Taze Ve Ticari Vişne Sularının Antioksidan Kapasitesi Ve Kapiler Elektroforez Yöntemi İle Organik Asit İçeriklerinin İncelenmesi

Abstract

Son yıllarda, vücudumuza alınan doğal antioksidan maddelerin kalp hastalıkları ve kanser riskini azalttığı yönündeki bilimsel çalışmalar, meyve ve meyve suyu tüketimine verilen önemi arttırmıştır. Latince adı Prunus cerasus olan vişne, yapısında bulunan yüksek miktarda polifenolik bileşikler ve meyveye rengini veren antosiyaninler nedeniyle antioksidan aktivitesi en yüksek meyvelerden biridir. Türkiye vişne üretiminde dünyada birinci sıradadır. Vişne ; meyve suyu, dondurulmuş ve kurutulmuş meyve olarak tüketilmekte ve ihraç edilmektedir. Antioksidanlar vücudu, metabolizmada oluşan serbest radikallere karşı koruyan maddelerdir. Serbest radikaller; DNA, protein ve lipit gibi makromolekülleri etkileyerek yaşlanmaya, doku hasarına ve bazı hastalıklara neden olmaktadır. Antioksidanlar, serbest radikallerle tepkimeye girerek bunların başlattığı zincir reaksiyonu durduran ve böylece vücudumuzdaki hayati bileşenlerin zarar görmesini engelleyen moleküllerdir. Bu nedenle antioksidan kapasitesi yüksek gıda ürünleri tüketmek kanser gibi hastalıkları önlemede ve yaşlanma sürecini geciktirmede önem kazanmaktadır. Meyveler bu bakımdan en önemli doğal antioksidan kaynaklarıdır. Öte yandan, meyve sularının organik asit profilleri onların koku ve tazeliğinin karakteristikleridir. Organik asit profilleri her meyve türüne göre farklılık gösterdiği için, meyve sularının organik asit içerikleri aynı zamanda bir meyve suyunun daha ucuz bir meyve suyu ile hilelendirilmesinde önemli bir indikatördür. Ülkemizde çok sayıda meyve suyu üretilmekte ve ihraç edilmektedir. Meyve sularının içeriklerinin kabul edilen standartlara uymaması ihracatta önemli sorunlara neden olmaktadır. Günümüzde meyve sularının antioksidan aktivitesi ve organik asit miktarları meyve suyunun kalitesinin önemli göstergesi olduğundan, bu yönde bir çok analitik yöntem geliştirilmiştir. Antioksidan aktivite tayinleri, daha çok spektrofotometrik yöntemlerle gerçekleştirilirken, organik asit içerikleri kromatografik yöntemlerle yapılmaktadır. Bu çalışmanın amacı ülkemizde önemli miktarda üretilen, taze ve ticari vişne sularının antioksidan aktivitelerinin ve organik asit içeriklerinin tayinidir. Vişne sularının antioksidan aktivite tayinleri için FRAP ve DPPH yöntemleri kullanılmıştır. Her iki yöntemle bulunan antioksidan aktiviteleri istatistik yöntemle karşılaştırılarak, sonuçların birbirleriyle uyumlu olduğu görülmüştür. Vişne sularının organik asit içerikleri kapiler elektroforez yöntemiyle incelenmiştir. Kapiler elektroforez iyonların yüksek elektrik alan altında yük/büyüklük oranlarına göre ayrılmasını sağlayan bir ayırma ve analiz yöntemidir. Yöntemin, çok hızlı ayırımı, çok az madde tüketimi ve yüksek ayırma gücü en büyük avantajıdır. Kapiler elektroforezde itici güç, kapiler içinde oluşan elektroosmotik akıştır. Organik asitlerin elektroforetik mobiliteleri çok yüksek olduğundan, elektroosmotik akış ile sürüklenmeleri güçtür. Bu nedenle bu çalışmada kapiler kolon dinamik olarak pozitif yüklü bir yüzey aktif madde ile kaplanarak, kapiler iç duvarı pozitif olarak yüklenmiştir. Organik asit standartları, polarite tersine çevrilerek negatif kutuptan enjekte edilmiştir. Böylece elektroosmotik akışın ve iyonların elektroforetik mobilitelerinin aynı yönde olması sağlanarak 3 dakika gibi çok kısa sürede ayırım ve analiz gerçekleştirilmiştir. Ayrıca, organik asitler UV aktif olmadıkları için ayırma ortamına ilave edilen bir kromofor madde yardımıyla dolaylı dedeksiyonları gerçekleştirilmiştir. Denemeler sonucu, organik asitlerin ayrılmasında en uygun ayırma şartlarının, tampon ortamı olarak pH 6’da 0,1 mM CTAB ve 5 mM PDC çözeltisi, 50 mBar basınçla 6 saniye örnek enjeksiyonu ve -30 kV ayırma voltajı, olduğu tespit edilmiştir. Dedeksiyon 210 nm’de yapılmıştır. Taze vişne sularında organik asit olarak malik asit tespit edilmiş ve kantitatif miktarları tayin edilmiştir. Ticari meyve sularında malik asit yanında koruyucu olarak katılan, meyve sularının etiketinde de belirtilen sitrik asit bulunmuştur.

Recently, it is scientifically proved that natural antioxidants is associated with a lower risk of cardiovascular disease and cancer. Thus, the consumption of fruits and fruit juices, which are natural antioxidant sources, have gained increasing interest among consumers. Sour cherry, is a species of Prunus in the subgenus Cerasus. It is closely related to the wild cherry. Sour cherries contain fewer calories than the sweet varieties, due to their lower sugar content. Sour cherry is considered to be one of the richest sources of antioxidants. Turkey is the firts range in the production of sour cherry in the world. Sour cherry may be beneficial for the management and prevention of inflammatory diseases, including inflammatory pain. Beyond the anti-inflammatory benefits, many of the phenolic compounds in cherries may offer protection against heart disease and stroke. Anthocyanins in sour cherries also might lower blood lipids, thus reducing heart disease risk. Researchers believe sour cherries may have the potential to reduce the risk of colon cancer because of anthocyanins and cyanidin, another type of flavonoid found in cherries. Sour cherries and their compounds appear to aid in diabetes control and in reducing the complications associated with this disease. Antioxidants are substances that may protect human cells against the effects of free radicals. Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Free radicals, also known simply as radtheicals, are organic molecules responsible for aging, tissue damage, and possibly some diseases. Free radicals are highly unstable molecules that are naturally formed when you exercise and when your body converts food into energy. Human body can also be exposed to free radicals from a variety of environmental sources, such as cigarette smoke, air pollution, and sunlight. Free radicals can cause “oxidative stress,” a process that can trigger cell damage. Oxidative stress is thought to play a role in a variety of diseases including cancer, cardiovascular diseases, diabetes, Alzheimer’s disease, Parkinson’s disease, and eye diseases such as cataracts and age-related macular degeneration. Antioxidants react with free radicals and stop the chain reaction of them. Our body uses antioxidants to stabilize the free radicals. This keeps them from causing damage to other cells. Antioxidants can protect and reverse the damage caused by oxidation to some extent. Human body produces some antioxidants to fight off the free radicals formed by normal body processes. We can also get antioxidants by eating a healthy diet. Examples of antioxidant-rich foods include fruits and vegetables that are high in nutrients such as vitamins A, C and E, beta-carotene, lutein, lycopene and selenium. The best way to get antioxidants is by eating a diet with lots of vegetables, fruits, whole grains, seeds and nuts. Several assays have been used to estimate antioxidant capacities in fresh fruits and fruit juices DPPH, FRAP, ORAC, TRAP, TOCS, CUPRAC and ABTS. On the other hand, organic acid profiles in juices characterise flavours, freshness, or spoilage of juices. Because organic acid profiles are distinct to each type of fruit juice, their profiles also give important evidences about adulteration of a juice with cheaper juices. The purpose of this study is to find antioxidant activities and organic acid profiles of Turkish fresh and commercial sour cherry juices. The antioxidant capacities of juices were determined with DPPH and FRAP antioxidant assays. FRAP assay is based on the ability of phenolics to reduce yellow ferric tripyridyltriazine complex (Fe(III)-TPTZ) to blue ferrous complex (Fe(II)-TPTZ) by the action of electron-donating antioxidants. The resulting blue color measured spectrophotometrically at 593 nm is taken as linearly related to the total reducing capacity of electron-donating antioxidants. FRAP assay needs acidic (nonphysiologically low pH value=3.6) conditions to maintain the iron solubility. One FRAP unit is defined as the reduction of 1 mol of Fe (III) to Fe(II). The DPPH radical is a long-lived organic nitrogen radical and has a deep purple color. It is commercially available and does not have to be generated before assay. In this assay, the purple chromogen radical is reduced by antioxidant/reducing compounds to the corresponding pale yellow hydrazine. The reducing ability of antioxidants towards DPPH can be evaluated by electron spin resonance or by monitoring the absorbance decrease at 515–528 nm until the absorbance remains stable in organic media. The percentage of the DPPH (%DPPHrem) remaining is calculated as: %DPPHrem = 100 x %DPPHr=o %DPPHrem is proportional to the antioxidant concentration, and the concentration that causes a decrease in the initial DPPH concentration by 50% is defined as EC50. The time needed to reach the steady state with EC50 is defined as TEC50. The results obtained both method well correlated. ORAC, TRAP, TOCS, CUPRAC and ABTS can also be used determine antioxidant capacity of sour cherries but mostly used are DPPH and FRAP. Organic acid contents of juices were determined by capillary zone electrophoresis method. Capillary electrophoresis (CE) is an analytical separation and analysis technique. The main advantage of the method is its high separation efficiency, its speed, and its very low sample consumption. In CE ions are separated based on their electrophoretic mobility in an electrical field with the use of an applied voltage. A typical capillary electrophoresis system consists of a high-voltage power supply which generates potential from -30 Kv to +30 kV, a sample introduction system which can be hydrodynamics (driven by a pressure diffrence over the capillary) or electrokinetic (by applying voltage), a capillary tube, a detector and an output device. Each side of the high voltage power supply is connected to an electrode. These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube. The capillary is made of fused silica and is sometimes coated with polyimide. Each side of the capillary tube is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer. Before the sample is introduced to the column, the capillary must be flushed with the desired buffer solution. There is usually a small window near the cathodic end of the capillary which allows UV-VIS light to pass through the analyte and measure the absorbance. CE in free solution is called as capillary zone electrophoresis (CZE). CE has several modes like micellar electrokinetic capillary chromatography (MEKC), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focusing (CIEF), capillar electrochromatography (CEC). Micellar electrokinetic capillary chromatography (MEKC),which is based on solute partitioning between the micellar phase and the solution phase. This technique provides a way to resolve neutral molecules as well as charged molecules by CE. The samples are separated by differential partitioning between micelles (pseudo-stationary phase) and a surrounding aqueous buffer solution (mobile phase). The basic set-up and detection methods used for MEKC are the same as those used in CE. The difference is that the solution contains a surfactant at a concentration that is greater than the critical micelle concentration (CMC). Above this concentration, surfactant monomers are inequilibrium with micelles. Macromolecules like DNA can be separated by capillary gel electrophoresis (CGE). CGE uses separation based on the difference in solute size as the particles migrate through the gel. Gels are useful because they minimize solute diffusion that causes zone broadening, prevent the capillary walls from absorbing the solute, and limit the heat transfer by slowing down the molecules. Capillary isotachophoresis (CITP), is the only method to be used in a discontinuous system. The analyte migrates in consecutive zones and each zone length can be measured to find the quantity of sample present. Capillary isoelectric focusing (CIEF) is a technique commonly used to separate peptides and proteins. These molecules are called zwitterionic compounds because they contain both positive and negative charges. The charge depends on the functional groups attached to the main chain and the surrounding pH of the environment. During a CIEF separation, the capillary is filled with the sample in solution and typically no EOF is used (EOF is removed by using a coated capillary). When the voltage is applied, the ions will migrate to a region where they become neutral (pH=pI). The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). Compounds of equal isoelectric points are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection. Capillar electrochromatography (CEC) have a packed column similar to chromatography. The mobile liquid passes over the silica wall and the particles. An electroosmosis flow occurs because of the charges on the stationary surface. CEC is similar to CZE in that they both have a plug-type flow compared to the pumped parabolic flow that increases band broadening. The driving force of capillary electrophoresis is electroosmotic flow that formed in capillary. Because of the electrophoretic mobilities of organic acids is very high, it is difficult to drift with electroosmotic flow. That is why, in this study capillary coloumn is coated with dynamically positive charged surfactant, and inner wall of capillary is charged positively. Organic acid standards injected at the negative pole with reversed polarity. Thus, it is provided that electroosmotic flow and electrophoretic mobilities of ions are in the same direction, and seperation and analysis carried out in a short time like 3 minutes. In addition, because of the organic acids are not UV active, they can be detected indirectly with the help of adding chromophore to the seperation solution. Of trials, it is determined that appropriate seperation conditions for the seperation of organic acids are 0,1 mM CTAB and 5 mM PDC at pH 6 as buffer solution, 6 second for sample injection at pressure of 50 mbar and -30 kV separation voltage. Detection have been achieved at 210 nm. Malic acid is determined in fresh sour cherry juices as the organic acid and it is quantitatively determined. In commercial sour cherry juices, in addition to malic acid, citric acid is determined that adding as preservative for the juices. Citric acid is specified on the labels of commercial sour cherry juices.