Soğuk plazma uygulanmış soğan kabuğu tozunun çikolata üretiminde kullanımı
Utilization of cold plasma treated onion skin powder in chocolate production
Yazar: BERNA ŞENGÜLER
Danışman: DR. ÖĞR. ÜYESİ CELALE KIRKIN GÖZÜKIRMIZI
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı / Gıda Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Soğan (Allium cepa L.) dünyada en çok üretilen ikinci sebze türüdür ve dünya yemek kültüründe önemli bir yere sahiptir. Soğan tüketiminin çokluğu beraberinde soğan kabuğu atığı üretimini de getirmektedir. Soğan atıklarının %60'ını oluşturan soğan kabuğu içerisindeki fenolik madde ve flavonoidler sayesinde önemli bir antioksidan kaynağıdır. Soğan kabuğu atığı fitokimyasal ve besinsel özellikleri sayesinde çeşitli gıda ürünlerinde kullanılarak ürünün fonksiyonel özelliklerini geliştirmede ve böylece sürdürülebilir bir yan ürün olarak gıda endüstrisine kazandırılmalıdır. 
Soğan kabuğu sebzenin dış katmanında bulunması ve dış etkenlere direkt olarak maruz kalması mikrobiyal yükünü arttırmakta ve mikotoksin gibi kimyasal kalıntılar içermesine sebep olmaktadır. Soğan kabuğu atığının gıda endüstrisinde kullanılması için mikrobiyal yükünden arındırılması gerekmektedir.
Soğuk plazma teknolojisi yüzey dekontaminasyonu ve mikrobiyal inaktivasyon için gıdaların işlenmesinde kullanılan yenilikçi bir teknolojidir. Maddenin dördüncü halini temsil eden plazmanın soğuk plazma uygulamaları için iyonlaşma derecesi %10-4 ile %10-1 arasında değişmekte, gaz sıcaklığı ise 1000 K'dır. Isıl işlemlere kıyasla soğuk plazma teknolojisi uygulandığı gıda ürününün besinsel, fiziksel ve kimyasal özelliklerinin korurken mikrobiyal yükü ve toksin miktarını azaltma da daha etkili bir yöntemdir. 
Bu çalışmanın ilk aşamasında atık olarak çıkarılan soğan kabuklarına öğütme işleminden sonar dielektrik bariyer boşaltım soğuk plazma (DBDSP) teknolojisi 40 kV, 56 kHz frekans, 26°C ve 10 mA koşullarında 10 (CP10), 20 (CP20), ve 30 (CP30) dakika boyunca uygulanmıştır. Kontrol (CP0) olarak soğuk plazmaya maruz kalmayan örnekler kullanılmıştır. Örnekler, mikrobiyal kalite, renk, nem, toplam fenolik madde içeriği ve antioksidan aktivitelerine göre incelenmiştir. Antioksidan aktivite DPPH ve FRAP yöntemleri kullanılarak analiz edilmiştir. 
DBDSP uygulamasının tek başına SKT'nin mikrobiyal yükü üzerinde önemli bir etkisi olmadığı gözlemlenmiştir. Küf-maya ve toplam bakteri sayılarının 30 dakikalık işlem sonunda kabul edilebilir aralığa ulaşmadığı sonucuna varılmıştır. Uygulama SKT'nin nem oranında ve L*, a* ve b* renk değerlerinde önemli bir değişime sebep olmamıştır. Toplam fenolik madde içeriğinin maruz kalma süresinin artması ile artan bir eğilim gösterdiği gözlemlenmiştir (p<0,05). Fenol içeriği en düşük CP0 örneğinde en yüksek ise CP30 örneğinde tayin edilmiştir. Benzer olarak, soğuk plazma uygulamasının SKT'nin antioksidan aktivitesi üzerinde olumlu bir etkisi olmuştur. CP30 örneği en yüksek antioksidan aktiviteye sahip iken işlem süresinin azalması ile aktivitenin azaldığı gözlemlenmiştir. 
Çalışmanın ikinci aşamasında, DBDSP uygulanmış CP0 ve CP30 örnekleri %1 ve %2'lik oranlarla bitter çikolata formülasyonuna dahil edilmiştir. Formülasyondaki diğer içerik ve parametreler sabit tutulmuştur. Örnekler SKT içermeyen kontrol, %1 ilave örnekleri (CP0-1 ve CP30-1) ve %2 ilave örnekleri (CP0-2 ve CP30-2) olarak belirlenmiştir.  Hazırlanan örnekler, renk, duyusal özellikler, toplam fenolik madde ve antioksidan aktivitelerine göre incelenmiştir. 
Renk değerleri SKT ilavesi ile değişim göstermiştir.  SKT oranın artmasıyla örneklerin iç ve dış yüzeylerindeki L* değeri azalma, a* ve b* değerlerinde artış gözlemlenmiştir.  Toplam fenol içeriği örnekler arasında değişim göstermiştir. Kontrol en düşük (20,08 mg/GAE g) içeriğe sahip bulunurken en yüksek içerik CP30-2 (23,40 mg/GAE g) örneğinde elde edilmiştir. Benzer olarak antioksidan aktivite analiz sonuçları da çikolata formülasyonlarının değişime bağlı olarak farklılık göstermiştir. SKT ilave oranın artması bitter çikolatada antioksidan aktivitenin artmasına neden olmuştur. DPPH ve FRAP analiz sonuçları CP30 örneğinin en yüksek aktiviteye, CP30-2 örneğinin ise diğer formülasyonlardan daha yüksek aktiviteye sahip olduğunu göstermiştir. Geliştirilen çikolataların tüketici tarafından kabul edilebilirliği ve koku, tat, ürün rengi, doku, yağlılık, ve acılık parametreleri duyusal analizler ile tespit edilmiştir. CP0-1 örneği yağlılık parametresinde en yüksek değere sahip bulunurken kalan parametrelerde en yüksek değer kontrol örneğine verilmiştir. SKT'nin eklenmesi acılık (bitter tat) haricindeki tüm parametrelerde olumsuz bir etki göstermiştir. %1 SKT eklenmesi tüketiciler tarafından genel olarak kabul edilebilir bulunmuştur.

Abstract:
Onion (Allium cepa L.) is the second most produced vegetable and has an important place in world food culture. The high consumption of onion also leads to the generation of onion peel waste. Onion processing consists of peeling, slicing and dicing, and these processes generate peel waste, which forms the main part of processing waste. Onion peel, which represents 60% of onion waste, is an important source of antioxidants owing to the phenolic substances and flavonoids it contains. In Europe, more than 0.6 MT of onion skins waste are generated every year, and in India 300-500 kg of onion skins are wasted every day. Also, onion skins are rich in fiber and carbohydrates. The fatty acid profile is mainly composed of 76.79% unsaturated fat and 21.42% total saturated fat. Onion peel contains various bioactive compounds such as organosulfur compounds, thiosulfinates, polyphenols including flavonoids and fructooligosaccharides (FOS). Quercetin derivatives, which give onion skins different colors from yellow to purple, are found in high amounts in onion skins to protect the onion from soil microbes. Thanks to its phytochemical and nutritional properties, onion peel waste can be used in various food products to improve the functional properties of the product and thus should be brought to the food industry as a sustainable by-product.
Onion peel is located on the outer layer of the vegetable and is directly exposed to external factors, which increases its microbial load and causes it to contain chemical residues such as mycotoxins. The onion peel waste needs to be purified from its microbial load to be used in the food industry.
Cold plasma technology is an innovative technology used in food processing for surface decontamination and microbial inactivation. The degree of ionization of plasma, the fourth state of matter, for cold plasma applications varies between 10-4% ile 10-1% and the gas temperature is 1000 K. Corona discharges, microwave plasma, radio frequency plasma, induction coupled plasma, capacitance coupled plasma, pulsed plasma, arc discharge, atmospheric pressure plasma jet and dielectric barrier discharge plasma are systems used in industry. In agricultural and food industry research, dielectric barrier discharge and jet plasma are generally preferred due to their simple, adaptable and versatile design. Cold plasma technology plays an effective role in the inactivation of a wide range of microorganisms, including spores and viruses.  The biological inactivation process involves reactive compounds, high-energy electrons, ionized atoms and particles, and UV radiation. Compounds derived from oxygen or nitrogen come into contact with biological material, causing disruption of proper membrane function and oxidation of proteins and lipids, leading to cell membrane disruption. Cold plasma is applied in the fields of food safety and quality processing, including food decontamination, toxin degradation, modification of proteins, packaging modifications. Cold plasma is an effective non-thermal processing technology for the inactivation of various pathogenic and spoilage microorganisms present in food products. Compared to heat treatment, cold plasma technology is a more effective method of reducing microbial load and toxin content while maintaining the nutritional, physical and chemical characteristics of the food product.
For the first part of the study, the onion skin powder samples were placed on a glass petri dish with a thickness of 1.5 mm with a gap of 2 cm between the electrodes (diameter: 12 cm, thickness 0.4 mm) and applied at 40 kV, 56 kHz frequency, 26°C and 10 mA for 10 (CP10), 20 (CP20), 30 (CP30) minutes for the first part of the study. Non-exposed samples to cold plasma were used as control (CP0). Onion peel extraction was performed for the analyses. Weighed 1 g of 12 onion peel powder samples exposed to cold plasma for different durations were added to 25 mL of methanol (80% by volume) and stirred for 1 min using a vortex. The mixture was kept in an ultrasonic water bath at 40°C for 2 hours for incubation. The incubated samples were centrifuged at 4000 rpm for 10 minutes.  The resulting onion peel powder extracts were stored at -18°C for further analysis. 
Samples were analyzed for microbial quality, color, moisture, total phenolic content and antioxidant activity. Total bacterial count in the samples was determined by smear plate method. Total yeast and mold counts in onion peel powder samples were determined by ICMSF. Antioxidant activity was determined using DPPH and FRAP methods. The color change in chocolate and onion skins was measured with a colorimeter in terms of L*, a* and b* respectively.
It was observed that DBDCP treatment had no significant effect on the microbial load of the onion peel waste alone. It was concluded that mold-yeast and total bacteria counts did not reach the acceptable range at the end of the 30-minute treatment. The treatment did not cause a significant change in the moisture content and L*, a* and b* color values of onion peel waste. Total phenolic content showed an increasing trend with increasing exposure time (p<0.05). The lowest phenolic content was determined in CP0 sample and the highest in CP30 sample. Similarly, cold plasma treatment had a positive effect on the antioxidant activity of onion peel waste. While the CP30 sample had the highest antioxidant activity, it was observed that the activity decreased with decreasing treatment time.
In the second stage of the study, DBDCP-treated CP0 and CP30 samples were added to the dark chocolate formulation at 1% and 2%. Other ingredients and parameters in the formulation were kept constant. The samples were determined as control without DBDSP, 1% addition samples (CP0-1 and CP30-1) and 2% addition samples (CP0-2 and CP30-2).  Chocolate extractions were prepared for analysis. Weighed 1 g of 5 chocolate samples with different formulations and added 5 mL hexane. The mixture was kept in an ultrasonic water bath at 30°C for 10 minutes. The incubated samples were centrifuged at 4000 rpm for 10 minutes. This process was repeated 2 times and the fat was removed. To the defatted chocolate samples, 25 mL of methanol (80% by volume) was added and mixed for 1 minute using a vortex. The mixture was incubated in an ultrasonic water bath at 40°C for 2 hours. The incubated samples were centrifuged at 4000 rpm for 10 minutes.  The resulting chocolate extracts were stored at -18°C for further analysis. The prepared samples were analyzed for color, sensory properties, total phenolic content and antioxidant activity. 
Sensory Analyses were carried out in Istanbul Gelisim University Application kitchen. The 12 research assistants participating in the experiment were informed about the experiment before the analysis. 15 room temperature chocolate samples coded with 3 different numbers were placed in a different order in the experimental setup for each panelist. Panelists were instructed to record their responses as soon as the samples were placed on the tongue. The chocolate samples were evaluated by the panelists for odor, internal color, taste, bitterness, fattiness, and texture on a 9-point hedonic scale (1: extremely dislike, 9: extremely like). Samples were evaluated 3 times by each panelist and each panelist was given a glass of water to rinse their mouth before tasting the samples. Antioxidant activity was determined using DPPH and FRAP methods. 
Chocolate obtained from the cocoa (Theobroma cacao) fruit is a widely consumed product due to its flavor and health benefits rich in fat, protein, carbohydrates, polyphenols and other bioactive compounds. Chocolate varieties are categorized depending on the cocoa dry matter, milk fat and cocoa butter they contain. Chocolate classified as dark, milk and white chocolate differs in its content components, especially carbohydrate, fat and protein. Chocolate production is a complex process involving numerous chemical reactions. This process includes fermentation, drying, roasting, roasting, grinding, mixing, conching and tempering, which are important reactions for the development of the appropriate chocolate flavor and texture. 
The flavor and texture of chocolate are two important characteristics that determine the quality of the product. Flavor and texture are of primary importance in the processing of cocoa beans into chocolate. Although there are many different flavors of chocolate, they should all be free of undesirable flavors and contain pleasant tastes that the consumer associates with the product.
Chocolate has many health benefits, but its quality and salutogenic effects depend on its cocoa content. Dark chocolate, which is more than 80% cocoa bean solids and cocoa butter by total weight, has a higher cocoa content than milk and white chocolate and therefore has more positive health effects. Dark chocolate, or cocoa in its natural form, has been used for thousands of years to improve performance and alleviate illness. Regular consumption of dark chocolate reduces the formation of high blood pressure and delays the formation of cardiovascular diseases, as well as reducing insulin resistance, keeping blood vessels healthy and preventing impaired blood flow. In addition, dark chocolate has been proven to be protective against cancer and premature aging by eliminating free radicals thanks to its antioxidant properties.
In the second stage of research, addition onion skin powder to bitter chocolate, color values changed with the addition of onion peel waste.  L* value on the inner and outer surfaces of the samples decreased, a* and b* values increased with the increase in ratio. Total phenol content varied among the samples. While the control had the lowest content (20.08 mg/GAE g), the highest content was obtained in CP30-2 (23.40 mg/GAE g). Similarly, the results of antioxidant activity analysis also showed differences depending on the change in chocolate formulations. The increase in the addition rate of onion peel powder caused an increase in antioxidant activity in dark chocolate. DPPH and FRAP analysis results showed that CP30 sample had the highest activity and CP30-2 sample had higher activity than the other formulations. The consumer acceptability of the developed chocolates and the parameters of odor, taste, product color, texture, fattiness, and bitterness were determined by sensory analysis. The CP0-1 sample was found to have the highest value in the fattiness parameter, while the highest value in the remaining parameters was given to the control sample. The addition of onion peel powder had a negative effect on all parameters except bitterness (bitter taste). 1% onion skin powder addition was generally found acceptable by consumers.