Şaraplarda Spektrofotometrik Yöntemle Kükürt Dioksit Ve Toplam Antioksidan Tayini

Abstract

Bir çok meyve, sebze ve içeçekler farkı çeşitlerde antioksidan içerirler.Örneğin vitaminler (E ve C), flanoidler, karotenoidler. Antioksidanlar kardiyovasküler hastalıklar, kanser, ve yaşa bağlı olarak dejeneratif dönüşümlere karşı koruma sağlarlar. Son yıllarda antioksidant dışında gıdalara birçok katkı maddesi katılmaya başlanmıştır. Bunlardan biriside kükürtdioksittir. Kükürtdioksit ve çeşitli formları mikroorganizmaların büyümeleri kontrol etmek, yapay antioksidant olarak, enzim-kataliz reaksiyonlarını inhibe etmek için gıdalara katılır. SO2’nin yararı ise yiyeceklerin korunmasında kullanılır. Özellikle şaraplarda kükürtdioksit, zararlı mikroorganizmaların (sirke bakteri-yabani maya gibi) gelişmesini, şarabın oksidasyonunu ve esmerleşmesini engellemek amacıyla kullanılır. Ne kadar antioksidan gibi davransada fazlası çeşitli zararlara neden olur. Aşırı kullanım durumunda ise insanda baş ağrısı ve bazı alerjik durumlara yol açabilir.Bu yüzden bütün yiyeceklerde SO2 miktarında sınırlama getirilmiştir. Çalışmamızın amacı CERAC yöntemine interfere etkisi olan SO2 molecular atomic absorpsiyon yöntemiyle tayin etmek ve sonra şarapların gerçek antioksidan kapasitelerini bulmaktır. Molecular absorpsiyon metodunda P.Parvinen, L.H.J.Lajunen and K.Wieczorek-Ciurowa’nın yapmış olduğu çalışma referans alınmıştır. Bu çalışmada grafit fırın kullanarak SO2’nin moleküler absorpsiyonu ölçülmüş. Birçok katod lambasıyla çalışılmışlar (Mo,Ni,Ag,As) ve en uygun olanı As(206,98) katod lambasını kullanmışlar. Biz de çalışmamızda SO2’nin molecular absorpsiyonu için bir çok lamba ile çalışıldıktan (Mg,Se,Sb,As) sonra en uygun olan Se katod lambası kullandık. Daha sonra yöntemin doğruluğunu kanıtlamak için CI formunda DOWEX X4 anyonik reçine kullanılarak SO2 leri HSO3- şeklinde tutturularak atomik absorpsiyon yöntemiyle tayin ettik.En sonn çalışmamız ise reçineden geçirilmiş ve geçirilmemiş örneklere CERAC ve CUPRAC yöntemleri uyguladık.Böylece her iki yönteminde birbirine yakın değerler verdiğini gözlemedik.

Antioxidants ; Substances which prevent or delay oxidation of proteins, lipids, carbohydrates and DNA that in the living cells are named antioxidants. Because of decreasing properties of abnormalities and forming tumors, it providex life which has minimum effect of ageing. There is a lot of different substances which have antioxidant properties. These subtances can be taken from outside and can produce by body for defense system according to the free radicals. These antioxidants are enzymes such as catalase, glutathione peroxidase and SOD (superoxide dismutase). Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials with a limited number of antioxidants detected no benefit and even suggested that excess supplementation with certain putative antioxidants may be harmful. Antioxidants also have many industrial uses, such as preservatives in food and cosmetics and to prevent the degradation of rubber and gasoline As fruits, vegetables and beverages contain different varieties of antioxidant compounds such as vitamins (especially vitamins C and E), flavonoids and carotenoids, a diet rich in these is assumed to offer protection against cardiovascular diseases, cancer and age-related degenerative transformations. Total Antioxidant Capacity Dtermination Methods in Literature as below TEAC (Troloks Equivalent Antioxidant Capacity) Method ; Trolox equivalent antioxidant capacity (TEAC) measures the antioxidant capacity of a given substance, as compared to the standard, Trolox. is based on the suppression of the absorbance of radical cations of 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) by antioxidants in the test sample when ABTS incubates with a peroxidase (metmyoglobin) and H2O2. A reaction mixture containing H2O2 (100 μmol/L), metmyoglobin (6.1 μmol/L), ABTS (610 μmol/L), and Trolox (concentration range, 0–1.65 μmol/L) was incubated and subjected to spectrophotometry .The formation of ABTS radicals increased in proportion to the incubation period until a plateau was achieved after 30 min. The higher the concentration of Trolox used in the reaction mixture, the more the absorbance of ABTS radicals was suppressed. FRAP ( Ferric Reducing Ability Plasma) Method; The FRAP assay was done according to Benzie and Strain (1996) with some modifications. The stock solutions included 300mM acetate buffer (3.1 g C2H3NaO2 3H2O and 16mL C2H4O2), pH 3.6, 10mM TPTZ (2, 4, 6- tripyridyl-s-triazine) solution in 40mM HCl, and 20mM FeCl3 6H2O solution. The fresh working solution was prepared by mixing 25mL acetate buffer, 2.5mL TPTZ solution, and 2.5mL FeCl3 6H2O solution and then warmed at 37°C before using. Fruit extracts (150mL) were allowed to react with 2850 mL of the FRAP solution for 30 min in the dark condition. Readings of the colored product [ferrous tripyridyltriazine complex] were then taken at 593 nm. The standard curve was linear between 25 and 800 mM Trolox. Results are expressed in mM TE/g fresh mass. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve. CUPRAC (CUPRIC Reducing Antioxidant Capacity) Method ; The “parent” CUPRAC (CUPRIC Reducing Antioxidant Capacity) method of antioxidant measurement, introduced by our research group to world literature, is based on the absorbance measurement of Cu(I)-neocuproine (Nc) chelate formed as a result of the redox reaction of chain-breaking antioxidants with the CUPRAC reagent, Cu(II)-Nc, where absorbance is recorded at the maximal light absorption wavelength of 450 nm; thus this is an electron-transfer (ET)-based method. DPPH (2,2-diphenyl-1-picrylhydrazyl) Method ; The DPPH assay was done according to the method of Brand-Williams et al. (1995) with some modifications. The stock solution was prepared by dissolving 24 mg DPPH with 100 mL methanol and then stored at 20°C until needed. The working solution was obtained by mixing 10mL stock solution with 45mL methanol to obtain an absorbance of 1.170.02 units at 515 nm using the spectrophotometer. Fruit extracts (150 mL) were allowed to react with 2850 mL of the DPPH solution for 24 h in the dark. Then the absorbance was taken at 515 nm. The standard curve was linear between 25 and 800 mM Trolox. Results are expressed in mM TE/g fresh mass. Additional dilution was needed if the DPPH value measured was over the linear range of the standard curve. CERAC ( Cerium Reducing Antioxidant Capacity) Method ; A Ce(IV)-based reducing capacity (CERAC) assay was developed to measure the total antioxidant capacity (TAC) of foods, in which Ce(IV) would selectively oxidize antioxidant compounds but not citric acid and reducing sugars. The redox potential of the Ce(IV) oxidant was fine-tuned in 0.3M H2SO4 +0.7M Na2SO4 aqueous medium for selective oxidation. In the classical Ce(IV)-based assay for which the name CERAC was proposed, the presence of citric acid (at 1.5×10−5M) caused approximately 25% reduction in Ce(IV) (at 2.0×10−4M) recovery, whereas in the present method, the presence of citric acid (at 1.0×10−4M) caused negligible error in the TAC measurement of quercetin. The trolox equivalent antioxidant capacity (TEAC) values in the order of quercetin>rutin>gallic acid>catechin>caffeic acid≥ferulic acid>naringenin≥naringin>trolox≥ascorbic acid were established with the proposed method and were found to be compatible with those found with other antioxidant assays. It is noteworthy that naringin and rutin were also hydrolyzed in the acidic medium of the method so as to exert their full antioxidant capacity not measured by other TAC assays. The proposed TAC assay with Ce(IV) is simple, low-cost, rapid, and can be easily applied in modestly equipped conventional laboratories. In recent years there has been much publicity given to consumer concerns about the addition of chemicals to food. One area of concern is the interaction between additives and the possible health risks or joint effects of the cocktail of additives that is consumed each day. Sulphur dioxide, in its various forms, is added to food to inhibit and control the growth of microorganisms, to inhibit enzyme-catalysed reactions, to inhibit non-enzymic browning, and to act as an antioxidant and reducing agent. By providing stabilizing and conditioning functions, it improves the appearance and maintains the quality of foods and wines. However, it has drawn much attention recently because of its allergenic effect on those individuals who are hypersensitive. Nowadays, many countries have set strict limits on the residual amount of sulphur dioxide in different types of food. Therefore, sulphur dioxide content of wine should be precisely determined along with their total antioxidant content. The aim of this work was to measure the CERAC antioxidant capacity and sulphur dioxide amount of wine samples. There are an interference effect of sulphur dioxide on the CERAC method. Therefore, sulphur dioxide in wine samples was established with molecular atomic absorption method. For this purpose, several hollow cathode lamps (As, Se, Sb, Mg) were used and as a result of in this study, Se, which was used hollow cathode lamp at a wavelength of 204 nm, was choosen for the analysis of sulfur dioxide. molecular atomic absorption method were used to determination of sulphur dioxide in the sulphited wine samples and antioxidant capacities of the same samples were analysised by CERAC and CUPRAC. Real antioxidant capasity of wine samples were found substracting the quantity of interferent sulphur dioxide. CERAC (Ceric ion reducing antioxidant capacity) assay, is based on the room temperature - oxidation of antioxidant compounds with Ce(IV) sulfate in dilute sulfuric acid solution, and measuring the absorbance of unreacted Ce(IV) at 320 nm. CUPRAC method was used for determining the sulphite levels of sulphited wine samples by prior separation of monohydrogensulphite at pH 3 on an anion exchanger followed by spectrophotometry. The desulphited wine solutions were analysed for their antioxidant content by CUPRAC. The CERAC method results were highly reproducible, and correlated well with those of CUPRAC. Turkish wines generally showed higher antioxidant capacity than those reported in the literature from other varieties of different geographical origin.