Gıda Renklendiricilerinin Tayini İçin Yeni Yöntemler Geliştirilmesi

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Tarih
2015-10-23
Yazarlar
Olgun, Fatoş Ayça Özdemir
Süreli Yayın başlığı
Süreli Yayın ISSN
Cilt Başlığı
Yayınevi
Fen Bilimleri Enstitüsü
Institute of Science And Technology
Özet
Yüzyıllardır gıdalar birçok farklı amaçlarla renklendirilmektedir. Milattan önceki yıllarda, şarabın renklendirilmesiyle başlayan bu süreç 1800’lü yıllarda sentetik renklendiricilerin kullanılmasıyla devam eder. Günümüzde ise renklendiricilerin kullanılması sonucu ortaya çıkan sağlık problemleri ve gelişen teknoloji doğrultusunda doğal renklendiricilerin kullanımı tercih edilmektedir.  Gıda pazarlamasında rengin ürüne kattığı cazibe nedeniyle gıdaların renklendirilmesine ihtiyaç duyulmuştur. Renklendiriciler sentetik ve doğal kaynaklı olmak üzere iki temel grupta toplanır. Bazı sentetik renklendiricilerin toksik ve karsinojenik etkilerinden dolayı kullanımları gıda mevzuatları tarafından sınırlandırılmıştır. Tarım ve Köy İşleri Bakanlığı tarafından yürütülen ve Avrupa Birliği uyum çerçevesinde hazırlanan “Gıdalarda kullanılan renklendiriciler” tebliğinde 2002/55 Ek 1 olarak verilen tabloda bulunan renklendiriciler gıda maddelerine katkı olarak eklenebilirler. Son 25 yıldır tüketiciler sağlık endişeleri nedeniyle gıdalarda doğal katkı maddelerinin kullanımını tercih etmektedirler. Gıda renklendiricileri tebliğinde kullanımına izin verilen renklendiricilerden bazıları karoten, klorofil ve antosiyanin gruplardır. Bu pigmentler gıdalara renklendirme özelliğinin yanısıra antiokisdan özellikler de kazandırırlar. Epidemolojik çalışmalar, antioksidanların kanser ve kardiyovasküler hastalıkları önlediğini göstermektedir. Antioksidanlar, insan vücudunda bulunan serbest radikalleri temizleyerek hücre hasarının önüne geçerler.  Serbest radikaller, aerobik solunum sonucu süperoksit, hidroksil, hidroperoksil, peroksil ve alkoksil gibi radikallere dönüşürler. Doğal antioksidan  enzimler bu serbest radikalleri uzaklaştırdığından oksidatif stresi azaltmak için antioksidan açısından zengin gıdalar tüketmek faydalıdır. Bu bağlamda gıdaların antioksidan kapasitelerinin belirlenmesi zorunludur. Gıda pazarlama endüstrisinde oldukça önemli yere sahip olan doğal veya sentetik renklendiriciler için tayin ve ayırma yöntemlerinin geliştirilmesi oldukça önemlidir. Sentetik renklendiriciler gıda proseslerinde kararlı yapıları ve ucuz maliyetleri nedeniyle tercih edilseler de hatalı kullanımları sonucu insan sağlılğını bozucu etkilere neden olabilirler. Bunun yanında, gıdalarda kullanılan renklendiricilerin seçimi tebliğ rehberliğinde olmalıdır. Doğal renklendiriciler ise gıdalara renk yanında antioksidan özellik katmalarından dolayı anioksidan kapasitelerinin belirlenmesi ve tüketicinin bilinçlendirilmesi sağlanmalıdır.  Bu çalışmanın kapsamında gıdaların renklendirilmesinde kullanılan sentetik renklendiricilerin hızlı, kolay ve uygulanabilir tayin yöntemleri ile analiz edilmeleri hedeflenmiştir. Bu amaç doğrultusunda, literatürde antioksidan tayin yöntemleri olarak kullanılan CERAC (Seryum(IV) indirgeyici antioksidan kapasite) ve CUPRAC (Bakır (II) indirgeyici antioksidan kapasite) yöntemleri sentetik renklendiricilere uygulanmış, toz içecek örneklerinin toplam sentetik renklendirici içeriği bulunmuştur.  Renklendirici tayininde oldukça tercih edilen kromatografik yöntemde ise 5 farklı sentetik renklendirici standartının ayırımı için dereceli elüsyon programı geliştirilmiş ve literatürde çok kullanılan asetonitrilin bu teknikte kullanılmamasıyla çözücü maliyetinin düşürülmesi sağlanmıştır.  Çalışmanın üçüncü bölümünde in-vitro antioksidan kapasite tayin yöntemleri ile HPLC tekniği birleştirilerek hat-üstü HPLC sistemi oluşturulmuştur. Hat-üstü HPLC yöntemi sayesinde, karmaşık matriks yapısında olan örneklerin parmak izi yapısı oluşturulmuştur. Tez kapsamında hat-üstü HPLC-CUPRAC yöntemi ilk defa sentetik renklendirici standartlarına uygulanmıştır. Hat-üstü HPLC-CERAC yöntemi ise tez çalışması kapsamında geliştirilmiştir. Akış hızı ve hat-üstü CERAC reaktif derişimi optimize edilmiştir.  Sonuç olarak sentetik renklendiricilerin tayini için yeni yöntemler geliştirmek amacıyla yapılan tez çalışmasında, literatürde in-vitro antioksidan kapasite tayin yöntemleri doğrudan sentetik renklendiricilere uygulanabilir olduğu saptanmıştır. Ayrıca kullanılan iki yöntem arasındaki sonuçların birbiriyle uyumlu olduğu gözlemlenmiştir. Bunun yanında beş farklı sentetik renklendiricinin HPLC yöntemi  ile ayırımı için dereceli elüsyon programı oluşturulmuştur. Bu ayırım programından faydalınılarak, hat-üstü HPLC-CUPRAC yönteminin uygulanabilirliği gözlemlenmiştir. Hat-üstü HPLC-CERAC yönteminin ise optimizasyonu sağlanmıştır.
Color has been added to our foods for centuries. Coloring food was first started as coloring wine before Christ and continued by the usage of synthetic colorants in 1800s. Today, the health concerns about the food colorants lead the food industry to focus on natural colorants. The appetitive and attractive appearance of colored food affecting the choice of the consumers direct the food marketing. Colorants are basically divided into two main groups as synthetic and natural food colorants. As a result of the toxic and carcinogenic effects of some synthetic food colorants, their utilisation is limited by the regulations defined by the governments. The food colorants allowed for usage are found in appendix 1 2002/55 given by regulations carried out by the Ministry of Agriculture, may be used as food additives. Synthetic colorants are preferred for their stability and low-cost in food processes. But their misusage may cause damage in human health. When choosing the right colorant, the regulations should guide.  For the last 25 years, the consumers prefer the usage of natural colorants as a consequence of health concerns. Colorants belonging to carotenoid, chorophyll or anthocyanin groups are found in the regulations as the allowed natural colorants. These pigments contribute antioxidant properties to the food beside the color benefits. The recent epidermological studies prove that antioxidants prevent cancer and cardiovasciular diseases. Antioxidants scaverging free radicals inhibit the cell damages. The free radicals found in human body are transformed into superoxides, hydroxyles, hydroperoxyles, peroxyles and alcoxyles as a result of aerobic respiration. Foods with rich antioxidant content should be consumed due to their benefit over removing free radicals and decreasing oxidative stress. Therefore, the determination and identification of these substances reveal a great importance. Apart from this, the antioxidant capacities of natural colorants must be evaluated for their antioxidant affects as well as their color properties. In this way, the consumers are informed about the subject. The aim of this study is to determine the synthetic colorants used as the food additives by a fast, accurate and appliable methods by developing and investigating novel methods for the determination of synthetic food colorants, analyzing synthetic colorant content of food products, providing food control by informing consumers about the limitations of these substances. Aiming the issues above, in-vitro antioxidant assays (CERAC and CUPRAC) were adapted for the determination of synthetic food colorants, proposed method results were correlated with HPLC findings and combination of in-vitro antioxidant assays with HPLC technique (application of online HPLC-CUPRAC technique and development of online HPLC-CERAC technique) were performed. CERAC method is ceric reducing antioxidant capacity (CERAC) reagent, successful in finding the antioxidant capacity of flavanoids, phenolics, vitamins C and E. The formal potential of the Ce(IV)/(III) redox couple in the presence of dilute sulfuric acid containing Na2SO4 is decreased from the standard potential of +1.61 V down to a value less than that of citric acid. Colorants with a redox potential less than this value can easily react with the CERAC reagent and be determined at the maximum absorption wavelength of Ce(IV). The CUPRAC method is a simple and versatile antioxidant capacity assay useful for a wide variety of polyphenols, including phenolic acids, hydroxycinnamic acids, flavonoids, carotenoids,anthocyanins, as well as for thiols, synthetic antioxidants, and vitamins C and E. The chromogenic oxidizing reagent bis(neocuproine)copper(II) cation (Cu(II)-Nc) is used as an outer-sphere electrontransfer agent and by reduction of this reagent with antioxidants, bis(neocuproine) copper(I) cation (Cu(I)-Nc) is formed. The main idea of these two in-vitro antioxidant capacity assays were used for the determination of synthetic food colorants. The term “Ponceu 4R equivalent colorant content” (PERI) coefficient which was found by the ratio of the molar absorption coefficient of a given synthetic food colorant with respect to CERAC and CUPRAC assays was introduced in order to calculate the total colorant content of yynthetic powder beverages. Most preferred methods for the determination of synthetic food colorants are still chromatographic techniques coupled with ultraviolet (UV) or diode array detectors (Serdar and Knezevic, 2009; Culzoni et al., 2009; Kirschbaum et al., 2006). There are two main problems with the use of single-wavelength UV detectors; various UV–visible (UV–Vis) spectra with different maximum absorbance wavelengths and long seperation time. Also in some cases, possible overlap of colorant peaks or the presence of other organic compounds such as flavors in the sample may occur. Both problems can be solved in the case of DADs. All dyes can be detected near to their maximum wavelength with the aid of multisignal detection capability, and peak identity can be easily confirmed. In the content of this study, a new gradient elution program was optimized for the analysis of synthetic food colorants. As an advantage, solution costs were minimized, not using the conventional solvent acetonitrile. Post-column derivatization involves the modification of the chromatographic system to allow the reaction to take place prior to entering the detector by inserting a post column reactor between the column and the detector. In order to achieve full resolution of all colorants, a variety of gradient elution programs were tested, using different mobile phases and changing retention times. But in all optimization experiments, the flow rate and injection volume were kept constant as 1 ml min−1 and 20 μL, respectively. The post-column reactor is required to fulfill the following functions: 1)Provide a source of reagent and a means of mixing it efficiently with the column eluent. 2)Ensure the reaction is complete before the derivatized product enters the detector. 3)Minimize the dispersion that takes place in the reactor so that the integrity of the separation achieved by the column is maintained. For post column derivitization technique, in-vitro antioxidant capacity assays (CUPRAC and CERAC) were combined with HPLC-PDA technique. The elution program used for the seperation of five synthetic colorants were employed. On-line HPLC-CUPRAC method assayed by Celik et. al. (2010) was applied directly to synthetic food colorants separating with the related gradient elution program (Celik et. al., 2010). Colorants were let to react with CUPRAC reagent in a time period of 1 minute. On-line HPLC-CERAC technique was used for the first time for the determination of synthetic food colorants. Therefore optimization experiments were required. For the flow rate optimization of derivatization (CERAC) reagent a fixed concentration of Ponceau 4R solution was used for the flow rate optimization experiments. CERAC reagent (kept in a flask away from air and light) was pumped with increasing flow rates between 0.8-0.2 mL/ min. Chromatograms were displayed for every flow rate and S/N ratios were calculated. Optimization of derivatization (CERAC) reagent concentration was performed wit a fixed concentration of Ponceau 4R used for the flow rate optimization experiments. CERAC reagent was pumped to the reaction coil with a flow rate of 0.5 mL/ min. S/N ratios of Ce(III) peak was detected at 320 nm. In this study, determination of five synthetic food colorants was investigated using spectrophotometric and chromatographic methods. By adapting the novel spectrophotometric CERAC and CUPRAC  assays of total antioxidant capacity to the determination of total food colorant content, certain beverage samples were easily and accurately analyzed. The total colorant content was found at low reagent and instrumentation costs with the use of a UV–vis spectrophotometer easily found in a conventional laboratory equipped with simple instruments. Moreover, simple sugars and citric acid, which are not colorants, are not oxidized in the CERAC method and therefore do not yield errors in colorant content calculations. Among CERAC and CUPRAC assays, experimental results showed that molar absorption coefficients of synthetic food colorants with respect to CERAC assay are similar with those of CUPRAC assay. HPLC analysis of colorants was performed with two different techniques. In conventional HPLC method, PDA detector system was used to monitor each colorant at its maximum absorbance wavelength. The selected mobile phase for the gradient elution program enabled the shortening of total analysis time when compared to other chromatographic methods. Furthermore, since acetonitrile as the conventional solvent was not used in the eluent, solution costs were minimized. On-line HPLC-CUPRAC method was adapted for the determination of synthetic food colorants. Optimized gradient elution program was used for the separation of colorants. Retention time periods were increased due to additional installation for derivatization coil. However, LOD values were decreased with on-line HPlC-CUPRAC method. Optimization experiments of on-line HPLC-CERAC method were performed. Considering the results, the method was found to be applicable for the determination of synthetic food colorants. Further experiments will be carried out for the method development.
Açıklama
Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2015
Thesis (PhD) -- İstanbul Technical University, Institute of Science and Technology, 2015
Anahtar kelimeler
hplc, post kolon, gıda renklendiricileri, CERAC, hplc, post column, food colorants, CERAC
Alıntı