Yerfıstığından enzimatik ekstraksiyon ile yağ eldesinde yüzey aktif madde kullanımının etkisi
Effect of surfactants on the enzymatic extraction of oil from groundnut
Yazar: BAHAR DEĞERLİ
Danışman: PROF. DR. GÜLDEM ÜSTÜN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / İleri Teknolojiler Ana Bilim Dalı / Moleküler Biyoloji-Genetik ve Biyoteknoloji Bilim Dalı
Derece: Yüksek Lisans, 2012
Konu: Biyoteknoloji = Biotechnology
Dizin: Enzimatik yöntemler = Enzymatic methods ; Yağ asitleri = Fatty acids

Özet:
Bu çalışmanın amacı, genellikle yağ ekstraksiyonunda kullanılan çözücüekstraksiyonu yöntemine alternatif olabilecek, çözücüsüz ortamda yüksek yağverimi ile yağın elde edilebileceği bir yöntemin geliştirilmesidir. Deneylerimizdeiçerisinde % 49,5 yağ içeren yerfıstığı tohumları kullanılmıştır. Yerfıstığı yağındaise % 59,44 oleik asit ve % 24,38 oranında linoleik asit bulunmaktadır. Alternatifekstraksiyon yöntemi olarak enzimatik sulu ekstraksiyon seçilmiştir. Enzimatik suluekstraksiyonda ortama hücre çeperlerini degrade etmesi için proteaz enzimikatılmıştır. Proteaz enzimi için optimum çalışma koşulları (pH, enzim miktarı vesüre) belirlenmiştir. Enzimatik sulu ekstraksiyonda, yağ verimi üzerine yüzey aktifmadde ve yüzey aktif madde-enzim kombinasyonlarının etkisi de incelenmiştir.Yüzey aktif madde kullanımında optimum koşullarının belirlenmesi için ise farklıyüzey aktif madde ve tuz miktarları denenmiştir.Enzimatik sulu ekstraksiyonda 0,6?1,0 mm tane büyüklüğündeki yerfıstığıfraksiyonunu ile çalışılmıştır. Alcalase 2.5L proteaz enzimi kullanılan deneyler, 50°C'de 6-24 saat sürede, 1:7 tohum:tampon çözeltisi oranında pH 5-8 aralığında vegram tohum başına 0,3-1,5 mL enzim kullanılarak yürülmüştür. Alcalase 2.5Lenzimi ile belirlenen optimum koşullarda, daha sonra 100 mL çözeltiye ağırlıkça %5'lik NaCl ve hacimce % 0,5 mL yüzey aktif madde eklenmiştir. Kullanılan yüzeyaktif maddeler non-iyonik Disponil NP10 ve anyonik Labsa 101'dir.Enzimatik sulu ekstraksiyon yöntemi ile yerfıstığı tohumlarından yağ eldesi proteazile 50°C'de uygun koşullar pH 7'de, 0,5 mL/gr tohum enzim miktarında, 24 saatolarak tayin edilmiştir. Yerfıstığı yağı ekstraksiyon verimi, sulu ortamdan gerikazanılan yağ miktarı üzerinden %81 olarak saptanmıştır. Ortama katılan yüzey aktifmaddelerden Disponil NP10 ile daha yüksek verim elde edilmiştir ve deneylerearttırılan yüzey aktif maddede (1,0-2,0 mL/100 mL çözelti) ve NaCl'deki miktarartışları (ağırlıkça %5 - %15) ile devam edilmiştir. Maksimum değerler % 2 DisponilNP10 kullanıldığında % 88,5 ve % 2 Disponil NP10 - % 15 NaCl kullanıldığında ise87,5'dir. Ayrıca enzimle beraber yüzey aktif madde kullanıldığında Labsa 101'degörülen verim artışı Disponil'e göre oldukça fazladır. Böylece yüzey aktif maddekullanımının yağ eldesi üzerindeki etkisi belirgin bir şekilde görülmektedir.

Abstract:
Lipids are essential substances as carbonhydrates and proteins that have vital value and play important role in nutrition. Vegetable oils have high nutritional value with the properties such as especially low saturated oil rates; solvability of vitamins dissolving in oil A, D, E, K and containing necessary free saturated fatty acids for cell structure.Oily seeds are the crops that the greater part of energy is stored. Some oily seeds can be consumed as food but the others are cultivated for oil and pulp obtainment specifically. The production of oily seeds is increased depending upon non-stop growing world population and life standards. Also, technological improvements has resulted in higher production levels and developments with increasing quality and variety.In our country; soya, canola, peanut, corn and sunflower oil agriculture has take place significantly.Peanut is from family of leguminosae and it has 45-60% oil, 20-30% protein, 18% carbohydrate, vitamins and minerals in its seeds. India, China, Nigeria, Senegal and United States of America are the countries where the most cultivation of peanut is carried out. The regions of maximal cultivation in our country are Mersin, Silifke, Adana and Hatay in Mediterranean Region. Also, the cultivation is made in Aegean, Thracian and Southeastern Regions. All parts of the peanut plant can be used. The peanut, grown primarily for human consumption, has several uses as whole seeds or is processed to make peanut butter, oil, and other products.Peanuts are consumed chiefly as roasted seeds or peanut butter and oil in the world. Salted and shelled peanuts, candy, and roasted-in-shell peanuts are the next most common uses for peanuts. The remaining production is used for seed, feed, production of oil, or exported as food or oil.Nonfood products such as soaps, medicines, cosmetics, and lubricants can be made from peanuts. The vines with leaves are an excellent high protein hay for horses and ruminant livestock. The pods or shells serve as high fiber roughage in livestock feed, fuel (fireplace "logs"), mulch, and are used in manufacturing particle board or fertilizer.Flavorful peanut oil is organic edible oil obtained from peanut kernels. Peanut oil comprises crucial nutritional value with rich composition of fatty acids oleic and linoleic acids. Cold pressed peanut oil has the physical properties of deep yellow color with pleasant nutty aroma and sweet taste. In addition, the refined oil has light yellow color and has neutral taste. However, highly refined oil makes it virtually devoid of impurities and allergens. Its specific gravity at 25 °C is 0.912-0.920, iodine value of 84?100, and saponification value between 185and 195. Pleasant and sweetflavoured peanut oil is low in saturated fats, free from cholesterol, contains essential fatty acid linoleic acid (omega-6) making it as one of the healthiest cooking oils. Being a vegetable oil, it is a good source of plant sterols, especially ß-sitosterol. Phyto-sterols competitively inhibit cholesterol absorption in the gut and thereby can reduce cholesterol levels by 10% to 15%. Peanut oil is high in calories. The high calorie content is because of its fats. However, it is especially rich in mono-unsaturated fatty acids (MUFA) like oleic acid (18:1) that helps to lower LDL or "bad cholesterol" and increase HDL or "good cholesterol" in the blood.Peanut oil contains resveratrol, a polyphenol antioxidant, which has been found to have protective function against cancers, heart disease, degenerative nerve disease, Alzheimer's disease, and viral/fungal infections.Peanut oil is used to lower cholesterol and prevent heart disease. It is also used to decrease appetite as an aid to weight loss. Peanut oil is sometimes applied directly to the skin for arthritis and joint pain, dry skin, eczema, scalp crusting and scaling without hair loss, and other skin disorders that cause scaling. Rectally, peanut oil is used in ointments and medicinal oils for treating constipation. Pharmaceutical companies use peanut oil in various products they prepare for internal and external use.Vegetable oils may be produced by the conventional methods such as mechanical pressing and solvent extraction. Hexane is used as solvent and generally the oil production efficieny is obtained but hexane is a toxic substance and have explosive property. Additionally, it releases hazardous and volatile substance. Conventional processes for the extraction of oil involve mechanical treatment, by pressing seed meat, and a solvent step, that submit the cake for further extraction with n-hexane. Although these technologies are economically justifiable, its well-known drawbacks: damage to the environment and quality loss of finished products (e.g. high free fatty acids and lower resistance to rancidity) are causes to search a new method.Enzymatic extraction uses enzymes to degrade the cell walls with water acting as the solvent. This makes fractionation of the oil much easier. The costs of this extraction process are estimated to be much greater than hexane extraction. The enzymatic extraction can be supported by ultrasonication. Therefore, the aqueous and enzyme-assisted aqueous extraction as alternative come into prominence to cease the use of solvents and obtain oil with high efficiency both environment-friendly and safely. The aqueous extraction method is carried out at lower temperature with respect to solvent-based extraction method and higher quality of oil is obtained but the difficulty of this process is not to able to extract oil with high efficiency. Therefore, there are studies of aqueous enzymatic extraction to overcome this problem. The aqueous enzymatic extraction process provives important advantages from the viewpoint of environment and safety. The extraction efficiency may be increased by the aqueous enzymatic extraction process as well.The aim of all extraction procedures is to separate cellular or fluid lipids from the other constituents, proteins, polysaccharides, small molecules (amino acids, sugars...) but also to preserve these lipids for further analyses. The preservation of proteins involves special precautions found in delipidation procedures.There is a great diversity of methodologies because biological tissues are not similar when considering their structure, texture, sensitivities and lipid contents. Removing the non-lipids without losing some lipids is a complex challenge, extracting some specific lipids is not always reliable for other kinds of lipids.The high sensitivity of the analytical methods needed for low amounts of extracted lipids requires the use of very pure solvents and clean glassware. The chemical nature of the extracted lipids must also be taken into consideration.Furthermore, all lipids must be protected against degradation through oxidation by solvent, oxygen, enzymes in combination with temperature and light.The aim of this study is the investigation of a method intended for elimination of toxic effects, economical issues and high yield of oil obtainment that is an alternative to solvent extraction by the use of generally hexane. The peanut seeds comprising 59.44% oleic acid and 24.38% linoleic acid were used in the experiments. The aqueous enzymatic extraction was selected as alternative. In addition, the effects of surfactants and the enzyme-surfactant combinations were tried.In aqueous enzymatic extraction, the optimum conditions (pH, enzyme amount and duration) were determined for the enzyme of proteaz that degrades the cell walls. In use of the surfactants, different amounts of surfactants and salt were tried.In aqueous enzymatic extraction, the seed fraction of 0.6 mm ? 1.0 mm was worked with. The experiments by using proteaz enzyme Alcalase 2.5L were conducted in 50°C, pH 5-8 with 1:7 seed:buffer solution rate during 6-24 hours. After the determination of optimum conditions for Alcalase 2.5L enzyme, % 5 NaCl by weight and %0.5 surfactant by volume to 100 mL of buffer solution. The surfactants used in the experiments were non-ionic Disponil NP10 and anionic Labsa 101.Alcalase 2.5L Type-DX is a protease obtained from Bacillus. It has a high proteolitic activity of 2,5 AU/gr (Anson Units/gram). The conditions that the enzyme is active are the temperature between 55 °C and 70 °C and pH 4-8 depending upon the substrate type.Labsa, the linear alkylbenzene sulfonic acid is the largest-volume synthetic surfactant because of its relatively low cost, good performance, the fact that it can be dried to a stable powder and the biodegradable environmental friendliness as it has straight chain. Labsa is an anionic surfactants with molecules characterized by a hydrophobic and a hydrophilic group. Surfactants are widely used in the industry needed to improve contact between polar and non-polar media such as between oil and water or between water and minerals. Linear alkylbenzene sulfonic acid is mainly used to produce household detergents including laundry powders, laundry liquids, dishwashing liquids and other household cleaners as well as in numerous industrial applications like as a coupling agent and as an emulsifier for agricultural herbicides and in emulsion polymerization. Also, Disponil NP10 is nonionic and shows stabilizing properties in emulsions and dispersions. It is used as emulsifier, dispersing agent and wetting agent in various applications.The determined suitable conditions at 50°C of peanut oil obtainment by aqueous enzymatic extraction with proteaz were designated as pH 7, enzyme amount of 0.5 mL/g seed and duration of 24 hours. The extraction yield of peanut oil was determined as 81% over oil amount recovered from aqueous medium. Higher yield was obtained from the surfactant Disponil NP10 and thus the experiments was continued with the increases in amounts of surfactants (1.0-2.0 mL/100 mL buffer solution) and salt (%5 -15 by weight). The maximum yield values are 88.5% when 2% Disponil NP10 was used and 87.5% when % 2 Disponil NP10 and % 15 NaCl were used. Additionally the increase in yield in case of the enzyme-surfactant combination is higher with Labsa 101. Thus, the effect of surfactant use in oil obtainment is clearly seen.