Süperkritik Akışkan Ekstraksiyon Yöntemi İle Elde Olunan Adaçayı Ekstraktının Antioksidan Özellikleri Üzerine Bir Çalışma

Abstract

Endüstrileşme süreci içerisinde, hazır gıda tüketimine yönelimin yaygınlaşması, katkı maddelerinin kullanımını zorunlu hale getirmiştir. Önceleri sonuçlan düşünülmeden katılan bu koruyucular içerisinde yeralan, sentetik antioksidanlar, toksik etkileri nedeni ile günümüzde tartışma konusudur. Bu bağlamda, ülkemizde ve dünyada tüketici eğilimleri doğrultusunda birçok sentetik koruyucu yerini doğal ekstraktlara bırakmaktadır. Bu çalışma, Türkiye 'de doğal olarak yetişen, halk arasında her derde deva olduğu düşünülen adaçayının, özellikle sıvı bitkisel yemeklik yağlarda antioksidan amaçlı kullanımını desteklemek amacı ile yapılmıştır. Adaçayı, ISCO Süperkritik Akışkanlarla Ekstraksiyon (SKA) cihazı kullanılarak ekstrakte edilmiş, bu amaçla çözücü olarak karbondioksit (CO2) ve modifier olarak ta farklı oranlarda etanol (% 5-15) kullanılmıştır. Ekstraksiyon koşullan içerisinde farklı basınç (100-500 bar) ve sıcaklık (40-60°C)değişimi önerilmiş ve bunlar dışındaki parametreler sabit tutulmuştur. Bu aşamadan sonra ayçiçek yağlarına belirli oranda katılan ekstraktların antioksidan aktiviteleri saptanarak, antioksidan aktivitesinin en yüksek olduğu koşullar belirlenmeye çalışılmıştır. Yağlann raf ömrünü belirlemek amacı ile hızlandırılmış oksidasyon testlerinin yapılabildiği Diferansiyel Taramalı Kalorimetre cihazından (DTK) yararlanılmıştır. Doğal adaçayı ekstraktları ile sentetik bazı antioksidanların antioksidan gücü aynı deney koşullarında karşılaştınlmıştır. Sentetik antİoksidanlardan BHT karşılaştırma amacı ile seçilmiştir.

Key Words: Sage, antioxidant, supercritical fluid extraction, thermal analysis, induction period. Spices and herbs have been used in seasoning of foods since time immemorial. They are aromatic vegetable materials which enhance savouries of foods, and are valued not only as flavouring agents but also for other properties like stimulation of appetite by increasing salivation, their carminative, preservative and antioxidant action in some foods. Their use in the commercial and domestic preparation of foods dates early, but recently they are gradually being substituted by some synthetic products like nature-identical aroma compounds. However, their use in food manufacture is still increasing due to demand by consumers for natural ingredients. They also have important uses in pharmaceutical industry and in cosmetics. It is well known that antioxidants retard oxidative rancidity which is caused by atmospheric oxidation in foods, and thus protecting the oils, fats, and fat soluble food components, such as vitamins, carotenoids, and other nutritive elements. In addition, they delay undesirable tastes and odor changes brought about by oxidation reactions in foods. Antioxidants may be added directly to the food system or as a solution in the food's oil phase, in a food grade solvent or in an emulsifier form which may be sprayed onto the food product. The type of food to which antioxidant may be added is variable ranging from dry (cereal-based product) convenience and snack foods (such as instant potato granules and crisps), biscuits, nuts, mayonnaise, fruit, drinks, chewing gum and meat products, oils and fats. It should be pointed out that they must not be added above a certain level not only due to the legal strains, but also because a pro-oxidant effect may then occur. To be most effective, antioxidants must be added to a fresh product as soon as possible because they can not reverse any oxidation that has already occurred. To avoid or delay autoxidation processes, commercial antioxidant preparations are being used for over 50 years all over the world(CUVELIER, 1994). Today the most commonly used antioxidants are synthetic chemicals. Because of possible toxic effects of butyllated hydroxy toluene(BHT), and butyllated hydroxy anisole (BHA) and together with consumers' preference for natural products, much research has been undertaken during the past ten years on natural antioxidant (CUVELIER, 1996). When foods are subjected to processing, natural indigenous antioxidants are often depleted either physically from the nature of the process itself, or by chemical degradation. Ideally, food producers would like their products to keep better. This objective can often be achieved by blending a natural product rich in antioxidants with processed foods, or by using well-recognised antioxidants as food additives (HUDSON, 1990). The continuing trend towards new forms of food processing, long term food storage, added to informed consumer concern,ensures that antioxidants as protective food ingredients will assume increasing importance for the food industry, in food distribution and at the point of sale. In order to apply antioxidants intelligently in food product formulation, some knowledge on their mechanisms of action is essential. Food antioxidants are functional in very small IX quantities, perhaps in 0.01% or lower concentrations. At higher concentrations, since they themselves are susceptible to oxidation, they can behave as pro-oxidants. At low levels they have no adverse effect or undesirable toxic effects. On the other hand, identification and estimation of small quantities demand sophisticated analytical techniques(HUDSON, 1990). Recently, the food industry has been shifting to using natural antioxidants from plant raw materials. Extracts from herbal plants like sage have been shown to have strong antioxidant characteristics in foods and food model system (CUVELIER et all., 1994). Major antioxidant components identified in sage are phenolic compounds ( carnosic acid and its derivatives) which are thought to be responsible for the antioxidative efficiency of sage (CUVELIER et al., 1994; SCHULER, 1990). This study was undertaken to investigate the possibilities of preparation of an odorless and flavourless natural antioxidant extract from a natural Turkish spice, sage, using supercritical fluid extraction, as well as to evaluate the antioxidative power of this extract by applying a rather unconventional analytical technique, differential scanning calorimetry. The plant material, sage, from the family Salvia officinalis was obtained from Gebze, Turkey, during its flowering period, in May 1997. Water content of plant was measured immediately after harvesting by Ohaus moisture determination apparatus. Sage plant was kept in refrigerator until the experimental stage. Ground and steam distilled sage herb was extracted with ethanol (%96) at room temperature. During extraction, it was left on a shaker- mixer overnight. The mixture was filtered and the solid residue was re-extracted with ethanol again at 60 °C under the same conditions. After the third extraction, the residue was discarded and the filtrates were combined together and were concentrated with Buchi rotary evaporator to 350 ml. After that the column was filled with active carbon(approximately 65 gr.). Firstly the column was washed with ethanol, then the concentrated filtrate was passed through the column. The filtrate's color changed to light brown. The light brown filtrate was concentrated by rotary evaporator to 350 ml again. Before supercritical fluid extraction, "response surface" experimental conditions were selected with an attempt to identify the output or the response of the system as a function of explanatory variables (THOMSON, 1982). The response can be thought of as a surface over the explanatory variables in experimental space. Consequently, the term "response surface" has been associated with experiments intended to identify or evaluate one or more response variables as a function of independent variables (THOMSON, 1982). Most of the literature on "response surface" experimental designs focuses on polynomial models. Experimental conditions were regulated by "central composite design ". This type of design includes there types of points. Number of independent variables is k = 3; number of parameters in the 2nd order model is p =10; number of cube points is 2 }Ma=n0, 8; star points number, na, 6, value a, 1,682; number of center points no, 9; total number of points, 23; number of center points, no, 6. The experimental design parameters created for this study are summarized in Tables 1 and 2. Table 1 Identification of parameters for this design Table 2 Central Composite Design Conditions for SF Extractions According to this experimental design, the exact conditions selected for variations of the three critical parameters are summarized in Table 3. Supercritical fluid extraction (SFE) refers to the extraction of a material with a solvent above its critical point. SFE has emerged as a viable sample preparation technique for analytical chemistry because of the need to speed up the analytical sample preparation protocols. It has been reported that two thirds of the analysis time in an industrial laboratory is being spent on le preparation. Potential advantages of SFE are speed of operation, selectivity of extraction, minimal solvent handling, ease of automation, capabilities of "off-line" or "on-line" analysis and reproducible extractions. These advantages are being achieved because of beneficial properties of supercritical fluids which include high diffusion rates, variable solvent strengths and low viscosities. Carbon dioxide has emerged as the most amenable supercritical fluid owing to its low critical temperature, moderate critical pressure, relative inertness, non-toxicity and its ready availability in a high purity at low cost. SFE with excellent recovery has been described using organic analytes such as hydrocarbons, for analyses of pesticides, herbicides, fats, food additives and flavors from the samples such as soil, fly ash, air, water, foods, and many other industrial solids a Table 3 SF Extraction Conditions In this study, the supercritical extraction from sage leaves was realized by using carbon dioxide and ethanol as the modifier for increasing the solubility of the fluid. The effect of experimental parameters such as pressures, temperatures and percentage of ethanol on the extraction yields were investigated. Except these parameters, everything else was kept constant, as shown in the three tables above. Using these experimental conditions, 23 different sage extracts in two parallels were obtained. Each of these 23 extracts was added into sunflower oil, which had zero initial peroxide value. The concentration was kept constant at % 0.02(0.02gr extract in 100ml sunflower oil). A mixer was used to homogenize the sample materials. Synthetic antioxidants BHT (% 0.01) were added to parallel sunflower oil samples at the same condition, to provide means of comparison with the antioxidative power of the sage extracts. One component of the thermal analyzer equipment, namely the differential scanning calorimeter (DSC), was employed to measure the antioxidative power of extracts by calculation of the induction period "IP" and the "onset" point.. DSC is the most widely used of all thermal analysis techniques. It is defined by the ICTA as: "a technique in which the difference in energy inputs into a substance and a reference material is measured as a function of a temperature whilst the substance and reference material are subjected to a controlled temperature program"(HALWARKAR, 1990). "Oxidative Induction Time" is an accelerated test used as a qualitative evaluation of stability of a material in general. An antioxidant is a promoter of stability and prevents the propagation of oxidation reactions; but it is consumed in the process and when completely depleted, the oxidation reactions will then proceed very rapidly. Since these reactions are highly exothermic, DSC can easily determine the "onset" and "trigger" points. The time during which the oil 's resistance to oxidation is still effective is called the 'Induction Period" (IP), a time which can in many cases be measured with a fair degree of reproducibility with thermal analysis (Hudson, 1990). The DSC analytical procedure was carried out to determine the antioxidant power of sage. This method involves heating the sample to a preset temperature in a nitrogen atmosphere and xu i equilibrium there. The atmosphere is then switched from nitrogen to oxygen and the time to the onset of degradation is then measured. Sample preparations were realized with maximum care since consistent surface area and sample weights are very important for adequate reproducibility in thermal studies. After the samples were weighed accurately, the sample pan was placed in an open condition. The temperature range during the experiment was programmed from 40 °C to the selected temperature (160 °C) with isothermal temperature at 40 C/min in the nitrogen atmosphere. We waited for sample equilibration at the selected isothermal temperature for 2 minutes before changing to the oxygen atmosphere. According to the statistical evaluation of analytical results of SFE studies, the temperature (T), pressure (P) and Ethanol (E) were all quite significant factors affecting % recovery of sage extracts, The effects of T, P and E factors on extraction were modelled using the reduced form of third degree of polynomial. The results of all SFE extraction studies are summarized in Table 4. As can be seen from the table, increasing the temperature had a clear positive effect on % recovery of sage extracts, but this effect was diminished with an increase in concentration of ethanol as the modifier. The higher the % ethanol in extracting phase, the lower was the effect of increasing the temperature. Thus it can be concluded that ethanol too had a positive quadratic effect on recovery. It is a known fact that increasing the pressure increases the amount of carbon dioxide in the supercritical phase. On the other hand, a higher temperature would decrease the amount of supercritical carbon dioxide, but the solute vapor pressure would then increase, thus increasing the solubility of sage extracts, and % recovery. The effect of this increase in solubility had a stronger effect than the increase in supercritical carbon dioxide density caused by increases in pressure. Thus it can be said that there was an interaction between the effects of temperature and pressure, where it was concluded that effects of pressure were maximized by effects of temperature increases. It is a known fact that increasing the pressure increases the amount of carbon dioxide in the supercritical phase. On the other hand, a higher temperature would decrease the amount of supercritical carbon dioxide, but the solute vapor pressure would then increase, thus increasing the solubility of sage extracts, and % recovery. The effect of this increase in solubility had a stronger effect than the increase in supercritical carbon dioxide density caused by increases in pressure. Thus it can be said that there was an interaction between the effects of temperature and pressure, where it was concluded that effects of pressure were maximized by effects of temperature increases. Ethanol concentration as modifier in the carbon dioxide phase was also an other factor which contributed to the increase in recoveries. It is also a known fact that solubility of materials with low volatility is increased when a modifier is used when compared to the use of a supercritical gas alone. The high temperature dependency in this study could be explained by the presence of ethanol. In a similar study, where recovery was defined as the percent of extract obtained from experiment compared to the theoretical yield calculated as the difference of carotenes in the initial sample and the supercritically extracted sample, it was Xlll also reported that increasing the percent of ethanol added to the supercritical fluid yielded clearly higher recoveries of carotenes (VEGA et al, 1996). Table 4 SF Extraction yields (% recovery) and DSC Results of antioxidative effects Concerning the studies on relative antioxidative effects of the 23 sage extracts on induction periods of sunflower seed oils trated with them, the Mathematica computer program was used for the 23 points where multiple regression equations were calculated. Based on these analyses, it can be concluded that when the solubilities were increased, there was a parallel lengthening effect on the induction periods of the oils treated with sage extracts. Based on investigations made in this study, the optimal conditions were 419 bar of pressure, at 44°C and with 13% ethanol addition, yielding 97.45 % recovery. This extract also had the most influential effect on extending the induction period (to 8.9 minutes as compared to 6.8 minutes in the one showing the lowest antioxidative effect). A high recovery of a natural product with significant antioxidative activity was obtained by subjecting the sage leaves to supercritical carbon dioxide-ethanol extraction. This extract was used as an antioxidant in treating sunflower seed oil for delaying the onset of its induction period, thus extending its shelf-life. XIV This study has shown the optimal conditions for obtaining such a sage extract and has given an indication of the extent of its protection against oxidation reactions in a highly unsaturated edible oil, namely sunflower seed oil..