Susam küspesinden ultrason ve enzim destekli protein ekstraksiyonu
Enzyme and ultrasound supported protein extraction from sesame meal
Yazar: AYŞEGÜL AKBAĞ
Danışman: DOÇ. DR. DERYA KAHVECİ KARINCAOĞLU
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:
Son yıllarda hayvansal gıda ağırlıklı beslenmenin sürdürülebilirlik açısından çeşitli olumsuz yönlerinden dolayı bitkisel protein üretimi önem kazanmıştır. Gıda endüstrisinde bu amaçla çeşitli hammaddeler kullanılabilmekte ve proses atıklarından bitkisel protein elde edilmesinin sürdürülebilir gıda üretimi hedefleri için oldukça uygun olduğu bilinmektedir. İyi bir bitkisel protein kaynağı olan yağlı tohumlar, bu amaçla kullanılan oldukça önemli ürünlerdir. 
Susam (Sesamum indicum L.), dünyadaki en önemli yağ bitkilerinden biridir. %50-60 yağ ve %25 protein içerir. Yüksek besin değeri, lezzeti, geniş kullanım alanı ve ekonomik değeri nedeniyle dünyada susam üretimi artarak devam etmektedir. Susamdan yağın uzaklaştırılması sonucunda kalan küspe, ham protein oranı açısından oldukça yüksek değerlere sahiptir. Yağ endüstrisinde atık olarak elde edilen susam küspesi, yaklaşık %40 oranında protein içermektedir. Günümüzde daha çok hayvan yemi olarak kullanılan susam küspesi, gıda katkı maddesi olarak da kullanılmaktadır. Bu çalışma kapsamında soğuk sıkım olarak elde edilen susam küspesi kullanılmıştır. Küspeler çalışma süresi boyunca +4 oC'de muhafaza edilmiştir. 
Susam küspesinin dezavantajı düşük protein çözünürlüğüdür, bu amaçla enzimlerin ve ön işlemlerin, protein verimini artıracağı ve proteinlerin fonksiyonel özelliklerinin iyileştirilebileceği belirlenmiştir. Enzim ve ultrason destekli protein ekstraksiyonu, çevre dostu olması ve geleneksel yönteme göre daha yüksek verim sağlaması nedeniyle son zamanlarda tercih edilmekte ve çalışmalara konu olmaktadır. Bu yöntemleri birleştirmenin daha yüksek verim sağladığı düşünülmektedir. Susam küspesinde bulunan protein ve fenolik maddeler üzerine ultrason ve enzimatik yöntemlerin farklı dizilimlerle kombinasyonu dahil herhangi bir çalışma bulunmamaktadır. 
Protein, önce alkalin ekstraksiyonu ile elde edilmiştir. Toplam protein değeri Kjeldahl yöntemiyle belirlenip, %23,3±0,48 g protein; toplam fenolik içeriği Folin-Ciocalteu yöntemiyle 0,43±0,15 mg/ml olarak bulunmuştur. Çözünebilir protein miktarı Bradford yöntemi ile belirlenip ve %62,18 bulunmuştur. Enzim ve ultrason uygulamaları için geniş literatür taraması yapılmış, olabilecek optimum koşullar belirlenmiştir. Enzim kullanılarak yapılan ekstraksiyonda farklı enzim miktarları denenmiştir. 0,01 ml/g, 0,05 ml/g, 0,1 ml/g Alkalaz enzimi 30, 75 ve 120 dakika olarak uygulanmıştır. Enzim ekstraksiyonunda %29,0±0,39 g ile en iyi sonuç 0,05 ml/g enzim örneğinden 30 dakika işleme süresiyle elde edilmiştir. Yine aynı koşullarda enzim ile yapılan analizlerde çözünebilir protein %6,09±1,1, fenolik madde içeriği 2,30±0,3 mg/ml bulunmuştur. Ultrason destekli protein ekstraksiyonu, ultrason kullanılarak 50, 70, 90 ve 110 dakika süreyle gerçekleştirilmiştir. Ultrason wolt gücü ve uygulama sıcaklığı sabit tutulmuştur. En iyi sonuçlar 70 dakikalık ultrason uygulamasında görülmüştür. 70 dakika ultrason uygulaması sonucunda %68,21±7,19 çözünebilir protein; %0,36± 0,03 mg/ml fenolik madde ve %37,5± 0,17 g toplam protein sonuçları elde edilmiştir. 
Belirlenen optimum koşullar sırasıyla önce enzim, sonra ultrason ve önce ultrason, sonra enzim olarak uygulanmış ve önce ultrason uygulanan numunelerden daha yüksek protein verimi elde edilmiştir. Önce enzim uygulamasında toplam protein %43,6±0,29 g bulunurken önce ultrason uygulamasında toplam protein %48,6±0,9 g bulunmuştur. Kombine uygulamada çözünebilir protein %10,648±4,01; toplam fenolik madde ise 0,43±0,36 mg/ml bulunmuştur. Ardından elde edilen susam küspesi, protein izolatına yapılan kalite analizlerinde, çözünebilir protein oranı %1,059 olarak pH 10 iken bulunmuştur. Su tutma kapasitesi 1,765 g/g protein; yağ tutma kapasitesi 3,56 ml/g protein; emülsiyon aktivitesi 44 m2 /g; emülsiyon stabilitesi 31,6 dakika ve antioksidan aktivitesi 0,183 μmol/g olarak bulunmuştur. Bu sonuçlar da uygulanan ön işlemlerin protein izolatının kalitesini düşürmediğini göstermiştir.

Abstract:
In recent years, plant protein production has gained importance due to various negative aspects of animal-based nutrition in terms of sustainability. The interest in plant proteins is increasing day by day due to consumption differences resulting from religious preferences, personal preferences such as veganism-vegetarianism, and changes in nutrition styles due to some health concerns. Various raw materials can be used for this purpose in the food industry, and it is known that obtaining vegetable protein from process wastes is quite suitable for sustainable food production targets. Cereals such as wheat gluten, soybeans and corn, along with oilseeds, are the most commonly used primary sources of plant protein. When added to food, proteins confer desirable functional properties such as solubility, viscosity, foam formation, emulsification, and water and oil retention ability. Vegetable proteins also play an important role in the nutritional, sensory, physical-chemical and organoleptic (color, texture, taste, etc.) properties of food, while they have great potential in industrial-scale applications in adhesion properties and film formation. Oilseeds, which are a source of vegetable protein, are very important products used for this purpose. Sesame plant, which is a good example of oilseed, has become one of the alternative sources of vegetable protein to animal protein sources due to its different usage areas and the fact that it can be produced in our country.
Sesame (Sesamum indicum L.) is one of the most important oil crops in the world. It contains 50-60% fat and 25% protein. It is an excellent source of nutrients with the oil, proteins, unsaturated fatty acids, vitamins, minerals and folic acid it contains. Sesame production continues to increase in the world due to its high nutritional value, taste, wide usage area and economic value. Sesame protein is high in methionine (3.2%), which is low in most plant proteins, and these unique properties make sesame seed an excellent protein source to nutritionally supplement soybean, peanut, and other plant proteins that do not contain sufficient methionine. The pulp remaining after removing the oil from sesame has very high values in terms of crude protein content. Sesame meal, obtained as waste in the oil industry, contains approximately 40% protein. Sesame meal contains 7.92% moisture, 27.83% fat, 30.56% protein, 6.22% fiber, 5.27% ash and 28.14% carbohydrates. The pulp, which constitutes approximately 50% of sesame, contains iron, zinc, sodium, potassium and calcium. Sesame meal, which is mostly used as animal feed today, is also used as a food additive. In this study, cold-pressed sesame meal was used. The meals were stored at +4 oC throughout the study period.
Alkaline methods are generally used in protein extractions. High alkaline concentration breaks down hydrogen bonds and enables proteins to dissolve in water. Generally, studies are carried out with a pH of 8 and above. However, alkaline methods remain inefficient in protein extraction. The disadvantage of sesame meal is low protein solubility, for this purpose it has been determined that enzymes and pretreatments will increase protein yield and improve the functional properties of proteins. Applications such as enzyme, ultrasound, microwave, pulsed electric field and supercritical fluid can be used to increase protein yield in protein extraction from different food wastes.
Enzyme and ultrasound-assisted protein extraction has recently been preferred and the subject of studies because it is environmentally friendly and provides higher efficiency than the traditional method. Ultrasound treatment causes partial unfolding and reduces the intermolecular interactions of proteins. In addition, it is also used as a modification method to improve the emulsification and solubility properties of proteins. With the protease enzymes used, the size of the proteins was reduced, allowing them to be extracted more easily, thus increasing the efficiency. Alkalase enzyme, which was chosen as a protease, has a high protein hydrolysis degree. Combining these methods is thought to provide higher efficiency. There are no studies on the protein and phenolic substances in sesame pulp, including the combination of ultrasound and enzymatic methods with different sequences.
The protein was first obtained by alkaline extraction. Total protein value was determined by the Kjeldahl method, 23.3%±0.48 g protein; Total phenolic content was found to be 0.43±0.15 mg/ml by the Folin-Ciocalteu method. The amount of soluble protein was determined by the Bradford method and was found to be 62.18%. An extensive literature review was conducted for enzyme and ultrasound applications and the optimum conditions were determined. Different enzyme amounts were tried in the extraction using enzyme. 0.01 ml/g, 0.05 ml/g, 0.1 ml/g Alkalase enzyme was applied for 30, 75 and 120 minutes. The best result in enzyme extraction with 29.0±0.39 g% was obtained from 0.05 ml/g enzyme sample with a processing time of 30 minutes. In the analyzes performed with enzyme under the same conditions, soluble protein was found to be 6.09±1.1% and phenolic substance content was 2.30±0.3 mg/ml. Ultrasound-assisted protein extraction was performed using ultrasound for 50, 70, 90, and 110 min. Ultrasound wolt power and application temperature were kept constant. The best results were seen in 70 minutes of ultrasound application. As a result of 70 minutes of ultrasound application, 68.21±7.19% soluble protein; Results of 0.36%± 0.03 mg/ml phenolic substance and 37.5%± 0.17 g total protein were obtained.
Combination of ultrasound and Alkalase enzyme processes were carried out by taking different parameters into consideration. While the ultrasound power was kept constant, the effect of application time was measured. In enzyme use, the effects of both enzyme amount and application time were examined, and the pH and process temperature in the applications were kept constant under specified conditions. Ultrasound application temperature is 45 oC and 375 W power; enzyme application temperature 55 oC; The pH value of both processes was determined as 9. The determined optimum conditions were applied first as enzyme, then ultrasound, and first as ultrasound, then as enzyme, and higher protein yield was obtained from samples that were treated with ultrasound first. Total protein was found to be 43.6±0.29 g% in enzyme application first, while total protein was found 48.6±0.9 g% in ultrasound application first. Soluble protein in combined application: 10.648±4.01%; Total phenolic substance was found to be 0.43±0.36 mg/ml. Hydrolysates obtained from sesame protein isolates have improved functional properties. Then, quality analyzes were performed on the protein isolate of the sesame meal obtained. The soluble protein rate was found to be 1.059% at pH 10. Solubility at various pH values is a sign of how well protein isolates will perform when included in food products. Water retention capacity 1,765 g/g protein; The fat retention capacity was found to be 3.56 ml/g protein. Water retention capacity measures the ability of proteins to absorb water. 
While emulsion activity represents the ability of proteins to adsorb at the water and oil interface, emulsion stability is expressed as the residence time of proteins at the oil–water interface after emulsion storage. Emulsion activity 44 m2/g; emulsion stability was determined as 31.6 minutes. Antioxidant activity was measured at four different pH values, and the best result was found to be 0.183 μmol/g at pH 9. These results showed that the applied pretreatments did not reduce the quality of the protein isolate.
With all these studies, protein extraction from sesame pulp was successfully carried out using enzyme and ultrasound pre-treatment. De-oiled sesame meal, which consists of approximately 50% protein, has been accepted as a good source of vegetable protein. Extraction efficiency was increased with the pre-treatments. It has been determined that this valuable by-product, which is generally used as animal feed, can be easily used in the food industry and will be a good example of alternative and vegetable protein sources.