Mercimek diyet liflerinin izolasyonu, karakterizasyonu ve fonksiyonel özelliklerinin incelenmesi
Isolation of dietary fibers from lentils, characterization and evaluation of their functional properties
Yazar: SİNEM KAMİLE GEDİK
Danışman: YRD. DOÇ. DR. DİLARA ERDİL
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2016
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Baklagiller Leguminosae ailesine mensup, yüksek besleyici değerleri nedeniyle dünyada tahıl ürünlerinden sonra en önemli gıdalar arasında ikinci sırada yer alan ve insanlar tarafından 3000 yıl önce yetiştirilmeye başlanan gıda kaynaklarıdır. Baklagillerin yüksek protein, karbonhidrat, diyet lifi, vitamin,  mineral ve fitokimyasal madde içerikleri son yıllarda önem kazanarak, söz konusu bileşenlerin izole edilip, farklı gıda kaynakları ile birleştirilerek kullanımı öne çıkmaktadır. 
Baklagillerin içerdikleri diyet lifi sağlıklı bir gıda bileşeni olarak sindirim sistemini destekleyici, kolesterol düşürücü ve kandaki şeker oranını düşürücü fonksiyonel özelliklere sahiptir. Mercimek de (Lens culinaris) diyet lifi içeriği açısından sağlık üzerine söz konusu olumlu etkileri gösteren bir bakliyat çeşididir. Mercimek; mercek-şekilli tohumları, sarı, yeşil, kırmızı, kahverengi ve siyah gibi farklı renklerle geniş bir yelpazeye sahiptir ve ülkemizde en çok yetişitiriciliği yapılan baklagillerdendir. Mercimeklerde uzun zincirli çözünebilir polisakkaritler ve çözünmeyen polisakkaritler başta olmak üzere, galakto oligosakkaritler ve dirençli nişasta gibi birkaç farklı diyet lifi çeşidine rastlanmaktadır. 
Bu çalışmada; kırmızı ve yeşil mercimeklerin içerdikleri diyet lifi fraksiyonlarının taneden izole edilmesi ve mercimek diyet liflerinin gıda ürünlerinde fonksiyonel bileşen olarak kullanılabilirliğinin belirlenmesi amaçlanmıştır. Bu kapsamda; öncelikle mercimeklerin diyet lifi içeriği belirlenmiş ardından en düşük düzeyde kimyasal kullanımı hedeflenerek, enzim kullanımı ile üç farklı tip diyet lifi fraksiyonu izole edilmiştir. Daha sonra izolatların kompozisyonu ve fonksiyonel özellikleri belirlenmiştir. 
Çalışmaya konu olan kırmızı ve yeşil mercimekler Tarım Ürünleri Hububat Bakliyat İşleme ve Paketleme Sanayicileri Derneği (PAKDER) tarafından temin edilmiştir. Mercimeklerin öğütülmesi ile elde edilen mercimek unlarını tanımlamak amacıyla kimyasal kompozisyonları; kül, nem, protein, yağ, çözünür diyet lifi ve çözünür olmayan diyet lifi içerikleri analiz edilmiştir.  Kırmızı mercimek unu için nem içeriği  % 8,29 ; kül içeriği % 2,26; protein içeriği %23,85; yağ içeriği %2,18; çözünür olmayan diyet lifi içeriği % 15,21; çözünür diyet lif içeriği % 1,68 olarak bulunmuştur. Yeşil mercimek unu için ise; nem içeriği  % 8,21; kül içeriği % 2,50; protein içeriği %22,43; yağ içeriği %1,67; çözünür olmayan diyet lifi içeriği %13,07 ; çözünür diyet lif içeriği %1,63 olarak bulunmuştur.
Diyet liflerinin izolasyon işlemleri sırasında üç farklı yöntem uygulanmış ve sonuçta kabuk diyet lifleri, çözünür olmayan kotiledon diyet lifleri ve çözünür kotiledon diyet lifleri olarak üç çeşit diyet lifi elde edilmiştir. Mercimek unlarından kabuk diyet lifleri mekanik yöntemlerle ayrıldıktan sonra, çözünür olmayan diyet lifleri ve çözünür diyet lifleri tanenin kotiledon bölümünden partikül boyutuna göre ve enzimatik yöntemler kullanılarak izole edilmiştir. İzolatların kullanılabilirliği açısından çözünür kotiledon diyet lifleri püskürtmeli kurutucu ile kurutulurken, çözünür olmayan kotiledon diyet lifleri dondurularak kurutulmuştur. Sonuçta yeşil mercimek ununun %6,83'ü kabuk diyet lifi, % 1,61'i çözünür olmayan kotiledon diyet lifi, %7,23'ü çözünür kotiledon diyet lifi olarak izole edilmiştir. Kırmızı mercimek ununun ise %5,16'sı kabuk diyet lifi, % 0,56'u çözünür olmayan kotiledon diyet lifi, %6,45'i çözünür kotiledon diyet lifi olarak izole edilmiştir. Bu sonuçlar doğrultusunda; kırmızı mercimek kotiledon unundan çözünür olmayan diyet lifi eldesinin yeşil mercimek kotiledon unundan çözünür olmayan diyet lifi eldesine göre daha düşük verimli olduğu görülmüştür. 
Diyet liflerinin izolasyon aşamalarında partikül boyutuna göre ayırmanın etkinliği gözlenmiştir. Ayırma işlemlerinde en önemli aşamaların kompleks nişasta içeriği ve yüksek protein içeriği içerisinde tutulan diyet liflerinin ayrılması olarak değerlendirilerek, bu problemi bertaraf etmede kabuk liflerinin ayrıştırılması, protein çöktürme ve nişastanın enzim ile parçalanması aşamalarınıın önemi görülmüştür. 
Elde edilen izolatların kimyasal kompozisyonları; kül, nem, protein ve yağ içerikleri de analiz edilmiştir. Yeşil mercimek kabuk diyet lifleri, çözünür kotiledon diyet lifleri ve çözünür olmayan kotiledon diyet lifleri için sırasıyla nem içeriği %6,33, %3,48, %13,73; kül içeriği % 2,28, %20,74, %1,80; protein içeriği %24,64, %21,71, %15,49; yağ içeriği %2,74, %6,52, %0 ve toplam diyet lifi içeriği %23,76, %2,51, %72,81 olarak bulunmuştur. Kırmızı mercimek kabuk diyet lifleri, çözünür kotiledon diyet lifleri ve çözünür olmayan kotiledon diyet lifleri için sırasıyla nem içeriği % 6,30, %3,93 %11,98; kül içeriği % 2,15, %19,29, %1,06; protein içeriği %19,49, %23,09, %19,29; yağ içeriği %3,25, %7,25, %0; toplam diyet lifi içeriği %20,30, %1,06, %43,68 olarak bulunmuştur. 
Öğütülerek elde edilen kırmızı ve yeşil mercimek unlarının ve izole edilen diyet liflerinin şeker profilleri, toplam fenolik madde miktarları, fenolik madde profilleri, antioksidan aktiviteleri ve karotenoid içerikleri ayrıca incelenmiştir.  Bu kapsamda, fenolik madde, antioksidan aktivite ve karotenoid içeriğinin örnekler arasında farklılık gösterdiği tespit edilmiştir. İzolasyon işlemlerinin söz konusu içeriğin diyet lifine geçişini etkilediği görülmüştür. 
Fonksiyonel özelliklerin incelenmesi kapsamında diyet liflerinin; su bağlama kapasitesi, yağ bağlama kapasitesi, şişme kapasitesi, emülsiyon oluşturma kapasitesi ve stabilitesi belirlenmiştir. Çözünür olmayan kotiledon diyet lifleri diğer izole diyet liflerine kıyasla daha yüksek su tutma, yağ tutma ve şişme kapasitesi göstermiştir. Emülsiyon oluşturma kapasiteleri mercimek diyet lifleri için genel olarak zayıf bulunmuştur, ancak çözünür kotiledon diyet liflerinin emülsiyon oluşturma kapasitelerinin çözünür olmayan kotiledon diyet lifleri ve kabuk diyet liflerine kıyasla daha yüksek olduğu görülmektedir. Bu özellikleri ile mercimekten elde edilen diyet liflerinin fonksiyonel bileşen olarak gıdalarda kullanılabilirliği uygun görülmüştür. Ayrıca öğütülerek elde edilen kırmızı ve yeşil mercimek unlarının ve izole edilen diyet liflerinin termal özellikleri DSC ile incelenerek mercimek diyet liflerinin jelleşme özellikleri de tespit edilmiştir.

Abstract:
Pulses, which are the members of the Leguminosae family are the second most important food source in the world after cereal grains due to their high nutritional values. Legumes are high in protein, carbohydrates, dietary fiber, vitamins, minerals and phytochemicals. That content gaining importance in recent years, isolation of these nutritions and their useage in combination with different food sources became preminant.
Legumes contain dietary fiber as a healty food component, supporting the digestive system, lowering cholesterol and lowering blood sugar which means that they are functional foods. Also lentils (Lens culinaris) as a variety of pulses show these healthy effects. Lentils has lens-shaped seeds with diffferent colors like red, brown and black. Lentils are also the most cultivated legume plant in our country, Turkey.  Galactooligosaccharides, high moleculer weight soluble polysaccharides, resistant starches all types and especially insoluble polysaccharides are found in appreciable levels in pulses.
The purpose of this research; was to develop methods to isolate water-soluble and water-insoluble dietary fiber from red and green flour fractions. Following isolation, the fiber compositions were characterized and functional properties were evaluated to determine practical uses of the fibers. The fiber isolated from lentil flours characterized in three different groups as; hull dietary fiber, insoluble cotyledon dietary fibers and soluble cotyledon dietary fibers. During isolation using minimal chemicals was targeted by using enzymes to be able to evaluate the isolates as a food ingredient.
Red and green lentils was obtained by the help of Association of Agricultural Products, Grains Processing and Packaging Industries (PAKDER). In order to identify the chemical composition of lentils, ash, moisture, protein, fat, soluble dietary fiber and insoluble dietary fiber contents of lentils were analyzed. Red lentil flour was found  to have moisture content of 8,29%; ash content of 2,26%; protein content of 23,85%; fat content of 2,18%; insoluble dietary fiber content of 15,21% and soluble dietary fiber content of 1,68%. The moisture content of green lentil flour was 8.21%; the ash content was 2,50%; protein content was 22,43%; fat content was 1,67%; the insoluble dietary fiber content was13,07%; and soluble dietary fiber content was found to be 1,63%.
During the isolation process of the dietary fiber, three different methods were applied and three different dietary fiber fraction obtained such as; hull dietary fiber, insoluble cotyledon dietary fiber and soluble cotyledon fiber. After seperating lentil hulls by mechanical means from lentil flours, insoluble cotyledon fibers and soluble cotyledon fibers were seperated from cotyledon part by particle size seperation and enzymatic digestion methods.
Insoluble fiber was separated from tailings starches by sieving through sieves with openings of 53µm. This indicates that the yield of insoluble fiber was mainly a function of its size in relation to the size of sieve openings, since increases in yield were observed by decreasing sieve opening size. The basic problem for purity of insoluble fiber was the availability of starch, which was believed to be entrapped in the fiber matrix as shown previously in common bean dietary fiber. Legume starches are noted for an irregular granular structure and adherance to protein. Insoluble fibers separated using the sieve with 53 µm openings were purified by reducing residual starch levels with  alpha-amylase. Purified insoluble fibers were freeze dried whereas soluble fibers were spray-dried. 
To isolate soluble fiber from the water soluble fraction, protein was precipitated and the supernatant spray dried as soluble fiber concentrate. By increasing the recovery and purity of protein from water solubles, the fiber concentration in the supernatant also increased. The inability to completely separate protein during precipitation was probably due to interactions of protein with soluble fiber or differences in isoelectric points of the different types of legume proteins.
The yield and purity of legume cotyledon fibers isolated by our methods are generally higher than those reported by other researchers working with legume cotyledon fibers.The resulting green hull lentil was 6.83% , 1.61% insoluble dietary fiber, 7,23% soluble dietary fiber of flour were isolated. The red lentil flour hull dietary fiber was 5,16%, 0,56% cotyledon insoluble dietary fiber and 6,45% of soluble cotyledon was isolated as dietary fibers. As a result of these findings;  the efficiency of obtaining cotyledon insoluble fiber from red lentils were found to be lower than the efficiency of cotyledon insoluble fiber obtained from green lentils.
The chemical composition of the different fiber types such as; ash, moisture, protein, fat content were analyzed. Green lentil hull dietary fiber, soluble cotyledon dietary fiber and insoluble cotyledon dietary fibers were found as; moisture content, respectively, 6,33%, 3,48%, 13.73%; The ash content of 2,28%, 20,74%, 1,80%; protein content 24,64%, 21,71%, 15,49%; fat content 2,79%, 6,52%, 0% and finally total dietary fiber content as 23,76%, 2,51%, 72,81%, respectively. Red lentils hull dietary fiber, soluble cotyledon dietary fibers were found to be ; moisture content, respectively, and insoluble dietary fibers 6.30% 3,93% 11.98%; the ash content of 2,15%, 19.29%, 1,06%; protein content of 19,49%, 23,09%, 19.29%; fat content was found 3,25%, 7,25%, 0% and finally total dietary fiber content as 20,30%, 1,06%, 43,68%, respectively. Also sugar profiles were analyzed by HPLC, total phenolic contents, antioxidant activities and total carotenoids were examined for isolated dietary fibers obtained from red and green lentil flours. Neutral compositions of the isolated fibers indicated that sucrose as a dissaccharide and raffinose as a oligosaccharide are common sugars of hull and cotyledon fibers. 
Fibers chemical composition indicates their purity. Because of the high protein and ash contents of the isolated fractions naming the fiber fractions as isolates considered as wrong. Defining soluble cotyledon fibers and hull fibers as concentrates will be better. The other component percentages of insoluble cotyledon fibers are considered to be much lower due to succesful process for obtaining insoluble cotyledon fiber. The study of the functional properties and the use of those ingredients obtained in the food industry should be taken into consideration when it comes to the classification of isolates and concentrates. The presence of those major components in fiber fractions were considered important. 
Within the scope of the examination of functional properties of dietary fibers; water binding capacity and the oil binding capacity, the swelling capacity, emulsifying capacity and the emulsion stability were determined. Insoluble cotyledon dietary fiber shows higher water holding, fat retention and swelling capacities compared to other isolated dietary fibers. Oil binding capacities of insoluble fibers were much higher than those of hulls and soluble fibers. The ability of fibers to swell and to bind water is highly indicative of their physiological role in gut function and control of blood glucose levels. Emulsion forming capacity is generally weak for lentil fibers. 
In the extent of functional component evaluation in lentil fibers and lentil flours, total phenolic content, total carotenoid content, phenolics profile and antioxidant activities were investigated by different methods. Total phenolic content determined using Folin- Ciacalteu method, phenolic profile determined using reversed phase HPLC methods, total carotenoids extracted with petroleum ether and measured with spectrophotometric methods. Antioxidant activities were analyzd by DPPH and CUPRAC methods.
Total phenolic content of the samples found between 21,0 – 531,0 mg gallic acid/100 g sample. 3-4 dyhydroxybenzoic acid,catechin and kaempherol derivatives are found higher in composition in fibers and lentil flours. Kaempherol derivatives determined by HPLC as the predominant phenolics in lentil flours and fibers. Green lentil; flour, hull dietary fiber, soluble cotyledon dietary fiber and insoluble cotyledon dietary fibers kaempherol content per 100 g sample were found as 13,35, 12,76, 5,78, 1,76 µg. Red lentil; flour, hull dietary fiber, soluble cotyledon dietary fiber and insoluble cotyledon dietary fibers kaempherol content per 100 g sample were found as 14,31, 31,79, 7,73, 1,81 µg. 
The highest amount of carotenoids in the green lentil sample groups were lutein but in the red lentil sample group it has changed as β-caroten. Green lentil; flour, hull dietary fiber, soluble cotyledon dietary fiber and insoluble cotyledon dietary fibers lutein content per g sample were found as 9,91, 1,44, 2,81, 1,64 µg. Red lentil; flour, hull dietary fiber, and soluble cotyledon dietary fibers β-caroten content per g sample were found as 3,81, 2,01, 2,31 µg.
Antioxidant activities were related to the phenolic and carotenoid compositions. Relations between the methods and functional components were evaluated using simple regression analysis. However,we noted that antioxidant activity assays DPPH and CUPRAC showed different results. Red lentil group samples shows higher antioxidant activity with CUPRAC method. These results related with high β-caroten content of red lentils. Also these values indicates that dietary fibers positive effects on health are related with their antioxidant activity.
Finally thermal properties of lentil flours and isolated fibers examined with DSC analyses. In the endothermic peaks soluble dietary fibers shows gellation in the junction zones.  Green lentil flours and fibers gellation temperatures were analyzed between 62,50 – 76,72 °C.  In green lentil sample group the highest entalphy change obtained for green lentil insoluble cotyledon dietary fibers where the lowest entalpy change obtained in  green lentil soluble cotyledon fibers gelling. Red lentil flours and fibers gellation temperatures were evaluated between 62,50 – 76,72 °C. The highest entalpy changes seen in red lentil sample group obtained for red lentil insoluble cotyledon dietary fibers where the lowest entalpy chang obtained in red lentil flours.
Lentils rich in functional food components and these findings indicates the usability of isolated fiber from lentils as a food ingredient. Methods to further purify lentil fiber fractions may studied in the future. Lentil fibers isolated in this study have functional compounds as; phenolics, carotenoids and shows antioxidant activities. Also isoluble fibers has higher swelling capacity and soluble lentil fibers has gelling ability. Therefore lentil fibers may be used as additives to health promoting foods and beverages in addition to increasing the understanding of their true functionality.