Chia tohumu yağı ve kürküminin mikroenkapsülasyonu ve sindirim davranışının incelenmesi
Microencapsulation of chia seed oil and curcumin and investigation of in-vitro digestion behaviour
Yazar: BURCU FIRTIN
Danışman: DOÇ. DR. NEŞE ŞAHİN YEŞİLÇUBUK
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı / Gıda Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2018
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Sağlık üzerine olumlu ekileri olan chia tohumu yağı, elzem yağ asitlerinden olan alfa linolenik asiti (ALA) yüksek oranda içermektedir. ALA, sağlık üzerine olumlu etkileri ile bilinmektedir. Omega-3 yağ asitleri fetus ve yeni doğan bebeklerin gelişimine, hipertansiyon ve kardiyovasküler hastalıklarını önlemede etkilidir. Omega-3 yağ asitleri ayrıca antiinflamatuar, antitrombotik ve antiaritmik özelliklere sahiptir.
Fonksiyonel gıdalara olan yönelim nedeniyle gıda endüstrisi probiyotik, elzem yağ asitleri, vitaminler ve antioksidanlar gibi fonksiyonel bileşenleri kullanmaya yönelmiştir. Ancak fonksiyonel bileşenlerin ışık, hava, sıcaklık, nem gibi çevresel faktörlere maruz kalması durumunda dayanımları düşüktür ve bozunmaya elverişlidir. Bu nedenle fonksiyonel bileşenlerin proses ve depolama sırasında fonksiyonel özelliklerini kaybetmemeleri için farklı yöntemler kullanılmaktadır.
Çoklu doymamış yağ asitleri oksidatif bozunmaya elverişlidir. Antioksidan kullanımının çoklu doymamış yağ asitlerinin raf ömrüne olumlu etkileri olduğu bilinmekte ve bazı sentetik antioksidanlar gıda endüstirisinde bu amaçla kullanılmaktadır. 
Enakpsülasyon yöntemi, fonksiyonel bileşenlerin duvar materyaller ile kaplanarak çevresel faktörlere maruz kalmasını engelleyerek dayanımlarını arttırmaktadır. Enkapsülasyon yöntemi gıda endüstrisinde yaygın olarak kullanılmaktadır.
Bu tez çalışmasında, Omega-3 yağ asidi olan ALA'yı yüksek oranda içeren chia tohumu yağının, doğal bir antioksidan olan kürkümin ile zenginleştirilerek mikroenkapsülasyon çalışmalarının yapılması amaçlanmıştır. Mikroenkapsülasyon çalışması dondurarak kurutma yöntemi ile yapılacak, böylece yaygın olarak kullanılan spreyle kurutma gibi ısıl işlem gerektiren enkapsülasyon yöntemlerinin çoklu doymamış yağ asidinin oksidasyonuna etkisi minimum indirilecektir. Bu çalışmada mikroenkapsülasyon işleminin hem de doğal antioksidan olan kürküminin chia tohumu yağının oksidatif stabilitesine etkisi gözlenecektir. Ayrıca in-vitro sindirim sisteminde sindirim davranışı incelenecek, kürkümin kullanımının chia tohumu yağının sindirimine bir etkisi olup olmadığı araştırılacaktır.
Bu amaçla ilk önce literatür araştırması yapılarak kullanılacak duvar materyaline karar verilmiştir. Duvar materyali olarak maltodekstrin ve akasya gamı kullanılmıştır. Emülsiyon stabilitesine etki eden faktörlerden kuru madde oranı, maltodekstrin/akasya gamı oranı ve yağ/duvar materyali oranı seçilmiş, 3x2x2 full faktöriyel dizayn ile emülsiyon stabilite testleri yapılarak bu faktörlerin stabiliteye etkileri araştırılmıştır. Her bir faktörün ayrı ayrı emülsiyon stabilitesi üzerine etkisi olduğu gözlenmiştir. Stabilite belirlenmesi için zeta potansiyel ölçümleri yapılmış, ölçümler -30 ile -40 arasında çıkmıştı. Bu sonuçlara bakılarak emülsiyonların orta halde stabiliteye sahip olduklarını söylemek mümkündür. Stabilitesi en yüksek çıkan numune ile dondurarak kurutma yöntemi kullanılarak enkapsülasyon elde işlemi yapılmıştır. Kurutma işlemi öncesi -80°C'de 12 saat ön işleme tabi tutulan emülsiyonlar ardından -35°C'de 0,1 mbar başınçta 36 saat boyunca kurutulmuştur. Elde edilen mikrakapsüllerin karakteristik özelliklerini belirlemek için nem, su aktivitesi, yüzey yağı, toplam yağ analizleri yapılmış ve enkapsülasyon verimi hesaplanmıştır. Nem analiz sonuçları %3,5 civarında çıkmıştır, gıda endüstirisinde toz malzemelerde istenilen nem analizi %3-4'tür. Ayrıca nem analizinin %3-4'ten az olmasının mikrokapsüllerin oksidatif stabilitesine olumlu etkisi olabileceği söylenmektedir. Su aktivitesi sonuçları 0,045 civarında çıkmıştır. Su aktivitesinin 0,6'dan düşük olması raf ömrü boyunca mikrobiyal riskin az olmasıyla ilişkilendirilmektedir. Yüzey yağ analiz sonuçları %2,5 civarında, toplam yağ ise %7,5 civarında bulunmuştur. Yüzey yağ ve toplam yağ değerleri ile enkapsülasyon verimi hesaplaması yapılmış, enkapsülasyon verimi %67 çıkmıştır. Dondurarak kurutma yöntemiyle yapılan çalışmalarda benzer enkapsülasyon verimi sonuçları bulunmuştur. Ardından chia yağı kapsülleriyle chia yağı ve kürkümin kapsüllerinde depolama analizleri yapılmış, haftalık periyotlarla peroksit sayısı tayinleri spektrofotometrik yöntemle yapılmıştır. Enkapsüle örneklerin peroksit sayısının periyodik artışı enkapsüle olmayan örneklere göre daha düşük çıkmıştır. Kürkümin ilavesinin depolama boyunca peroksit sayısı artışına bir etkisi görülmemiştir. 
Enkapsüle numunelerde salım davranışı incelenmiştir. Ağız, mide ve bağırsak suyunda sırayla 2 dakika, 2 saat ve 2 saat bekletilen enkapsüle numunelerde yarım saatlik periyotlarda numune alınıp salami gerçekleşen chia tohumu yağı miktarına spektrofotometrik yöntemle bakılmıştır. Ağız ve mide suyunda gerçekleşen salım çok düşükken bağırsak fazında gittikçe artan bir salım eğrisi görülmüştür. Kullanılan duvar materyallerinin salıma etkisi olduğu gözlenmiştir.
Enkapsüle numunelerde biyoyararlılık analizleri gerçekleştirilmiştir. Kürküminli olan örneklerde toplam fenolik madde ve antioksidan kapasitesi daha yüksek çıkmıştır. Enkapsüle numunelerin geri kazanım oranları %40 civarındadır. Enkapsüle numunelerin antioksidan özelliklerinden vücutta yararlanılamadan çoğu dışarı atılmaktadır.

Abstract:
Consumers are tended to buy functional foods which have health promote effects. Probiotics, essential faty acids, vitamins and antioxidanst are widely known as functional components. Because of consumer tendency, food industries have  started to search for and use functional components in their products which brings several difficulties for industries. It is hard to store, process and keep the functionality during the shelf life of products. Functional components are sensitive to light, heat, air and humidity and can be easily spoiled. Different methods have been researched to preserve the functionality of functional components.
Encapsulation is a method that is used for preserving the functional components surrounded with wall materials and protecting them from environmental conditions such as temperature, light and air. Encapsulation technology gives controlled release characteristic to encapsulated functional components. There are several techniques to encapsulate the functional components, such as spray drying, freeze drying, electrospinning and complex coacervation. Spray drying is a technique which is caused by evaporation through hot air in emulsion. This technique is commonly used in food industry due to the fact that cheapness in production, feasibilty and low operational cost features. Freeze drying is the method which uses sublimation to evaporate the water. The method can be explained like freeze the emulsion below 0°C and decrease the pressure of medium and this situation causes the sublimination of the water from emulsion and obtain dry matters. This technique has high investment and high operation cost therefore it is used for sensitive components like probiotics and poly unsaturated fatty acids commonly. Electrospinning is the technique which is used for obtain nanoencapsulated components. The logic of this method is using high electrical field. The emulsion or solution is behave like electrode due to the fact that high electric field. The other commonly used encapsulation technique is complex coacervation technique. Encapsulation methods are widely used in food industry.
Poly unsaturated fatty acids (PUFAs) have low oxidative stability. There are different methods for preventing PUFAs from oxidation. One of them is using antioxidants which is widely used in food industry nowadays. These antioxidants are synthetic and their effects on human health and metabolism are in debate. Therefore usage of natural antioxidants is being researched recently.
Chia seed oil includes alpha-linolenic acid (ALA) which is an essential fatty acid. The advantages of ALA on human health and metabolism are proven. Omega-3 fatty acids boost the brain and eye development of fetus and newborns. Omega-3 fatty acids prevent the cardiovascular diseases and hypertension. It is known that omega-3 fatty acids have antiinflamatory, antitrombotic and antiaritmic properties. 

One of the PUFAs is linolenic acid, which has 18 carbon atoms and three cis bonds. It is a carboxyllic acid and obtained from different plants. It has two different izomers; alpha-linolenic acid (18:3n-3) and gamma-linolenic acid (18:3n-6). Alpha-linolenic acid (ALA) is an essential fatty acid, it founds in seed oils, beans, nuts and leafy vegetables. ALA is the precursor fatty acid for synthesis of two important omega-3 fatty acids; docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). DHA and EPA is essential for brain development, cardiovascular diseases and inflamatuar diseases. DHA and EPA is only available with consumption of fish. ALA can be converted to EPA and DHA in finite, only 8% of ALA can be converted to EPA and only 4% of ALA can be converted to DHA. Consumption of ALA is important because of it's plant base sources for vegan and vegetarian consumers. ALA consumption reduces risk of cardiovascular diseases,cholesterol, inflamatuar diseases due to it's antioxidant property. ALA reduces the reactif oxygen stress.
Curcumin is one of the natural antioxidants. It is known that it's colorant features in turmeric. It has been used even for color the buddhistic qown. Turmeric has been used in food because of it's yellowish color. Beside of colorant property, interest in turmeric has been stared to increase when it was observed that the food which turmeric was added also shows longer shelf life. Antioxidant properties of turmeric is caused of curcuminoids. Curcuminoids are based on –O-methoxyl and phenolic compounds bonded with polien bond to two aromatic components. This polien bond can be found as keto-enol or di-keto bond. It is observed that keto-enol bond or di-keto bond is affected from pH of medium. The bond is keto form in polar, acidic and nautral medium, enol form is dominant in apolar and alcaline medium. The aromatic form results of hydrophobic form of curcuminoids, the bond is results of flexibility and support the hydrophobic and polar presence. 
Curcumin is the most important curcuminoid. Curcumin is the reason of the yellowish color of turmeric. Turmeric is based on three main curcuminoids and it comprise of 3-5% curcuminoids. The other curcuminoids are known as demethoxycurcumin and bisdemethoxycurcumin. Distribution of curcuminoids are; curcumin is between 50 and 60%, demethoxycurcumin is between 20 and 30% and bisdemethoxycurcumin is between 7 and 20%.
Antioxidant features of curcumin consists of it's phenolic form and diketone bond. Phenolic form has it's own antioxidant features independent from the bond. Curcumin can react with two free radicals because of two phenolic form. The bond is react with free radicals based on transfer of H atom in their diketone form, transfer of electron in their keto-enol form. It gives the curcumin reactive feature to degradation of free radicals in every condition of bond. It is also observed that the antioxidant feature of bond is lower than the peholic forms of curcumin.
In this thesis study, it is aimed to observe the effects of using curcumin as a natural antioxidant and microencapsulation methods on oxidative stabilty od chia seed oil. It is planned to microencapsulate on chia seed oil which contains omega-3 fatty acids in high proportion enriched with curcumin which is a natural antioxidant. Freeze drying method is used to obtain microencapsulated chia seed oil to minimize the negative effects of heat treatment on PUFAs in chia seed oil. The shelf life controls were made by based on peroxide value. It is aimed that investigate the effects both usage of natural antioxidant and encapsulation process on shelf life of chia seed oil which contains high quantity of ALA. The digestion behaviour of curcumin enriched chia seed oil microencapsules will be investigated.
First of all, it was researched that the most suitable wall material substances based on literature searches. Maltodextrin and gum arabic were choosen as wall materials in this study and preperation of emulsion process was applied. Three factors were choosen to investigate; maltodextrin:gum arabic ratio, total solid content and core:wall material raito. It was investigated that the stability effects of three different factors in 2x2x3 full factorial disagn method. It was observed that each factor has effects on stability of emulsions. Creamy stability tests and zeta potantial measurements were performed. The result of zeta potantial measurements were between -30 and -40. Based on these results it can be said that the emulsions had moderate stability. The most stabil emulsion was choosen based on these analyses, it was the 6th sample. It has 40% total solid content, 1:3 maltodextrin:gum arabic ratio and 1:5 core:wall material ratio. The gain of microencapsulation was performed with 6th sample with followed steps; emulsion was prepeared with 1800 rpm and 5 minutes with UKA homogenizator. Than the emulsion is pretreated with -80°C while 12 hours. After that, freezedrying process was applied with -35°C, 0,1 mbar while 36 hours. 
Characteristic identification of microencapsuled chia oil and microencapsuled chia oil enriched with curcumin was practised after microcapsulles were obtained. Moisture, water activity, surface oil and total oil analyses were performed in order to identificate the characteristics of microcapsulles and encapsulation efficiency was calculated with usage of surface oil and total oil analyses results. Moisture analysis results are about 3,5%, it makes the capsulles are suitable for food industry because the moisture value of powder materials wanted to be 3-4% in food industry. It can be said that lower moisture value prevent the microcapsulles from oxidative damage. Water activity analysis result were found approximately 0,045. When water activity is lower than 0,6, it can be associated with lower microbiological risk while all of the shelf life. Surface oil analysis results were found like 2,5% and total oil analysis results were approximately 7,5%. Encapculation efficiency was calculated with usage of the results of surface oil and total oil analyses. Encapsulation efficiency was found about 67%. Similar results were obtained from microencapsulation of PUFA studies which were use freeze drying as the method. 
The microcapsulles and bulk oils were examined while their shelf life. In order to understand the effect of encapsulation process and curcumin addition on shelf life, hydrogen peroxide values determined in weekly period. Spectrophotometric method was used in order to determine the hydrogen peroxide values. Increase of peroxide value in microcapsulled samples is lower than bulk oil samples. It can be said that encapsulation process has positive effect on oxidative damage while shelf life.There is no observed difference on peroxide values between curcumin added samples and without curcumin samples. It can be said that as a natural antioxidant, curcumin has no effect on oxidative stability during shelf life. The reason of not observed difference between curcumin added samples and without curcumin samples can be the spectrophotometric method. Curcumin has colorant properties and that property can be resulted with deviated results. 
In-vitro release behaviour was investigated in this study. In this purpose, simultaed salivary fluid, gastric fluid and  intestinal fluids were prepeared. Encapsulated samples were placed in salivary fluid for 2 minutes, gastric fluid for 2 hours and intestinal fluid for 2 hours respectively. The releasion of chia oil amount was determined with usage of spectrophotometry. The samples were collected in 30 minutes period. The results showed that the release of chia oil is lower in salivary and gastric fluid but increases in intestinal fluid. The wall materials affected the releasion behaviour.
Bioavalability analyses were performed for microcapsulled samples. Chia oil encapsule enriched with curcumin has higher total phenolic content and antioxidant capacity. The recovery rates of encapsulled samples wew about 40%. It is observed that the antioxidant properties of capsulled samples were throwed from body without absorption.