Kara nohut nişastasından enzimatik yöntemle elde edilen dirençli nişastanın fizikokimyasal ve fonksiyonel özellikleri
Physico-chemical and functional properties of resistant starch prepared from black chickpea (Cicer arietinum L.) starch by enzymatic method
Yazar: HİLAL DEMİRKESEN
Danışman: YRD. DOÇ. DR. DİLARA NİLÜFER ERDİL
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2015
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Nişasta = Starch ; Nohut = Chickpea

Özet:
Nohut  Leguminosae (baklagiller) familyasının Papilionaceae (kelebek çiçekliler) alt familyasına ait Cicer cinsinden bir baklagil türüdür. Baklagiller insan beslenmesinde önem taşıyan bitkisel kaynaklı besinlerdir. Diğer bitkisel kaynaklı besinler ile karşılaştırıldıklarında besin değeri bakımından birçok üstünlüğe sahiptirler. Nohut (Cicer arietinum L.) tohum rengi ve coğrafik dağılımına göre genellikle desi ve kabuli olarak iki grup oluşturmaktadır. Desi; küçük, kırışık yüzeyli, köşeli ve koyu renkli tanelere sahiptir. Kara nohut da desi grubunda Malayta yöresine ait oldukça koyu renkli ve küçük taneli yöresel bir nohut çeşididir. Yöre halkı tarafından kara çorba, döğme çorbası gibi çeşitli çorbaların yapımında ve yarmaca denilen kabuklarından ayrılıp ikiye bölünmüş haliyle bir diğer adı da muhaşer olan yarmacalı pilav yapılarak tüketilmektedir.
Bu çalışmada kullanılan kara nohut; yöresel ürünler satan yerel bir marketten temin edilmiştir. Nohuttan izole edilen nişastaya ve nohutun öğütülmesiyle elde edilen una ilk aşamada temel kompozisyon analizleri yapılmıştır. Nişastaya ve una ait nem içeriği sırasıyla %11,64 ve %10,19  bulunurken aynı sıralamada kül içerikleri %0,07 ve %2,86; protein içerikleri %0,85 ve %20,99; yağ içerikleri %0,44 ve %4,28 bulunmuştur.
Kara nohuttan izole edilen nişastadan enzimatik  modifikasyon yöntemiyle Tip-3 EDN formunda enzime dirençli nişasta üretilmiş ve çalışmanın devamında, elde edilen Doğal nişastanın modifikasyonu ile elde edilen ürün (DNE), jelatinize nişastanın modifikasyonu ile elde edilen ürün (GNE) ve işlem etkisini karşılaştırmak amacıyla  elde edilen ürün retrograde kontrol nişastası (RNK)'nın ve ayrıca işlem uygulanmamış doğal nişasta ve unun fizikokimyasal ve fonksiyonel özelliklerinin belirlenmesi amaçlanmıştır. Enzimatik modifikasyon işlemi; doğal ve jelatinize nişastaların 40U/g pullulanaz enzimi ile 10 saat  60 °C'de  inkübasyonunu takiben 121°C 30 dakika otoklavlama işlemiyle devam etmiş ve sonrasında 24 saat-4°C buzdolabında retrogradasyon ve liyofilizasyon ile son bulmuştur. Çalışma kapsamı için önemli bir bileşen olan dirençli nişasta miktarları enzimatik olarak belirlenmiştir. Enzimatik ve termal uygulamalar ile elde edilen DNE ve GNE örneklerinde enzime dirençli nişasta (EDN) miktarları sırasıyla; %16,35 ve %12,34 bulunmuştur. RNK ile DNE karşılaştırıldığında EDN miktarı işlem etkisiyle %40,58 artmıştır.

Doğal nişasta ve una ait EDN içeriği ise sırasıyla %15,19 ve %4,99 olarak bulunmuştur. Doğal nohut nişastasına yapılan uygulamalar hızlı sindirilebilen nişasta miktarını artırırken, yavaş sindirilebilen nişasta miktarını azaltmıştır. Bu uygulamalar jelatinize nişastanın amiloz içeriğini  önemli oranda artırmış, amiloz içerikleri DNE'de %24,37 RNK'da %20,91, GNE'de %54,33 olarak bulunmuştur. 
Enzimatik ve termal uygulamalar su tutma kapasiteleri ve suda çözünürlük indislerinde artışa neden olmuştur. Şişme gücü ise örneklerin EDN içeriği ile ters orantılı olarak artmıştır. Uygulamalar aynı zamanda nişasta örneklerinin yağ tutma ve emülsiyon oluşturma kapasitelerinde de artışa neden olmuştur. Sinerezis ve bulanıklık özellikleri incelendiğinde ise ölçüm yapılan günler arasında kararlı bir artış ya da azalış görülmemiştir. RNK örneğinin bulanıklık değerleri diğer nişasta örnekleriyle karşılaştırıldığında beş gün boyunca yapılan ölçümlerde en az olarak bulunmuştur.
Nişasta örneklerinin ve unun termal özellikleri diferansiyel taramalı kalorimetre (DSC) ile incelenmiştir. Otoklavlama ve retrogradasyon işlemleri uygulanan örneklerde jelatinizasyon başlangıç ve pik sıcaklıkları ve entalpi değerleri azalmıştır. Mikro-visko analizör (RVA) ile ölçülen viskozite değerlerinde ise enzimatik ve hidrotermal uygulamalar sonucunda elde edilen nişasta örneklerinin doğal nişastaya göre pik viskozitesi ve son viskozitelerinde düşüş gözlenmiştir.
Nişasta örneklerinin ve unun X ışını difraksiyon dağılımları incelenmiş, kuvvetli pikleri 2ϴ=17°'de verdiği görülmüştür. Bu da baklagil nişastaları için karakteristik olan C tipi kristalizasyon olduğunu göstermiştir. Granül yapısı ve dağılımı ise taramalı elektron mikroskobu (SEM) ile incelenmiş doğal nişastada oval granüler yapı ağırlıktayken diğer nişasta örneklerinde retrogradasyonun etkisiyle bu yapının dağıldığı görülmüştür.
Bu çalışmada, kara nohut nişastasından enzimatik yöntemle Tip-3 EDN üretimi başarı ile gerçekleştirilmiştir. Kara nohut nişastasıyla, nişasta örnekleri EDN içerikleri ve diğer özellikleri bakımından karşılaştırıldığında, teknolojik olarak kullanımına bağlı olmak üzere farklı özellikleri karşıladıkları görülmektedir. Daha yüksek yağ tutma ve emülsiyon kapasitesi istenilen kalite özelliklerinde ürün üretilmesini daha mümkün hale getirecektir. Elde edilen nişasta örneklerinin bu özellikleri sayesinde fonksiyonel bir ingrediyen olarak ve EDN miktarını yükseltmek için doğal nişasta yerine kullanılabileceği düşünülmektedir.

Abstract:
Chickpea is a member of Leguminosae (legumes), Papilionaceae is a leguminous species of the genus Cicer subfamilies. Legumes are plant-derived nutrients that are important in human nutrition. They have many advantages in terms of nutritional value when compared with other plant-derived foods. Chickpea (Cicer arietinum L.) form two groups according to seed color and geographic distribution and acceptance. Desi: small , wrinkled surface , square and has a dark spots. Black Chickpea is also in the desi family and is closely connected to the Malayta culture, Black Chickpea has a dark color and is a part of the small grained local region type. Locals consume it with a variety of soups such as dark, and yogurt soup and often called the divided, the shells separates into two parts which is put into rice, and are ready to be served. 
Legumes are plant-derived nutrients that are important in human nutrition. In terms of nutritional value when compared with other plant-derived foods have many advantages. There are many edible protein ratio of 18-36 % of the composition of dry grain legumes and digestibility degree of protein is also very high (78%). Essential amino acids shows values in terms close to the animal protein . The chickpea seeds have high amount of vitamins (A, B, C and D ) and minerals (Fe, P, Ca ).
Chickpea (Cicer arietinum L.) is in terms of acreage and production in edible grain legume species with agriculture in the world after dry bean ( Phaseolus vulgaris L.) and pea ( Piston sativum L.) in the third place. According to FAO data of the last five years (2009-2013),  chickpea producing countries ranks: 1) India 2) Australia 3) Pakistan 4) Turkey 5) Myanmar. Chickpeas in Turkey in 2013, planted 4.235.570 acres and produced a total of 506 000 tonnes of peas. The average yield was 121 kg per hectare in 2013 . This thesis used cultural/local black chickpeas as its material. Black chick pea is a legume of Malatya region produced in the dark and small grain group.
Starch ; The main carbohydrate source in the human diet. The starch plants is found to be in the form of granules. The starch granules, plant seeds , roots and tubers of further stem, leaf, fruit and even pollen are energy storages. Chemically, starch glucose units α -D- (1,4) and / or α -D- (1,6) are formed by bonding the polysaccharide linkages. Digestible starch overall α - amylase, sucrase - isomaltase and glucoamylase enzymes that are cleaved to the free glucose in the small intestine and is absorbed there. However, not all of the starch can not be digested and absorbed in the small intestine. Resistant starch consumed within 120 minutes after D- glucose in the small intestine but starch fraction of fragmentation becomes fermented in the colon. Resistant starch; the starch or starch products can also be defined as being resistant to gastrointestinal digestion progresses.
The digestibility of starch in the food varies depending on the food 's native state. Therefore the digestion of starch in the human body at different rates and classification in terms of nutrition degree of fragmentation during in vitro hydrolysis by enzymes in the digestive system has gained importance. Accordingly, starch in diet is composed of three separate factions; including rapidly digestible starch (RDS), slowly digestible starch (SDS) and enzyme- resistant starch (RS). Englyst et al were classified RS under 4 groups.
Type-1 RS , the food,  is a term used for starch which resists digestion because it is not physically accessible. Foods in normal cooking conditions shows resistance to heat. For example, grains , present in the cell walls of seeds and tubers include starches in the inside. The type-2 RS is indigestible because of structural features are the natural starches. The crystal structure does not permit hydrolysis. They contain high levels of amylose, for example starches can be found in raw potatoes and green bananas. Type-3 RS digestion resistance is defined as non- granular starch derivatives. Typically, these are formed during the retrogradation of the starch granules. A portion of the molecule in the starch retrogradation state is resistant against digestion enzymes. Type-3 RS α -1,4- D- glucan consisting of short-chain units have a flat structure and are highly heat resistant molecules. More interest is seen because of thermal stability and the conventional ingredients in foods that are possible. Heat and moisture -containing food processes Type-1 and Type-2 while destroying the RS , RS also contribute to the formation of Type-3 . Type-3 shows more water holding capacity compared to starch granules. For example, chilled cooked potatoes and corn flakes. Type - 4 RS etherification , esterification and chemical structure modified by methods such as crosslinking and resistance are imparted starch enzymes. Modified starches including bread , cakes, are examples of foods which include this type of RS. 
RS, unlike traditional dietary fiber makes it possible to produce products of the desired quality characteristics. Foods containing a high proportion of dietary fiber, refined and processed foods by more coarse structure, are dense and flavorless. RS is the texture and flavor of food as a food ingredient and provides very little effect on the development of the sensory properties .RS has  small particle size , white -looking, dearomatized component. It has a low water binding capacity. For use as functional ingredients in food products to provide increased viscosity and gel forming properties is possible. Inability to be digested as normal starch, there are some benefits of RS on health. RS is member of the insoluble fibers class, but just like soluble fibers, there are physiological benefits to them.Outstanding features of RS has become a topic of interest. Researchers working in this field often use a variety of methods to obtain different sources of RS. RS  production and this new products functional properties and physicochemical properties have been concentrated heavily on. RS production methods as a basis ; physical modification (temperature –moisture applications , pregelatinisation etc . ) , Split ( acid -enzyme hydrolysis and oxidation processes such as the result of decomposition) and chemical modification (etherification , esterification, cross-linking , etc. ) can be grouped under three main headings.  Applications usually tried include, heat treatment, partial acid hydrolysis, enzyme modification, acid or enzyme modification with heat treatment, extrusion or chemical methods. RS formation and the amount of food after application ; heat treatments applied to the starch , amylose starch, amylopectin ratio,the chain length of the molecules is affected by various conditions such as the presence of amylose - lipid complexes. 
Black chickpea used in this study is obtained from local markets. Starch was isolated from chickpeas are ground to be used in analysis. Moisture content of 11.64 % and 10.19 %; protein content of 0.85 % and 20.99 %; fat content of 0.44 % and 4.28% were obtained for the starch and flour, respectively. Starch isolated from black chickpeas were modified by enzymes to form RS (DNE). sample obtained byapplying enzyme on the gelatinized starch is GNE and the retrograde control starch  is RNK. For all those samples physicochemical and functional properties were deternied and compared. Enzymatic modification process involves 40U/g pullulanase addition to both natural and gelatinized starch followed by incubation for 10 hours at 60° C with enzyme, autoclaving at 121 ° C for 30 minutes and after 24 hours keeping at 4 ° C refrigeration for retrogradation and lyophilized. 
Resistant starch (RS) contents found for DNE and GNE is respectively; 16.35 % and 12.34%. When DNE and RNK is compared RS content was found to increase about 40.58 % by the effect of enzyme. The RS content of the natural starch and the flour was found to be 15.19 % and 4.99 %, respectively. Treatments made to the natural black chickpea starch increased the amount of rapid digestible starch and reduced the amount of slowly digestible starch when compared with the uncooked starch. On the other hand these applications have increased the amylose content of the gelatinized starch (GNE) significantly, amylose content in DNE is found to be 24.37%, for RNK is 20.91%, and for GNE is 54.33%. 
Enzymatic and thermal applications led to an increase in water absorption capacity and water solubility index. The swelling power increased in inverse relation to the RS content of the sample. Applications also caused increase in the fat binding capacity and emulsifying capacity of the starch sample. Syneresis and turbidity properties had no distinct increase or decrease behavior. Compared with other starch samples for five days, RNK was found to have the lowest levels for those properties. For thermal properties of samples by differential scanning calorimetry (DSC); Autoclaving and retrogradation applied samples had lower initial gelatinization temperatures and the peak temperatures and enthalpy values of the applied sample were also reduced. Rapid visco analyzer (RVA) measurements showed that viscosity values for native starch reduced by enzymatic and hydrothermal applications by measn of decreases in peak viscosity and final viscosity. KNU and KNN were examined by X ray diffraction and strong peaks were found at 2θ = 17°. This showed C-type crystallinity which was characteristic for legume starches. The granular structure and distribution of the peaks obtained by scanning electron microscope (SEM) showed that for natural starch oval granular starch was dominant whereas for the other starch samples granules were found to collapse due to the effects of retrogradation. 
In this study; from black chickpea starch Type-3 RS by a pullulanase enzyme application was produced successfully. Starch samples and black chickpea starch  when compared in terms of RS content and other characteristics it was obvious that according to different technological uses these samples had different properties. The higher fat binding capacity and emulsion capacity of enzyme treated black chickpea starch samples will make it possible to produce different products with desired quality parameters. Thanks to these features, the starch samples obtained could be used as a functional ingredient instead of natural starch to increase the amount of RS.