya Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2019
Konu: Kimya Mühendisliği = Chemical Engineering

Özet:
Yağlar, insan beslenmesinde karbonhidrat ve proteinlerle birlikte alınması zorunlu olan temel besin öğelerindendir.  Toplumlarda ortaya çıkabilen sağlık sorunları ile beslenme rejimleri arasındaki ilişki araştırıldığında, en fazla sorgulanan gıda bileşeni yağlardır. Bu yüzden, beslenmede; yağ çeşidi seçiminden tüketim şekline kadar uzanan her aşamada daha bilinçli olmak gerekir.  Yağlar kökenlerine göre hayvansal ve bitkisel yağlar olmak üzere iki gruba ayrılmaktadır. Bitkisel yağlar, istenmeyen bileşenlerin uzaklaştırılması ya da miktarlarının kabul edilebilir düzeye düşürülebilmesi amacıyla rafine edilirler. Yapışkan maddeler, serbest yağ asitleri, oksidasyon ürünleri, renk maddeleri gibi istenmeyen bileşenlerin yağdan uzaklaştırılmasında, kimyasal ve fiziksel rafinasyon yöntemleri kullanılmaktadır. Rafinasyon işlemi reçine giderme, asitlik giderme, ağartma, koku giderme gibi aşamalardan oluşmaktadır. Ancak bu aşamalarda, işlem koşullarına da bağlı olarak tokoller, fenolik maddeler, bitkisel steroller, Squalen gibi insan sağlığı açısından önemli olan bazı biyoaktif bileşenlerde de nitel ve nicel kayıplar meydana gelmektedir. Bu kaybın gerçekleştiği esas kademe olan koku giderme aşamasında squalen ve tokoferol gibi önemli biyoaktif maddeler koku giderme atığına geçmektedir. Squalen ve tokoferolün koku giderme atığından geri kazanılması gıda, kozmetik ve ilaç sektörlerinde büyük önem taşımaktadır. 
Bu çalışmada, koku giderme atığından yüksek verimle ve yüksek saflıkta skualenin geri kazanılması üzerine çalışmalar yürütülmüştür. Modifiye edilmiş Soxhlet ektraktörü ile gerçekleştirilen deneysel çalışmalarda, zeytinyağı koku giderme atığında (KGA) bulunan  yağ asidi steril esterleri (FASE) ve skualen gibi polar olmayan bileşikler (NPLF) bir fraksiyonda; tokoferoller, serbest fitosteroller, triaçilgliseroller (TAG) ve serbest yağ asitleri (FFA) gibi polar bileşikler başka bir fraksiyonda verimli bir şekilde ayrılmıştır. Bu amaçla, öncelikle KGA silika jel üzerine absorbe edilmiş daha sonra ektraksiyon işlemi uygulanmıştır. Ekstraksiyona etki ettiği düşünülen yağ/silika jel oranı, sıcaklık, süre parametreleri incelenmiş, bu parametrelerin optimizasyonu Tepki Yüzey Metodolojisi (TYM) kullanılarak yapılmış ve  uygun proses koşulları belirlenmiştir.
Zeytinyağı koku giderme atığının içerdiği Skualen miktarını belirlemek için ince yüzey kromatografisi/alev iyonizasyon dedektörü (TLC-FID) kombine sistemi kullanılmıştır. Bu sistemle zeytinyağı KGA'nın ağırlıkça %1,5 skualen içerdiği bulunmuş ve konsantrasyonu 0.154 g/L olarak belirlenmiştir. TYM yöntemiyle gerçekleştirilen optimizasyon çalışmaları sonucunda en yüksek Skualen konsantrasyonu 0.149 g/L olup, 1/3 yağ/silika oranı, 65°C sıcaklık ve 8 saat çalışma koşullarında elde edilmiştir. 
Optimum koşullarda yürütülen Soxhlet ektraksiyonu sonucunda elde edilen ve 0.149 g/L skualen içeren NPLF fraksiyonuna kolon kromatografisi uygulanarak, skualenin başlangıca göre ne kadar arttığı belirlenmiştir.  Kolon kromatografisi sonucunda başlangıçta %1.5 olan skualen miktarının %18'e yükseldiği görülmüştür.

Abstract:
Lipids are essential nutrients that must be taken together with carbonhydrates and proteins in human nutrition. When the relationship between nutritional regimes and health problems in societies is investigated, the most questioned food component is fat. Therefore, in nutrition; it is necessary to be more conscious at every stage from the selection of oil type to the consumption. Lipids are divided into two groups as animal and vegetable oils according to their origin source. Vegetable oils are refined in order to remove undesirable components or reduce their amounts to an acceptable level. Chemical and physical refining methods are used to remove undesirable components such as adhesives, free fatty acids, oxidation products and odor components. In both chemical and physical methods, refining involves different processes in which degumming is the first stage process and is applied for removal of phospholipids (which are also known as resins or gums), waxes oxidation agents and some other impurities. Notralization is applied for removing free fatty acids or reduce to acceptable level. Bleaching is a process that increase the oxidative stability of the oil. In this process, many minor impurities are removed using bleach clays or charcoal. The final step is deodorization and free fatty acids (FFA) and other volatile odor and aroma substances are almost completely removed. he essential properties of human health (tocopherol, tocotrienol, squalene, sterol, acylglycerol and FFAs) are subjected to qualitative and quantitative losses while providing the desired properties to the oil at these steps. In the refining, depending on the process conditions, qualitative and quantitative losses occur in certain bioactive components such as tocopherols, phenolic substances, plant sterols, squalene, which are important for human health. The deodorization step is the main stage at which important bioactive substances such as squalene and tocopherol pass to deodorization distillate. The recovery of squalene and tocopherol from deodorization distillate is of great importance in the food, cosmetic and pharmaceutical industry. In the literature; chemical, enzymatic and physical methods have been investigated for the recovery of squalene which has a great importance for human health and has a wide range of industrial uses. Many of these studies use very expensive methods and require many pretreatments. However, modified Soxhlet extraction method could be performed more easily. 
Modified Soxhlet extraction method is one of the physical processes to isolate bioactive components from vegetable oil deodorizer distillates The normal Soxhlet extractor has been modified to control temperature of the jacket on the outer surface of the apparatus.
In this study, the extraction parameters were investigated for the recovery of squalene and the optimum conditions were determined using Response-Surface Methodology. The parameters were oil/silica gel ratio, temperature and extraction time were also selected for the efficient separation of squalene by modified Soxlet extraction method.
Thin layer chromatography/flame ionization detector (TLC-FID) combined system was used to determine the amount of Squalene contained in olive oil deodorization distillate. 
In first stage of this study olive oil deodorizer distillate (OODD) was seperated into two fractions based on differences in the polarity of the constituent compounds applying modified Soxhlet extraction method. Starting with OODD that contains 1.5% squalene, it was possible that a non-polar oil fraction can be obtained from the olive oil deodorization distillate using modified Soxhlet extraction method. Nonpolar lipid fraction (NPLF) is rich in fatty acids steryl esters (FASEs) and squalene, while the other fraction is rich in polar compounds such as tocopherols, free phytosterols, free fatty acids (FFAs) and acylglycerols. 
The nonpolar lipids were extracted with hexane which was put in a 500 mL round-bottom flask and then was heated. The hexane vapor travelled up the distillation arm and flooded into the chamber housing the thimble. The condenser ensured that any hexane vapor condenses and drips back down into the chamber housing the thimble. The city water was used as a refrigerant to cool the jacket and a cryostat was used to control the temperature of the city water.  
In the second part of the study Response Surface Methodology was employed to evaluate the effects of the oil/silica gel ratio, temperature, and time as the independent parameters. These parameters were thought to have an effect on squalen concentration and so optimization of these parameters were applied to determine appropriate process conditions. Consequently, the highest concentration of squalene yielded at conditions 65 °C, 1/3 oil/silica ratio and 8 hours. NPLF sample containing 0.149 g/L squalene was obtained under these conditions.
A silica gel column chromatography was then applied to purify the squalene from the squalene-enriched fraction. The 3 g NPLF sample was dissolved in hexane and loaded onto the column. In the column chromatography a 500 mL solvent system of a hexane/diethyl ether (95:5, v/v) mixture was used as a mobile phase and fractions were collected at a constant flow rate of 3 mL/min. 
Thin layer chromatography method was used to determine the fractions containing squalene. Hexane/ethylacetate /acetic acid (90/10/1 v / v / v) solvent system was used as the mobile phase for thin layer chromatograpy. After the phases progressed, the plate was kept in the iodine tank so that the spots on the thin layer could be seen clearly. The fractions containing Squalen were determined on the colored plate in the iodine tank by comparing with the Squalen standard. Then the squalen rich part was obtained with FASE in the first fractions. The squalen-rich fractions were collected in a balloon. After NPLF sample was subjected to column chromatography concentration of squalene was increased to 18% in the NPLF. This method has shown that valuable components from the KGA can be recovered at high purities
This study is also important in terms of avoiding the depletion of these organisms, which are protected by a simple process of an alternative source of industrial squalene from deep-sea fish liver fats.
Although the extraction process was carried out in a modified Soxhlet apparatus for temperature control, the squalene concentration obtained in the experiments carried out at system temperature was found to be the same as the constant 65 ° C. This has shown that this work can also be carried out with the classic Soxhlet system to save energy to keep the temperature constant. Thus, the use of the classical Soxlet assembly has shown that this process can contribute to the industry in terms of both ease and economy.
Consequently, the process and process conditions were evaluated economically and regulations were proposed for industrial application of this method.