Kabak çekirdeğinden enzimatik sulu ekstraksiyon ile yağ eldesi ve yüzey aktif madde kullanımının yağ verimine etkisi
Enzymatic aqueous extraction of oil from pumpkin seeds and effect of surfactants on the oil yield
Yazar: GÖZDE NUR DALKIRAN
Danışman: PROF. DR. GÜLDEM ÜSTÜN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Moleküler Biyoloji-Genetik ve Biyoteknoloji Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Biyoteknoloji = Biotechnology
Dizin: Endüstriyel yağlar = Industrial oils ; Heksanlar = Hexanes ; Tohum yağları = Seed oils ; Yağ endüstrisi = Oil industry ; Yağ verimi = Oil yield ; Çözücü özütleme yöntemi = Solvent extraction method

Özet:
Cucurbitaceae familyasının yaygın türlerinden biri olan kabak çekirdeği (Cucurbita pepo L.), sıcak iklimlerde ve nemli bölgelerde yetişmektedir. Kabak tohumlarındaki yağ oranı %40-60 arasında değişmekte ve bu yağın %98-99'unu yağ asitleri oluşturmaktadır. Bunların %80 civarı doymamış yağ asitlerinden olan linoleik ve oleik asitten oluşmaktadır. Kabak çekirdeği gıda, ilaç, kozmetik endüstrisinde ve medikal alanlarda, yapısında bulunan antioksidan maddeler, bol miktarda E vitamini ve zengin yağ asiti içeriği ile son yıllarda ilgi çeken bir bitkisel tohum haline gelmiştir.

Bu çalışmanın amacı, bitkisel tohumlardan yağ ekstraksiyonunda geleneksel olarak kullanılan çözücü ekstraksiyonu yöntemine alternatif olabilecek, yüksek kalitede ve verimde yağ elde edilebilecek bir yöntem geliştirilmesidir. Deneylerde içerisinde %44,1 oranında yağ içeren Ukrayna orijinli kabuksuz kabak çekirdeği tohumları kullanılmıştır. Alternatif ekstraksiyon yöntemi olarak enzimatik sulu ekstraksiyon seçilmiş olup, hücre duvarını degrade etmek için proteaz enzimi eklenmiştir. Proteaz enzimi için pH, enzim miktarı ve süre açısından optimum çalışma koşulları belirlenmiştir. Sulu ekstraksiyonda, yağ verimi üzerine tuz-yüzey aktif madde ve yüzey aktif madde-enzim kombinasyonlarının etkisi de incelenmiştir. Yüzey aktif madde katkısında optimum koşulları belirlemek amacıyla farklı konsantrasyonlarda tuz ve yüzey aktif madde kullanılmıştır. Daha sonra tohum miktarı artırılarak denemeler yapılmış ve yağ verimi değişimleri incelenmiştir. Bu kısımda doğrudan pipetle çekme, dekantasyon ve hekzanla ekstraksiyon yöntemleri karşılaştırılarak yağ verimine etkileri araştırılmıştır.

Enzimatik sulu ekstraksiyonda 0,6-1,0 mm tane boyutundaki kabak çekirdeği fraksiyonuyla çalışılmıştır. Alcalase 2.5L proteaz enzimi kullanılan deneyler 1:7 tohum:tampon çözeltisi oranında, pH 5-8 aralığında, gram tohum başına 0,25-1,0 mL enzim miktarı ile, 50°C'de, 6-24 saatlik sürelerde gerçekleştirilmiştir. Alcalase 2.5L enzimi ile belirlenen optimum koşullarda, daha sonra 100 mL sulu çözeltiye ağırlıkça %5'lik (5 gr) NaCl ve hacimce %1'lik (1 mL) yüzey aktif madde eklenmiştir. Kullanılan yüzey aktif maddeler non-iyonik Triton X-100, Tween 60 ve Tween 80, anyonik Labsa 101 ve Texapon N 70, katyonik Stepantex DC90'dır.

Kabak çekirdeği tohumlarından enzimatik sulu ekstraksiyon yöntemi ile yağ eldesinde proteaz ile 50°C'de optimum koşullar; pH 5'te, 0,75 mL/gr tohum enzim miktarında, 24 saat olarak tayin edilmiştir. Kabak çekirdeği yağı ekstraksiyon verimi, bu koşullarda sulu ortamdan geri kazanılan yağ miktarı üzerinden %57,2 olarak belirlenmiştir. Sulu ortama katılan yüzey aktif maddelerden ise, Triton X-100 ile daha yüksek verim elde  edilmiş ve deneylere artırılan tuz miktarı (ağırlıkça %5-20) ve farklı Triton X-100 konsantrasyonları (hacimce %0,5-2) ile devam edilmiştir. Maksimum değer %1 Triton X-100 - %15 NaCl kullanıldığında %56,5'tir.

Enzimatik sulu ekstraksiyon ve yüzey aktif madde katkılı sulu ekstraksiyon aşamalarında ulaşılan optimum koşullar, daha sonra enzimle beraber yüzey aktif madde kullanımında uygulanmıştır. Ayrı ayrı pH 5 fosfat tampon çözeltisi ve distile su varlığında, tuz, enzim ve yüzey aktif madde kombinasyonları test edilmiştir. En yüksek verime %61,4 ile dH2O - %15 NaCl - %1 Triton X-100 - 0,75 mL/gr tohum proteaz enzimi kombinasyonunda ulaşılmıştır. pH 5 tampon çözeltisi kullanılarak aynı kombinasyon ile yakın değerlere (%60,2) ulaşılmıştır. Böylece yüzey aktif madde katkısının yağ eldesindeki olumlu etkisi belirgin şekilde görülmektedir.

Yağ verimine etkisini gözlemlemek amacıyla tohum miktarı artırılarak çalışmalar denenmiştir. Bu amaçla, 3 katı miktarda (12 gr) kabak çekirdeği tohumları kullanılarak 3 farklı yöntem denenmiştir. Yağ ekstraksiyonu için, önceki denemelerde verimin %58,7 olduğu gözlemlenen, endüstriyel anlamda yüksek maliyetli proteaz enzimini içermeyen pH 5 - %15 NaCl - %1 Triton X-100 kombinasyonu seçilmiştir. Doğrudan pipetle çekme yöntemi, dekantasyon ve hekzanla ekstraksiyon yöntemleri ile yağ verimi belirlenmiştir. Hekzanla ekstraksiyon yöntemi ile %60,4'e yükselen bir verim artışı gözlenmiş olup, tohum miktarını artırarak çalışmanın endüstriyel boyutlarda üretim açısından bir potansiyel olabileceği düşünülmektedir.

Abstract:
In human nutrition, lipids are essential substances like carbohydrates and proteins that have vital roles in health. Vegetable oils obtained from oil seeds have A, D, E and K vitamins that dissove in oil and these vitamins have healthy effects on body. Vegatable oils also maintain rich fatty acid content and free fatty acids that are required for energy and cell integrity.

High amount of energy is stored in the oil seeds because of their high lipid content. While some oil seeds are consumed as food, the others are cultivated for oil production and pulp obtainment for animal feed. The production of oil seed crops has been increased depending upon non-stop growing world population and unhindered life standards. Moreover, technological developments has brought higher production levels and improvements with increasing quality and variety.

The climate and ground features of our country is convenient for many commodity agriculture. Most cultivated oil seed crops in Turkey include soybean, canola, peanut, cotton, olive, pumpkin, corn and sunflower.

Cucurbitaceae family has cultivated plants of the genus Cucurbita that belong to Cucurbita pepo, Cucurbita maxima and Cucurbita moschata species. Pumpkin (Cucurbita pepo L.), a very common member of the Cucurbitaceae family, grows widely in tropical, subtropical and temperate regions over the world. Thus, its seeds are cultured in many places of the world. China, India, Russia and United States of America, respectively are the countries where most of the production of pumpkin seeds is carried out. The regions of intensive cultivation in our country are Central Anatolia, Thrace and Marmara Regions. The most considerable areas where pumpkin seeds are produced are Nevşehir, Adapazarı, Kayseri, Edirne and Aksaray.

Pumpkin seeds are excellent sources of both oil and proteins. According to the variety and geographical cultivation condition of the pumpkin, it contains about %35-50 lipid, 25-40% protein, 25% carbohydrate, dietary fiber, vitamins and minerals in its seeds. Recently, pumpkin seed oil has gained attention as a potential nutraceutical with its broad range of health benefits. In our country, the pumpkin crop seeds that are used for extracting oil mostly belongs to the Cucurbita pepo L. species. 98-99% of the pumpkin seed oil comprise of fatty acids. About 80% of these are unsaturated fatty acids linoleic acid and oleic acid. Lately, due to its high qualified oil, pumpkin seeds became attractive in food, pharmaceutical, cosmetic industries and medical areas by its strong antioxidant activities, high vitamin E content and rich fatty acids ingredients. Hulls of the pumpkin seeds are innoxious, so they can be used in pulp production with its high protein content for animal feed.

Pumpkin seed oil have significant nutritive values and benefits for health. It is an edible oil with greenish brown color and strong smell and taste characteristics. Today, it is commonly used in Austria and Slovenia as export commodity and also in daily nutrition as salad oil. It provides the intake of both Omega 3 and Omega 6 essential fatty acids together that are needed for brain functionality and hormone metabolism. The oil is rich with regard to vitamin E, sterols and mineral elements. Tocopherols in vitamin E are good sources of antioxidants, therefore strengthen the ligaments and muscles of the organism. Also, phytosterols in the oil lower the cholesterol levels of the body. Various minerals in the oil enables it to be utilized in skin treatments by their anti-inflammatory effects and roles in enzyme activities.

Vegetable oils are mostly extracted from seeds by conventional methods such as mechanical pressing and solvent extraction. Generally n-hexane is used as solvent and high oil production yield is obtained. Although there is an advantage of good oil yield for the use of hexane, there occur low oil quality, high investment and management costs, and high energy requirement problems for its usage. Besides, organic solvent hexane is a toxic substance and has explosive property, it releases hazardous volatile materials to the atmosphere. Even though this traditional process for the extraction of oil is economically suitable, there are draw-backs like damage to the body and environment and quality loss of finished products which cause to search for new techniques. Thus, due to environmental safety regulations and public health risks associated with the use of hexane, alternative methods which are safe, environment-friendly, provide edible protein and qualified, highly efficient oil have been developed by researchers.
 
Aqueous extraction method is carried out at lower temperatures with respect to solvent-based extraction method and more qualified oil can be obtained by it. However, due to low oil efficiency, enzymes are added to the extraction medium to increase the yield and to minimize byproducts. Enzymatic aqueous extraction makes use of enzymes to degrade the cell walls with water acting like a solvent. This enables much easier oil release and refining of the oil. Aim is not only to separate cellular or fluid lipids from other constituents, proteins, polysaccharides and macromolecules, but also to preserve these lipids for further analyses. The preservation of proteins also permits the pulp to be rich in proteins. Removing the non-lipid molecules without losing some lipids is a complete challenge. By means of enzymes, these difficulties are tried to be reduced. Although cost of enzymatic extraction process is estimated to be much more than hexane extraction, this situation may be overcome by recycling of the enzymes and using immobilized enzymes to decrease enzyme cost. If the oil to be extracted has high market value, again investment cost could be compensated. Enzymatic extraction can also be supported by ultrasonication for increasing the oil efficieny. Therefore, aqueous and enzyme-assisted aqueous extraction substitute the use of solvents and lead to obtain oil with high efficiency, so there is no more need for organic solvents in extraction process.

The aim of this study is the investigation of a method intended for elimination of toxic effects, attainment of high oil yield and quality and diminished economical issues which is an alternative to conventional solvent extraction. Ukraine origin unhulled pumpkin seeds comprising 44,1% lipids are used in the experiments. Aqueous enzymatic extraction is selected as alternative extraction method and protease enzyme is used for degrading cell walls. Optimum conditions in respect to pH, enzyme amount and time for the enzyme of protease are determined. In aqueous extraction, effects of both salt-surfactant and surfactant-enzyme combinations on oil yield are examined. In the case of surfactant contribution, different concentrations of salt and surfactants are tried for exploring optimum conditions. Afterwards, scale-up trials are made to evaluate the pumpkin seed for industrial manufacture. Direct pipetting, decantation and extraction with hexane methods are compared for their contribution to the oil efficiency.

In aqueous enzymatic extraction, pumpkin seed fraction of 0,6 mm – 1,0 mm is worked with. The experiments with the use of Alcalase 2.5L protease ezyme are conducted at 50°C, pH 5-8 interval, with 1:7 seed:buffer solution ratio and 0,25-1,0 mL/g seed enzyme amount, during 6-24 hours. After the determination of optimum conditions for Alcalase 2.5L enzyme, 5% NaCl by weight (5 g) and 1% surfactant by volume (1 mL) are added to 100 mL of aqueous solution. Surfactants used in the experiments are non-ionic Triton X-100, Tween 60 and Tween 80, anionic Labsa 101 and Texapon N 70, and cationic Stepantex DC90.

Alcalase 2.5L Type-DX is a serine protease enzyme obtained from Bacillus licheniformis microorganisms. It has a high proteolitic activity of 2,5 AU/g (Anson Units/gram). The optimum conditions for enzyme activity are at the temperature between 55-70°C and pH 4-8 interval depending upon the substrate type.

Labsa 101 is an anionic surfactant with Lineer Alkyl Benzene Sulphonic Acid chemical structure and is used in detergent industries. Texapon N 70 is also anionic and shows sodium lauryl ether sulphate property. Due to its good foaming feature, it is one of the basic surfactants in cosmetic cleansings especially in soaps and shampoos. Triton X-100 is a non-ionic detergent and possesses the hydrophilic polyethylene oxide chain. It can show effective performance in broad temperature ranges. Stepantex DC90 is a cationic surfactant ve has cleaning properties. Quaternary ammonium salt Stepantex DC90 is widely used in industrial fields. Tween 60 is a non-ionic surfactant and displays detergent activities. Tween 80 is also non-ionic and is utilized in food industries, more actively in ice-creams. Polysorbate class members Tween 60 and Tween 80 are used as emulgators that are active in emulsion stabilization. They are also used in cosmetic areas for solubilizing essential oils into water-based products.

Suitable conditions at 50°C for pumpkin seed oil extraction by aqueous enzymatic extraction method with protease enzyme are defined as pH 5, enzyme amount of 0,75 mL/g seed and duration of 24 hours. The extraction yield of pumpkin seed oil at these conditions is determined as 57,2% over oil amount recovered from aqueous medium. In surfactant based aqueous extraction, Triton X-100 has the highest yield among other surfactants. Thus the experiments are continued with the increases in salt amount (5-20% by weight) and different Triton X-100 concentrations (0,5-2% by volume). Maximum yield value when 1% Triton X-100 – 15% NaCl used is 56,5%.

Optimum conditions that are achieved during enzymatic aqueous extraction and surfactant-based aqueous extraction, are later applied in the case of usage of both enzyme and surfactant together. In the presence of pH 5 phosphate buffer solution and distilled water separately, salt, enzyme and surfactant combinations are tested. Maximum efficiency is acquired with 61,4% value at dH2O – 15% NaCl – 1% Triton X-100 – 0,75 mL/g seed protease enzyme combination. By the use of same combination at pH 5 buffer solution, a close value is reached with 60,2% oil yield. Hence, positive effect of surfactant contribution on oil extraction is clearly seen.

With the purpose of determining the effect on oil yield, scale-up trials are performed. For this purpose, three different techniques are tried with the usage of 3-fold amount (12 g) of pumpkin seeds. For oil extraction, pH 5 – 15% NaCl – 1% Triton X-100 combination which has oil yield of 58,7% on previous experiments and doesn't include high costly industrial protease enzyme is chosen. Oil efficiency is determined by direct pipetting, decantation and extraction with hexane methods. By means of extraction with hexane, there is a yield increase to 60,4% which demonstrates the potential of the large scale trials to be utilized at industrial applications for production in the future.