Elektrodöndürme yöntemiyle elde edilen jelatin nanoliflerinin emülsiyonlarda stabilize edici olarak kullanılması
Using electrospun gelatin nanofibers as stabilizers in emulsions
Yazar: NAGİHAN OKUTAN
Danışman: YRD. DOÇ. DR. FİLİZ ALTAY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2013
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Damla büyüklüğü = Droplet size ; Difüzyon katsayısı = Diffusion coefficient ; Elektriksel iletkenlik = Electrical conductivity ; Jelatin = Gelatin ; Reolojik özellikler = Rheologic properties ; Spektrometrik analiz = Spectrometric analysis ; Stabilleştirme = Stabilization ; Türbidit = Turbidite ; Yüzey gerilimi = Surface tension ; Zeta potansiyeli = Zeta potential

Özet:
Nanoteknoloji, maddenin atom seviyesinde daha gelişmiş materyaller, araçlar ve sistemler geliştirilmesi amacıyla işlenmesi olarak tanımlanmaktadır. Yeni üretim işlemlerine dayalı olması, daha iyi ve daha yüksek standartlarda ürünlerin üretilebilmesine olanak sağlaması  nedeniyle nanoteknoloji günümüzün rağbet gören teknolojilerinden biridir. Diğer akademik alanlardan farklı olarak nanoteknoloji tek bir disipline ait değildir. Nanoteknoloji bilimi ve bunun uygulamalarında kimya, fizik, biyoloji, malzeme bilimi ve çeşitli mühendislik dalları yer almaktadır. Bu da bugüne kadar etkileşim içinde olmayan birçok bilim adamını yanyana getirmektedir. Atomik seviyede tasarlama yetisi klasik bilimleri ayıran sınırları bulanıklaştırarak, yepyeni disiplinler doğurmakta ve dünyada standartlaşmış işlem prosedürlerini derinden etkilemektedir. Gıda sanayinin nanoteknolojiden yararlanma potansiyeli yüksek olmasına rağmen  kullanımı çok sınırlı ölçülerdedir. Yine de özellikle son iki yıl içinde dünya gıda sanayi bu teknolojinin üstünlüklerinden yararlanma yollarının arayışı içine girmiştir. Bazı firmalar nanoteknolojik araştırma programlarını desteklemekte  ve gelecek 10 yıl içerisinde kullanmayı amaçlamaktadır. Günümüzde tüketiciler tarafından talep edilen gıda kavramı değişmekte ve buna bağlı olarak üreticiler tüketicilerin gereksinim ve istekleri doğrultusunda değişebilen interaktif gıda fikirleri üzerinde çalışmaktadır. Buna ilaveten nanoteknolojinin gıdalarda değişik amaçlarla uygulanması çalışmalarıyla ilgili yayınlar gün geçtikçe artmaktadır.
Nanoteknolojik uygulamalarda kullanılan nanoyapılar nanokapsüller, nanotüpler ve nanolifler olmak üzere temelde üç gruba ayrılmaktadır. Nanolifler çapları bir mikronun altında olan yapılardır. Başta elektrodöndürme yöntemi olmak üzere çeşitli tekniklerle üretilebilmektedirler. Elektrodöndürme yöntemi ile üretilen nanolifler tekstil, tarım, tıp ve ilaç uygulamaları, uzay uygulamaları, savunma endüstrisi, hava ve su filtrasyon uygulamaları gibi alanlarda kullanılmaktadır. Gıda endüstrisinde nanolifler özellikle ambalaj ve sensör uygulamalarında çalışılmıştır. Toksikolojik durumları ve bu konudaki yasal düzenlemelerin azlığı veya bazı ülkelerde yokluğu nedeniyle sadece nanoliflerin değil nanoyapıların gıdaya ilaveleri henüz yaygınlaşmamıştır.
Emülsiyonlar karışmayan iki sıvının (genellikle su ve yağ) birbiri içinde damlacıklar halinde dağıldığı sistemlerdir. Basit olarak su içinde yağ (Y/S) ve yağ içinde su (S/Y) olarak ikiye ayrılmaktadırlar. Y/S emülsiyonlarında sürekli faz su dağılan faz yağdır. Bu tip emülsiyonlara süt, mayonez ve salata sosları örnek olarak verilebilir. S/Y içinde su emülsiyonlarında ise sürekli faz yağ iken dağılan faz sudur. Bu tip emülsiyonlara örnek olarak margarin ve tereyağı gösterilebilir. Birçok gıda emülsiyonunda dağılan faza ait damlacık çapı 0,1 ve 100 µm arasında değişmektedir. Emülsiyonlar termodinamik olarak stabil olmayan sistemlerdir. Dağılan fazın sürekli faz içerisinde kinetik olarak anlamlı bir süre boyunca stabil kalabilmesi için emülsifiye edici veya kıvam artırıcı maddelere ihtiyaç vardır. Emülsifiye ediciler dağılan fazın çeperlerine adsorbe olarak emülsiyonu stabil hale getiren amfifilik (hem hidrofilik hem de hidrofobik kısma sahip) bileşenlerdir. Adsorbe olan bileşen sayesinde damlacıkların etrafında bir çeper oluşmakta ve bu çeper damlacıkların bir araya gelmesini engellemektedir. Gıda endüstrisinde emülsiyon stabilitesini sağlama ve arttırma için yeni tekniklerin ve/veya malzemelerin denenmesi çalışmaları devam etmektedir.
Bu çalışmada öncelikle doğal polimerlerden elektrodöndürme tekniği ile nanolif elde edilmeye çalışılmıştır. Denenen polimerler içinde sadece jelatin ile tek başına nanolif elde edilebilmiştir. Jelatin ile elde edilen nanoliflerde farklı elektrodöndürme koşullarının nanolif morfolojisine etkisi incelenmiştir. Elde edilen SEM görüntüleri sonucunda en ince nanolif çapına 28 kV, 0,1 ml/s debi, 10 cm plaka mesafesinin uygulandığı elektrodöndürme şartlarında ulaşıldığı görülmüştür. Daha sonra 18 kV, 1ml/s debi, 10 cm plaka mesafesi koşullarında üretilen nanolifler biriktirilerek emülsiyonların stabilizasyonunda kullanılmıştır. Jelatin nanolifleri farklı konsantrasyonlarda Y/S emülsiyonlarına ilave edilerek, emülsiyon stabiliteleri zeta potansiyeli, damlacık boyutu ve dağılımı, türbidite, yüzey gerilimi, reoloji ve kremleşme indeksi bakımından değerlendirilmiştir. Ölçüm sonuçları Y/S emülsiyonuna jelatin ilave edilmesi durumunda alınan sonuçlarla karşılaştırılmıştır. Jelatin nanolifleri ile stabilize edilen emülsiyonlarda jelatin ile stabilize edilen emülsiyonlara göre ortalama damlacık boyutlarının ve türbidite değerlerinin daha küçük olduğu, yüzey gerilimlerinin ve reolojik karakterizasyonda n değerlerinin daha yüksek olduğu, 15 gün boyunca 4o C?de ise kremleşme indekslerinin daha düşük olduğu gözlemlenmiştir. Bu karşılaştırmalar sonucunda Y/S emülsiyonlarına jelatin nanolifleri ilavesinin, jelatin ilavesine göre daha stabil emülsiyonlar oluşturduğu sonucuna varılmıştır.

Abstract:
Nanotechnology defined as the process for development of advanced materials, tools and systems. Nanotechnology which has taken as basis of having different properties at nanoscale has resulted sensitive, precise and applicable in daily life outcomes. Comparing to other areas, applications of nanotechnology in foods has been limited. Nanotechnology applications located in the centre of various engineering branches, it also brings many scientists together doesn't interact so far. The ability to design at atomic level nanotechnology creatinga new world and deeply affects standardized operating procedures.Their applications to the agriculture and food sector are relatively recent compared with their use in other areas. Nevertheless,in the last two years,the world has entered into the search for ways to take advantage of this technology in the food industry. Some companies support nanotechnology research programs and intends to useinthe next 10 years. Nowadays, the concept of food demanded by consumers is changing and food manufacturers are working on new interactivefoodideas. In addition, publications about food nanotechnology are increasing every day. 
Nanostructures used in nanotechnology applications divided into three groups including nanoparticles, nanotubes and nanofibers. Nanofibers are defined as fibers with diameters on the order of 100 nanometers. Nanofibers especially organic nanofibers constitute a particularly interestine and versatile class of one dimensional nanoaterial. Several techniques unrelated to electrospinning were reported in early literature for the laboatory preparation of nanofibers. Electrospinning is easier and more economical method comparing to other methods for obtaining nanomaterials and used in textile, agriculture, medical and pharmaceutical applications, aerospace applications, defense industry, air and water filtration applications. The first documented accounts of elctrostatic spinning of a polymer solution into nanofibers were described in 1902 by J.F. Cooley and by W.J. Morton. Anton Formhals patented an improved version of electrospinning process and appartus. His first patents on electrospinning of cellulose acetate from acetone used a fiber collection system that could be moved allowing some degree of fiber orientation during spinning. Taylor?s contribution in the 1960s towards the fundamental understanding of the behaviour of the droplets placed in an electric field helped further develop the technique. The minimum equipment requirements for dempnstration of simple electrospinning in the laboratory are; a viscous polymer solution or a melt, an electrode that is maintained in contact with the polymer solution, a high-voltage DC generator connected to the electrode and a grounden or oppositely charged surface to collect nanofibers. 
An emulsion consist of two immiscible liquids with one of the liquids dispersed as small spherical droplets in the other. In most foods the diameters of the droplets usually between 0.1 and 100 µm. Emulsions can be classified according to the distribution of the oil and aqueous phases. A system which consist of oil droplets dispersed in aqueous phase is called oil-in-water or O/W emulsions. A system which consist of water droplets dispersed in oil phase is called water-in-oil or W/O emulsions. Emulsions are thermodynamically unstable systems. It is possible to form emulsions that are kinetically stable for a reasonable period of time by substances known as emulsifiers. Biopolymers that have significant amount of both polar and non-polar groups tend to be surface active. Biopolymers such as proteins and polysaccharides prevent destabilization by combined mechanisms. They decrease interfacial tension, adsorb and form physical barrier between droplets, changes the viscosity of continuous phase, and induce static or electrostatic interactions. Gelatin is a relatively high molecular weight protein derived from annimal collagen. Gelatin is slightly surface active and can act as an emulsifier in oil-in-water emulsions. Characterization of emulsion properties is important for designing and manufacturing foods with improved properties. Zeta potential, average droplet size and droplet size distribution, turbidity, surface tension, rheological characteristics, and creaming index are some of the important parameters of emulsions.
In this study we investigate using gelatin nanofibers obtained by electrospinning technique for the stabilization of O/W emulsions. First we investigated the process factors affecting the morphology and diameter of gelatin nanofibers. 
The electrical conductivity of gelatin solutions increased with gelatin concentration. In  contrast  electrical  conductivity  results,  surface  tensions  decreased  with  gelatin concentration. The decrease in surface tension from 36.24 mN/m to 34.91 mN/m. In our study, at low concentration of 7%, we were unable to obtain nanofibers  low concentration with insufficient entanglements and high surface tension. Instead, the  mixtures  of  drops  and  some fibrous structures  were seen.  and this is due to the viscosity of the solution being too low to generate continuous fibers. When  the  applied  voltage increased from 28 to 35 kV, the fibrous structure formations started at feed rate of 1 mL/h. At lower feed rate (0.1 mL/h) the same fibrous structure formations  occurred  for  sample  4  when  the  applied  voltage  increased  from 28 to  35  kV. At the same applied voltage when the feed rate decreased from 1 to 0.1 mL/h  , the drops  became fibrous structures with bead. In addition, due to appearance of increasing fibrous structure the diameters of fibers were determined as in between ~ 23-26 nm. These results suggested that as the applied voltage increases and the feed rate decreases, electrospun samples  can be produced with  more fibrous structure, instead of drops at sufficient polymer concentration. SEM images of electrospun gelatin nanofibers  from solution at 20% (w/v) were showed that when gelatin concentration increased from 7% to 20% (w/v),  we  were  able  to  obtain  nanofiber  formations  under  all  electrospinning  process conditions,  meaning  the  amount  of  gelatin  in  the  solution  at  20%  was  enough  to  form nanofibers. When the applied voltage increased from 28 to 35 kV the diameters of nanofibers  had  a  tendency  to  increase  from  46-55  nm  to  47-62  nm. It should  also  be  noted  that  nanofibers  became  less  branching,  without  bead  and  more  homogenous as applied voltage increased. At lower feed rate (0.1 mL/h)  similar  results were obtained. When the applied voltage  increased from  28 to 35 kV  bead formation disappeared and more homogenous fiber formation occurred. The range of diameters increased from 45-49 nm to a wider range of 50-88 nm. When the feed rate decreased from 1 to 0.1 mL/h at 28 kV, the few beads appeared, the range of diameters slightly decreased from 46-55 nm to 45-49 nm. At higher applied voltage  (35 kV) as  the feed rate decreased from 1 to 0.1 mL/h , the range of diameters increased from 47-62 to 50-88 nm. 
It  appears  that  electrospinning  changed  the  zeta potentials from negative to positive  for  all samples.  The zeta potential values of nanofibers obtained at 28kV were higher than electrospun samples obtained at 35 kV. As applied voltage decreased from 35 kV to 28 kV, the zeta potential values of nanofibers increased.  The zeta potential values of nanofibers decreased with the feed rate at any given applied voltage. For keeping a suspension in a stable or in a dispersed state the zeta  potential values should be above +25 mV or below -25mV. Accordingly the closer value to the +25 mV belonged to the sample obtained from 28 kV and 0.1 ml/h feed rate, meaning these nanofibers  may  suspend in a dispersed state  longer  comparing to the other nanofiber samples. 
In case of diffusion coefficient, electrospun gelatin samples higher diffusivities than gelatin  powder meaning  electrospinning  may  increase  the  ability  of  samples  to  diffuse comparing to gelatin powder. The  diffusion  coefficient  values  of  nanofiber  samples  decreased  with  the  increasing concentration  of nanofibers  in  ethanol. Accordingly,  higher  diffusion  coefficient  means  higher  mobility  of  the  polymer  in  the suspension.  We  determined  that  the  sample  obtained from 28 kV and 0.1 ml/h feed rate,  had  the  highest  diffusion  coefficient  (1.19 µm2/s),  and  probably  the  highest  mobility  comparing  to  the  other  electrospun  samples. Furthermore, the diffusion coefficient values of nanofiber samples decreased as the applied voltage increased at any given concentration and feed rate. The gelatin-acetic acid solution at 7% (w/v) did not produce nanofibers but we were able to obtain gelatin nanofibers under all electrospinning process conditions with 20% (w/v) gelatin-acetic acid solutions. When the applied voltage increased from 28 to 35 kV the diameters of gelatin nanofibers had a tendency to increase when the other process parameters were constant.
We stabilized O/W emulsions by adding gelatin nanofibers obtained from 18 kV and 1 ml/h feed rate, at different concentrations. The stability of O/W emulsions determined by zeta potential, droplet size and distribution, turbidity, surface tension and rheological measurements. Measurement results of O/W emulsions compared with the results of gelatin stabilized emulsions. 
The zeta potential of 0.5% (w/w) gelatin stabilized O/W emulsion (-10.1 mV) is lower than 0.5% (w/w) gelatin nanofiber stabilized O/W emulsion. At 0.75% (w/w) concentrations gelatin stabilized emulsion (-14.3 mV) has higher zeta potential than gelatin nanofiber stabilized emulsion (-10.7 mV). Zeta potential of 1% (w/w) gelatin stabilized emulsion (-1.7 mV) is lower than 1% (w/w) gelatin nanofiber stabilized emulsion ( -14 mV). According to these results we suggested that changes in zeta potentials are independent from gelatin type and concentrations.   
In general at the same concentrations emulsions stabilized gelatin nanofibers produced smaller average droplet sizes and narrower droplet size distributions. Average droplet diameters of 0.5% gelatin stabilized emulsion is 762,6 nm, 0.5% gelatin nanofiber stabilized emulsion is 552.1 nm; 0.75% gelatin stabilized emulsion is 506.3 nm, 0.75% gelatin nanofiber stabilized emulsion is 411 nm; 1% gelatin stabilized emulsion is 560.5 nm, and 1% gelatin nanofiber stabilized emulsion is 498.3 nm.
Turbidity of 0.5% gelatin stabilized emulsion is 55876.54 m-1, 0.5% gelatin nanofiber stabilized emulsion is 41264 m-1; 0.75% gelatin stabilized emulsion is 56947.43 m-1, 0.75% gelatin nanofiber stabilized emulsion is 41799.45 m-1; 1% gelatin stabilized emulsion is 58484.69 m-1, and 1% gelatin nanofiber stabilized emulsion is 43361.20 m-1. Turbidity of emulsions are decrease as the average droplet diameters decrease, and increased as the gelatin concentrations increase.
Surface tension of 0.5 % gelatin stabilized emulsion is 56.1 mN/m, 0.5% gelatin nanofiber stabilized emulsion is 63.9 mN/m; 0.75% gelatin stabilized emulsion is 55.9 mN/m, 0.75% gelatin nanofiber stabilized emulsion is 60.1 mN/m; 1% gelatin stabilized emulsion is 64.9 mN/m, and 1% gelatin nanofiber stabilized emulsion is 59.3 mN/m.
According to the rheological characterizations ?n? values of gelatin nanofiber stabilized O/W emulsions are higher than gelatin stabilzed O/W emulsions. ?n? value of 0.5% gelatin stabilized emulsion is 0.6383, 0.5% gelatin nanofiber stabilized emulsion is 0.7066, 0.75% gelatin stabilized emulsion is 0.4947, 0.75% gelatin nanofiber stabilized emulsion is 0.7281, 1% gelatin stabilized emulsion is 0.6083, and 1% gelatin nanofiber stabilized emulsion is 0.7320.
Creaming rates are higher at gelatin stabilized O/W emulsions than other emulsions at all concentrations. Creaming index of 0.5% gelatin stabilized emulsion is 62%, 0.5% gelatin nanofiber stabilized emulsion is 20%; 0.75% gelatin stabilized emulsion is 19%, 0.75% gelatin nanofiber stabilized emulsion is 15%; 1% gelatin stabilized emulsion is 20%, and 1 % gelatin nanofiber stabilized emulsion is 14%.
In this study droplet size and droplet size distributions are major factors influencing the emulsion stability. Little droplet aggregation occured at emulsions stabilized by gelatin nanofibers during storage. According to the comparisons we conclude that gelatin nanofiber added O/W emulsions are more stable at 4o C for fifteen days. This study provided valuable information about the potential application of gelatin nanofibers as emulsifiers in O/W emulsions.