LEE- Gıda Mühendisliği Lisansüstü Programı

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http://hdl.handle.net/11527/19292

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Aquafabanın yanıt-yüzey metodolojisi ile optimizasyonu ve bitkisel bazlı mayonez üretiminde kullanımı

(Lisansüstü Eğitim Enstitüsü, 2023-02-21) Büker, Mine ; Karaça Can, Aslı ; 506201518 ; Gıda Mühendisliği

Aquafaba nohut gibi baklagillerin haşlama işlemi sonrasında tanelerin ayrılması ile elde edilen sıvı olarak adlandırılmaktadır. Yapısında bulunan protein ve karbonhidrat bileşenleri sayesinde köpük ve emülsiyon oluşturma özellikleri bulunmaktadır. Bu özellikleri sebebiyle aquafaba günümüzde vegan ürünler geliştirilirken yumurta alternatifi olarak kullanılma potansiyeli olan bir hammaddedir. Aquafabanın yumurtasız mayonez, mousse, mereng, dondurma, emülsifiye soslar, kokteyller ve fırıncılık ürünlerinde kullanılabilme potansiyeli yüksektir. Çorba ve keklerde emülsiyon, ekmek ve dondurmada su tutma kapasitesi ve tatlılarda yağ tutma kapasitesi sağlaması amacıyla bu tip ürünlerde aquafaba kullanılmaktadır. Aquafabanın fonksiyonel ve kimyasal özellikleri üretim koşullarına göre farklılık göstermektedir. Nohut-su oranı, süre, sıcaklık, pH, ilave edilen katkı maddeleri gibi parametreler aquafabanın bileşimini ve özelliklerini doğrudan etkilemektedir. Bu çalışma aquafabanın köpük oluşturma ve emülsifiye etme özelliklerini iyileştirmek için gereken proses koşullarını Yanıt-Yüzey Metodolojisi kullanılarak optimize etmeyi amaçlamaktadır. Bu kapsamda belirlenen koşullarda deney tasarımı oluşturularak aquafabanın fonksiyonel özellikleri incelenmiştir. Aquafaba numuneleri katı-sıvı oranı 0,20, 0,225 ve 0,25, haşlama süresi 60, 70 ve 80 dk olacak şekilde üretilmiştir. Aquafaba numunelerinin deney tasarımında belirlenen pH değerleri aquafabanın sıcaklığı 20°C'ye düştükten sonra sitrik asit ve trisodyum sitrat çözeltileri ilave edilerek 2, 4 ve 6'ya ayarlanmıştır. Deney tasarımına göre hazırlanan aquafaba örneklerinin °Brix, pH, köpük oluşturma kapasitesi, köpük stabilitesi ve emülsiyon aktivite indeksi değerleri ölçülmüştür. Elde edilen örneklerin °Brix değerleri 1,3-2,2 arasında değerler alırken ilk pH değerleri 6,0-6,1 arasında değişmiştir. Köpük oluşturma kapasitesi ve köpük stabilitesi incelendiğinde pH değeri 4 olan örneklerin köpük oluşturma özellikleri diğer örneklere göre daha iyi sonuç vermiştir. En yüksek köpük oluşturma kapasitesi ve köpük stabilitesi pH 4 olarak ayarlanan örneklerde kaydedilmiştir. Köpük oluşturma kapasitesi %58,5 -74,5 arasında değişirken; köpük stabilitesi % 4,5-83,8 arasında değerler almıştır. Emülsiyon aktivite indeksi köpük özelliklerinin aksine daha düşük pH seviyelerinde daha iyi sonuçlar kaydedilmiştir Emülsiyon aktivite indeksi ise % 96,8- 99,1 arasında değişmiştir. Katı-sıvı oranının emülsiyon aktivite indeksini önemli düzeyde etkilediği görülmüştür (p<0,05). Deney tasarımı ile elde edilen örneklere uygulanan analizler sonucunda optimum koşullarda bir örnek üretilmiş olup; validasyonu yapılmıştır. Yapılan optimizasyon sonucuna göre bu deney tasarımında pH 3,58, süre 80 dk ve katı-sıvı oranı 0,25 olduğunda optimum koşulların sağlandığı sonucuna ulaşılmıştır. Optimize koşullar ile üretilen örnek ile bitkisel kaynaklı bir mayonez üretilmiştir. Üretilen bu mayonez ticari olarak satılan standart ve vegan mayonez ile karşılaştırılmıştır. Mayonez örneklerinin pH, °Brix, asitlik, kıvam, raf ömrü ve ısı stabilitesi özellikleri incelenmiştir. Bunlara ek olarak duyusal analiz ile ürünler panelistler tarafından değerlendirilmiştir. Mayonez örneklerinin kimyasal özellikleri incelendiğinde aquafaba ile üretilen üründe °Brix ve kıvam değerleri daha düşük olarak ölçülmüştür. Bunun sebebi kullanılan formülasyonlar arasındaki farklılıklardır. Mayonez örneklerinin ısıl stabilitesi incelendiğinde 30 dk sonunda aquafaba içeren mayonez ve piyasadan temin edilen vegan mayonezde herhangi bir değişim gözlenmemiştir. Fakat piyasadan temin edilen standart mayonez örneğinde yağ fazının ayrılmaya başladığı ve örnek yüzeyinde sıvı birikmeye başladığı dikkat çekmiştir. Piyasadan temin edilen standart mayonezin ısıl stabilitesi bu çalışma kapsamında üretilen ve piyasadan ticari olarak temin edilen mayoneze göre daha düşük olduğu gözlemlenmiştir. Duyusal analiz sırasında panelistlerden hem demografik sorular içeren; hem de parametreleri puanlayabilecekleri bir form doldurmaları istenmiştir. Duyusal analiz sonucu elde edilen veriler incelendiğinde tüketicilerin %85'i "Bu ürünü satın alır mısınız?" sorusuna "Satın alırım" şeklinde yanıt vermiştir. Panelistlerden gelen yorumlara göre yapılan değerlendirmede piyasadan temin edilen vegan mayonezin ağızda yağlı ve yapay bir tat bıraktığı yönünde yorumlar iletilmiştir. Bu çalışma kapsamında üretilen mayonez için ise için 'Zayıf, ağızda çok çabuk kayboluyor' şeklinde geri dönüşler alınmıştır. Aquafabalı mayonezin renk olarak da geliştirilmeye ihtiyacı olduğu panelistler tarafından vurgulanmıştır. Ürünlerin genel kabul edilebilirliği incelendiğinde en düşük puanı vegan olan ticari ürün almıştır. Aquafabalı ürün hakkında gelen yorumlar incelendiğinde beklenildiği gibi yabancı tadın yoğun hissedilmediği ve düşünülene göre çok daha iyi olduğu ile ilgili yorumlar panelistler tarafından ek olarak iletilmiştir. Aqufabalı ürünün stabilitesi hakkında bilgi sahibi olabilmek için raf ömrü testi yapılmıştır. Bu analizde aquafaba ile üretilen mayonez örneği 5 gün boyunca 37°C'de depolanmıştır. Sürenin sonunda ürün incelendiğinde duyusal özelliklerin önemli ölçüde değiştiği, asitliğin yükseldiği gözlemlenmiştir. Yapılan bu teste göre 5 günün sonunda ürün kalitesinde kabul edilemez ölçüde düşüş meydana gelmiştir. Sonuç olarak aquafanın özellikleri pH, süre ve katı-sıvı oranına göre değişkenlik göstermiştir. Aquafabanın üretim koşulları optimize edilerek istenmeyen yabancı tadı baskılamak ve fonksiyonel özelliklerini geliştirmek mümkündür. Yabancı tat azaldığı durumlarda aquafabanın farklı ürünlerde kullanımı daha kolay olurken; aynı zamanda tüketiciler tarafından da daha kabul edilebilir ürünler elde etmek mümkündür. Bu çalışma kapsamında aquafaba bitkisel bazlı mayonez formülasyonunda başarılı şekilde kullanılmıştır. Fakat yapılan raf ömrü testi sonuçları gözlemlendiğinde ürün stabilitesinin düşük olduğu görülmüştür; bu nedenle ürünün raf ömrünün uzatılmasına ihtiyaç vardır.
Bakladan ultrases destekli alkali ekstraksiyon ile protein eldesi: Teknofonksiyonel, in vitro biyoerişebilirlik ve ade inhibisyon özelliklerinin incelenmesi

(Lisansüstü Eğitim Enstitüsü, 2022-05-30) Mertdinç, Zehra ; Özçelik, Beraat ; 506181516 ; Gıda Mühendisliği

Son yıllarda nüfus artışı, gıda kaynaklarının tükenmesi, iklim değişikliği ve hayvan hakları gibi birçok sebepten dolayı hayvansal gıda kaynaklarına alternatifler aranmaya başlamıştır. Bitkisel diyetlerin çevresel ve ekonomik katkılarının yanı sıra, kardiyovasküler hastalıklar, Tip-2 diyabet, obezite ve hipertansiyon gibi hastalıkların önlenmesinde de etkili olduğu bilinmektedir. Bakla, ülkemizde genellikle Ege, Marmara ve Akdeniz bölgelerinde üretimi yapılan, besleyici değeri yüksek ve biyoaktif bileşenler açısından zengin bir bitkisel protein kaynağıdır. Gıdaların besleyici ve fonksiyonel özelliklerinin geliştirilmesi için bakliyatlardan protein konsantresi/izolatı elde edilmektedir. Bu amaçla, özellikle alkali ekstraksiyon ve izoelektrik noktada çöktürme metodu sıklıkla kullanılan yöntemlerden biridir. Aynı zamanda bu yöntemler, ultrases, mikrodalga ve enzim destekli uygulamalar ile daha verimli hale getirilmektedir. Elde edilen proteinlerin, gıda matrisinde uygulamaları, depolanması ve işlenmesi noktasında teknofonksiyonel özelliklerinin (su/yağ tutma, köpürme kapasitesi/stabilitesi, çözünürlük gibi) incelenmesi gerekmektedir. Bu çalışmada da İzmir'de yetiştirilmiş bir bakla türünden alkali ekstraksiyon ve alkali ekstraksiyon öncesi ultrases uygulaması ile elde edilen protein konsantrelerinin teknofonksiyonel özellikleri incelenmiştir. Gıdalardan biyoaktif peptilerin eldesinde pepsin, alkalaz, tripsin gibi enzimler kullanılarak hidrolizasyonu gerçekleştirilebilir. Elde edilen hidrolizatlar, aminoasit dizilimleri, büyüklükleri ve bazı fizikokimyasal özelliklerine bağlı olarak anti-hipertansif, anti-oksidan ve anti-mikrobiyal etki gösterebilirler. Aynı zamanda hidrolize uğrayan peptitlerin insan vücudunda in vitro gastrointestinal sindirimin ardından biyoerişilebilirlikleri de etkilenmektedir. Bu çalışmada da alkali ve ultrases destekli alkali ekstraksiyonla elde edilen protein konsantrelerine pepsin ile enzimatik hidrolizasyon uygulanmış, ardından elde edilen hidrolizatlarda da in vitro protein biyoerişilebilirliği ve anti-hipertansif etkisinin belirlenmesi amacıyla ADE inhibisyon analizi uygulanmıştır. Bu çalışmada elde edilen sonuçlara göre, %27,32 protein miktarına sahip olan bakla unundan, alkali ve ultrases destekli alkali ekstraksiyonla sırasıyla %65,93 ve %75,04 protein içeriğine sahip konsantratlar elde edilmiştir. Ekstraktların teknofonksiyonel içerikleri de incelendiğinde, alkali ve ultrases destekli uygulamalar sonucunda ekstraktlarda protein çözünürlüğü, su ve yağ tutma kapasiteleri, köpürme stabiliteleri gibi özellikleri geliştirilmiş olup, çoğu uygulamada da ultrases uygulamasının alkali konsantrelerden istatistiksel olarak anlamlı bir farklılık gösterdiği tespit edilmiştir. Ayrıca hammadde, ekstraktlar ve enzimatik yöntemlerle elde edilen hidrolizatların, in vitro gastrointestinal sindirim sonrasındaki protein biyoerişilebilirlikleri incelenmiştir. Bu çalışmada da en yüksek protein biyoerişilebilirliği %46,39 ile ultrases destekli hidrolizatlarda tespit edilmiştir. Öte yandan, hidrolizatlarda ADE inhibisyon değerleri hesaplanarak baklanın potansiyel anti-hipertansif etkisi incelenmiştir. Analizler sonunda sırasıyla alkali ve ultrases yöntemlerinden elde edilen hidrolizatların IC50 değerleri 504,42 ve 222,89 μg/ml olarak hesaplanmıştır. Elde edilen sonuçlar göstermiştir ki, ultrases uygulaması ile ADE inhibisyon aktivitesinin arttığı gözlemlenmiştir. Özetle, bu çalışma ile Türkiye'ye özgü bir bakla türünden iki farklı yöntemle ekstrakte edilen proteinlerin teknofonksiyonel özellikleri tanımlanmış olup, enzimatik hidrolizasyonu ile de in vitro biyoerişilebilirlik ve ADE inhibisyon potansiyelleri incelenmiştir. Sonuç olarak, ultrases teknolojisinin, proteinlerin bazı teknofonksiyonel özellikleri, protein biyoerişilebilirliği ve anti-hipertansif etkileri üzerindeki olumlu etkileri incelenmiş ve gıda uygulamalarındaki potansiyeline dair bir bakış açısı geliştirilmesi hedeflenmiştir.
Bioactive components and protein content in black carrot leaves

(Graduate School, 2024-07-08) Çambel, Hatice ; Karıncaoğlu Kahveci, Derya ; 506211517 ; Food Engineering

Black carrot leaves are considered food waste, even though they may be a multifunctional food commodity. Black carrot leaves contain various bioactive compounds such as phenolics, flavonoids, and proteins, making them a valuable resource for multiple applications in the food industry. In this study, black carrot leaves (BCL) were cold pressed, and 357.47± 40.17 g BCL cold-pressed resulted in 159.51± 14.03 g pulp and 176.50± 28.79 g leaf juice. So, the yield was calculated as 49.22± 4.47 % g leaf juice/ g leaves. The obtained leaf juice was analyzed for TPC (total phenolic content), TFC (Total flavonoid content), CUPRAC (Antioxidant capacity), and DPPH (Antioxidant capacity measurement) analyses by using spectrophotometric methods. The measurements were recorded as 1.912± 0.209 mg Gallic Acid/ g BCL, 2.914± 0.592 mg Quercetin/ g, 6.586± 0.882 mg Trolox/ g BCL, and 0.273± 0.166 mg Trolox/ g BCL, respectively. Subsequently, the protein content in the juice was precipitated by two different methods: acid treatment and heat treatment. The protein yield was calculated as 0.0020± 0.0004 g protein per g BCL via acid-treatment. The protein yield was calculated as 0.0011± 0.0003 g protein per g BCL via heat treatment. The protein content in BCL was determined by the Kjeldahl method. Due to sustainability concerns, the heat-treatment method was chosen in the following steps. The proteins extracted via heat treatment were subjected to in vitro digestion, employing a laboratory setup mimicking the human gastrointestinal tract without symbiotic bacteria in the intestine. This simulation demonstrated that human digestive enzymes can break down Black Carrot Leaves (BCL) proteins, and the Bradford assay examined the results. Moreover, techno-functional properties critical for food and selecting the place to use it were examined. These are water-holding capacity, oil-holding capacity, emulsifying activity and stability, solubility, and foam capacity and stability properties. As a result, the water holding capacity is 4.62± 0.31 g water/ g Pdry, and the oil holding capacity is 5.38± 0.225 g corn oil/ g Pdry. On the other hand, Emulsifying Activity Index (EAI) and Emulsifying Stability Index (ESI) values were examined according to different pH values. EAI increases with increasing pH and reaches its highest value at pH 12. ESI gives very different low or high values with very little pH change. When the solubility was examined as a percentage, it was observed that it increased and as it approached neutral (pH = 7). And, the solubility reached the maximum at pH 12. Foam capacity (FC) and foam stability (FS) values were found to be 0.0516± 0.037% and 33.53± 18.27%, respectively. The evaluation of the antioxidant capacity of BCL, the extraction of protein, and the evaluation of techno-functional properties of BCL, which were commonly considered food waste post-harvest, hold significant importance from a sustainability point of view. Understanding the composition of BCL and its potential applications can contribute to minimizing food waste and exploring sustainable alternatives across multiple sectors.
Bioactive peptide encapsulation by electrospinning technique: Characterization of electrospun fibers and mathematical modelling of release kinetics

(Graduate School, 2023-06-12) Kırbaş, Zahide ; Altay, Filiz ; 506152509 ; Food Engineering

Bioactive peptides, which are biologically active amino acid groups in the sequence of proteins, exhibit a variety of beneficial effects including antioxidant, anti-inflammatory, antihypertensive, anticancer, antidiabetic, antimicrobial, antithrombotic, hypocholesterolemic, antiaging and opioid activities as well as prevention of cancer, osteoporosis, hypertension, cardiovascular disorders and neurodegenerative diseases such as Parkinson and Alzheimer's diseases. However, the bioactive peptides isolated from plants and animals may be lost during processing and storage. Furthermore, bioactive peptides have short in vivo half-lives, low bioavailability and poor stability against gastrointestinal conditions. Therefore, to use of encapsulation technologies such as coacervation, ionic gelation, electrospraying, microfluidic, emulsification, liposomal encapsulation, spray drying and electrospinning have been started to become widespread. Considering the above, the objectives of this Ph.D. thesis were (i) to produce a nanofibrous delivery vehicles for bioactive peptides without using any synthetic polymers or any hazardous solvents by using electrospinning, to characterize electrospun fibers to evaluate the effect of formulation and properties of feed solutions on electrospinnability and to examine the encapsulation efficiencies of produced nanofibrous delivery vehicles by using a model peptide; (ii) to produce carnosine (Car) loaded water-in-oil-in-water (W1/O/W2) double emulsions with different formulations using as feed emulsions in emulsion electrospinning study; (iii) to produce carnosine (Car), an antioxidative peptide, loaded pullulan (Pul)-sodium alginate (NaAlg) based composite nanofibers by uniaxial (blending), coaxial and emulsion electrospinning techniques and to characterize electrospun fibers to evaluate the effect of solution /emulsion properties and the role of emulsion parameters; (iv) examining the encapsulation efficiencies of electrospun fibers and to investigate the effect of encapsulation on antioxidant activity of Car; to determine the effects of electrospinning encapsulation and crosslinking on release behavior of carnosine from electrospun nanofibers during in vitro digestion and to analyse the release kinetics by establishing corresponding mathematic models.
Bioactivity and functionality of chickpea protein-spent coffee phenolic complex

(Graduate School, 2022) Vafa, Hamedreza ; Şeker, Dursun Zafer ; Food Engineering Programme

The demand for alternative protein resources is growing by the day, due to factors such as rising population and changing environmental conditions. Plant-based proteins are one of the most beneficial protein sources to consider in this regard. However, since the functional and nutritional properties of plant-based proteins are inadequate to those of animal-based proteins, several modification applications are being explored in order to improve these properties. Protein interaction is one of the promising protein modification methods that have the potential to improve the bioactive properties of plant-protein resources. The interaction of chickpea protein with spent coffee phenolics was investigated in this study under two conditions interaction conditions including pH 7 and pH 9. The binding properties, the effect of the interaction on the structure and functional properties of the chickpea protein and the in vitro bioaccessibility properties of the complex were then investigated to characterize the effect of interaction on protein and phenolic compounds. Although chlorogenic acid was found in the extract, it was not found in the protein phenolic solution. Besides that, the amount of catechin in the protein phenolic interaction solution was lower than in the phenolic extract. Therefore, it was concluded that these two compounds were bound with chickpea protein. The effect of protein phenolic interaction on the functional properties of chickpea protein was investigated regarding protein solubility, foaming and emulsifying properties. There was no significant difference on the solubility of chickpea protein after its interaction with phenolic compounds. Similarly, there was no significant difference between foaming properties and emulsion capacity and stability of chickpea protein after at pH 7 (p>0.05). However, these properties of chickpea protein are enhanced at pH 9. The foaming capacity of chickpea protein was 43.33%, while the phenolic addition ranged from 123.33 to 142.89% with no significant difference between concentrations. Similarly, interaction at pH 9 increased the emulsion capacity and stability of chickpea protein by 10% and 8%, respectively. The emulsion activity index and stability index of the samples increased after both interactions. The CPI + PE 1 example showed the highest increase in chickpea protein. The determined increase was 55 % for the interaction at pH 9 and 47% for the interaction at pH 7, respectively. In the same examples, the emulsion stability indexes increased by 70% and 6%, respectively. In vitro bioavailability analysis was performed to examine the bioactive properties of protein phenolic solution and its stability during gastrointestinal digestion. The total phenolic content and antioxidant capacity of the samples were increased with the addition of phenolic extract. After intestinal digestion, the total phenolic content in covalent samples decreased, however, no statistically significant difference was observed after the interaction at pH 7. The total phenolic content of the samples interacted at pH 9 decreased after intestinal digestion, but there was no statistically significant difference the samples interacted at pH 7. The ABTS method indicated that, while total antioxidant capacity was reduced in the gastric phase, total antioxidant capacity was increased by up to 33% only after the interaction at pH 9 in the intestinal phase. Although there was little or no increase in total antioxidant capacity of the samples in the gastric phase, an increase in antioxidant capacity of up to 68 and 18 %, respectively, was observed in the intestinal phase after interaction at pH 7 and pH 9 with the addition of phenolic. Zeta potential, FTIR spectrum, and fluorescence intensity analyses were used to investigate the effect of the interaction on the structural properties of the chickpea protein. While the samples' absolute zeta potential was low after interaction at pH 9, the absolute zeta potential increased after interaction at pH 7. The FTIR spectrum of the analysis showed that there was a change in the secondary structure of the chickpea protein due to the change in the amide bands. Also, change in the tertiary structure was detected due to the change in the fluorescence intensity after both interaction conditions. Consequently, spent coffee ground phenolics were found to cause changes in protein structure as a result of interaction with chickpea protein. The functional properties of the chickpea protein have been improved as a result of these changes. It was concluded that phenolics from spent coffee grounds could be an effective alternative protein modification for chickpea protein.
Biological control of Aspergillus flavus growth and its aflatoxin b1 production by antagonistic yeasts

(Graduate School, 2022-06-29) Dikmetaş, Dilara Nur ; Güler Karbancıoğlu, Funda ; Özer, Hayrettin ; 506181504 ; Food Engineering

The presence of mycotoxins in food and feed poses a risk to human health and animal productivity and result large economic losses. In the field and during storage period, Aspergillus flavus infect grains. Aflatoxin B1, is classified as group 1 carcinogen, having hepatotoxic, genotoxic, and teratogenic properties, may be produced by generally A. flavus and Aspergillus parasiticus in addition to grain deterioration and yield loss. Aflatoxins have been found in a variety of foods including oilseeds, nuts, dried figs and spices. Dried figs, pistachios, hazelnuts and groundnuts cultivated in Turkey are risky products in terms of aflatoxins. The application of synthetic fungicides is the most common method for controlling decay in most crops. However, because of the fungal resistance and detrimental impacts on human and animal health, as well as the environmental concerns in general, their use is being tried to diminished. Due to these concerns, researchers have tended to investigate more eco-friendly and healthy methods to manage fungal diseases. As a result, detecting and preventing Aspergillus species contamination, as well as lowering the level of aflatoxins in foodstuffs used in many agricultural products. To reduce usage of synthetic fungicide, biological control is an important strategy as a promising alternative with low environmental impact in reducing fungal infection and mycotoxin production in the field and during postharvest period. In addition, among microorganisms, yeast species have been extensively studied as antagonist due to simple nutritional requirements, able to colonize dry surfaces for long periods of time and able to grow rapidly in bioreactors with inexpensive substrates. Furthermore, yeast is simply adapted to microenvironments. The crucial and most important step is to develop biocontrol agent is isolation and screening of yeast isolates. Antagonistic yeasts have been showed several different mechanism to control of different moulds such as competition for space and nutrients, biofilm formation, parasitism, production of antimicrobial volatile organic compounds and production of lytic enzymes. The antagonist mechanism generally explained with cell wall-degrading enzyme synthesis including chitinases, β-1,3-glucanase, protease, cellulase and pectinase. Yeasts with high cell wall degrading enzyme activity, also showed high biocontrol efficacy. Biocontrol of aflatoxin has been generally documented by non-aflatoxigenic Aspergillus species. However, the studies to control Aspergillus flavus by yeasts limited. In various industrial processes, the Metschnikowia yeast has wide range of biotechnological application and generally isolated from fruits and flowers. Several Metschnikowia based biocontrol products have been industrialized to control postharvest diseases including Botrytis or Monilinia spp. However, only a few biocontrol agents are converted into industrial products. Meyerozyma, Moesziomyces and Metschnikowia sp. yeasts antifungal activity have been studied by several researchers. Among antagonistic microorganism mechanism of action, production of antimicrobial volatile organic compounds is one of the least studied. Primarily, volatile organic compounds produced by antagonistic yeasts have great potential used as biocontrol agents of filamentous fungi. In this study, four yeast isolates have been isolated and identified by different plant parts including hawthorn, hoşkıran, bean and grape leaf collected from Turkey different region. Four previously isolated and identified isolates from grapes, blueberry have been also included in this study. The eight antagonistic yeasts have been belong to Moesziomyces sp., Meyerozyma sp. and Metschnikowia sp. Yeasts secrete fungal lytic enzymes which are typically associated with biocontrol mechanism. Lytic enzyme activities of yeasts were examined with screening method. All of the isolates have β-glucosidase and chitinase activity, which are crucial for antifungal mechanism, however absence of the pectinase activity. Among antagonistic yeasts, only Metschnikowia pulcherrima (26-BMD) found as protease negative. Dual culture assay have been conducted to observe antagonistic effect of yeasts against aflatoxin B1 producer Aspergillus flavus. All of the antagonistic yeasts formed inhibition zones in dual culture assay against to Aspergillus flavus due to secretion of diffusible antifungal compounds. After that, yeasts antifungal and antiaflatoxigenic impact on aflatoxin producer Aspergillus flavus by spot inoculation method with different incubation period by in vitro studies. Different yeasts used to investigate to control Aspergillus flavus growth. In addition to that, origin of the yeast affect their biocontrol efficacy. All of the antagonistic yeasts formed inhibition zones in dual culture assay against to Aspergillus flavus due to secretion of diffusible antifungal compounds. While Aspergillus flavus mycelial growth of inhibition 86-97% after three days. All isolated and identified yeasts were effective to control Aspergillus flavus, as well as aflatoxin. Aflatoxin B1 production was reduced from 1773 ng/g (in control samples) to 1.26-10.15 ng/g with the application of antagonistic yeast. Metschnikowia aff. pulcherrima (32-AMM) was found as the most effective yeasts to inhibit mycelial growth of Aspergillus flavus among other yeasts. In addition, yeasts plant origin and incubation period also affect their inhibition potential (p<0.05). All of the yeasts might be used as biocontrol agent against Aspergillus flavus growth. Additionally, all of the yeast volatile organic compounds (VOCs) reduced sporulation however among antagonistic yeasts only Moesziomyces bullatus (DN-FY), Metschnikowia aff. pulcherrima (DN-MP) and Metschnikowia aff. pulcherrima (32-AMM) were reduced Aspergillus flavus mycelial growth with in vitro studies. But only VOCs produced by Metschnikowia aff. fructicola (1-UDM) was also found effective in reduction of Aflatoxin B1 production in in vitro experiments. This activity was associated to different volatile organic compounds. As a result, more investigation into the role of volatile organic compounds in Aspergillus flavus and aflatoxin B1 control is required. Further field experiments would indicate yeasts biocontrol potential on the products prone to contaminated from Aspergillus flavus. By the way, also isolation of volatile organic compounds from yeasts should be used to protect products from contamination without harmless to humans and environmentally friendly.
Bioprospection of antagonistic yeasts for biocontrolling postharvest pathogenic fungi and physicochemical characterization of a yeast exopolysaccharide

(Graduate School, 2023-05-04) Öztekin, Sabahat ; Güler Karbancıoğlu, H. Funda ; 506142509 ; Food Engineering

The leading cause of food loss or fruit and vegetable waste was associated with fungi-induced postharvest diseases. Since fruit is rich in water, sugar, and essential nutrients, phytopathogenic and/or mycotoxigenic fungi can easily spread and find an ideal habitat, especially in damaged fruits. At this point, synthetic fungicides appear to be the first way of protection. Although these fungicides are efficient and economical, they can develop resistance in fungi, and their toxic residues may pose a health and environmental threat. The European Food Safety Authority (EFSA) has set tight regulations for the maximum pesticide residue levels in crops. It also encourages using low-risk or non-chemical fungicides for health and the environment. Recently, climate change has reduced crop yields and triggered multi-mycotoxin formation. Therefore, there is a need for non-toxic, natural, and efficient bio-fungicides to be used in agroecosystems. Harnessing beneficial microorganisms, especially bacteria, yeasts, and moulds, has emerged as an alternative to synthetic fungicides. Among these microorganisms, antagonistic yeasts have a great potential for biopreservation with their GRAS (generally regarded as safe), biodegradable, non-toxic, genetically stable, and safety characteristics (non-pathogenic without producing antibiotics, mycotoxins, and allergenic spores). Additionally, antagonistic yeasts can use a variety of substances as carbon and energy sources, thereby limiting nutrients for spoilage fungi. They can also tightly attach to fruit and secrete hydrolytic enzymes, volatile organic compounds, mycocin, and biofilm. In line with the United Nations' Sustainable Development Goals of combating hunger and poverty, biocontrol yeasts have sparked much interest as a green and sustainable solution. The biocontrol yeasts can be a viable alternative to synthetic fungicides by bio-managing the notorious fungi and their mycotoxins, increasing agricultural productivity and food security. Regarding yeast metabolites, yeast-based exopolysaccharides (EPS) have received much attention due to their high yields, ease of extraction, and bioactive compounds. Considering the above, a research strategy has been developed to investigate the biocontrol potential of fruit-isolated yeasts against selected fungi on lemons, mandarins, and grapes, along with their exopolysaccharide production and characterization. The objectives of this PhD dissertation were (i) bioprospecting Metschnikowia sp. isolates as biocontrol agents against postharvest fungal decays on lemons with their potential modes of action; (ii) evaluating the combinatorial effect of antagonistic yeasts (Hanseniaspora uvarum, Meyerozyma guilliermondii, and Metschnikowia aff. pulcherrima P01A016) on the bio-management of green mould disease on mandarin fruits; (iii) recruiting grape-isolated Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hansenispora uvarum yeasts on the biopreservation of Botrytized grapes; (iv) characterization of a novel exopolysaccharide produced by a cold-adapted yeast Rhodotorula glutinis. Four research concepts (Chapters 3-6) were carried out to accomplish these objectives. Firstly, various fruits (grape, rosehip, hawthorn, blackberry, and cornelian cherry) and leaves were employed for yeast isolation. These yeasts were screened for their in vitro antifungal activity against various fungi. Since the highest antagonism was observed on Penicillium spp., lemons were selected as a substrate for further experiments. Then, pectinase-free antagonistic yeasts were applied to wounded lemon fruits to test their biocontrolling efficacy (Chapter 3). The compatible combinations of three antagonistic yeasts were employed to manage green mould decay on wounded and intact mandarin fruits. Furthermore, the findings were compared to the effectiveness of the synthetic fungicide, imazalil (Chapter 4). Grape-isolated yeasts Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hanseniaspora uvarum were used in the biopreservation of grapes against Botrytis cinerea, along with elucidating antifungal mechanisms of action (Chapter 5). Finally, yeast isolates were screened for their exopolysaccharide production capabilities. A blackberry isolate, R. glutinis, was the most promising one with its high yield, characterized by its structural, rheological, antioxidant, and antibiofilm properties (Chapter 6). The research framework and objectives of this PhD thesis are introduced in Chapter 1. Following that, Chapter 2 provided a comprehensive review of the exploitation of antagonistic yeasts for sustainable bio-management of postharvest phytopathogenic and mycotoxigenic fungi in fruits. The potential use of antagonistic yeasts for biocontrol purposes was mentioned, along with explaining their antifungal mechanisms of action. The current literature was screened to determine how antagonistic yeasts could be used with other agents or processes for improved biocontrolling activity. Subsequently, biocontrol yeasts' principles, advantages, disadvantages, and applications for commercial use were discussed. In Chapter 3, eleven distinct yeast cultures of Metschnikowia sp. belonging to six different species were identified using polymerase chain reaction (PCR) with sequence-based analysis of the D1/D2 domain of 26S rDNA. Yeast antagonism (1 × 108 cells/mL) against various fungi (Fusarium oxysporum, Botrytis cinerea, Penicillium digitatum, Penicillium expansum, and Alternaria alternata) was determined through a dual culture method on plates. The highest antagonism was obtained against Penicillium digitatum NRRL 1202 and Penicillium expansum DSM6284 (83.63-100 %). Chitinase activity was present in all of the examined yeasts, while others also exhibited protease, pectinase, cellulase, β-1-3 glucanase, and gelatinase activities. Due to the high pectin content in lemons, three pectinase-free cultures with strong in vitro antagonistic effects on Penicillium were chosen. The interaction of lytic enzyme secretion, biofilm formation, iron depletion, and volatile organic compound (VOC) formation determined the antifungal mechanism of action. Pectinase-free Metschnikowia sp. yeasts significantly reduced the disease incidences and lesion diameters on lemons. M. aff. fructicola had the best biocontrol efficacy against Penicillium on lemons. In Chapter 4, the antagonistic yeasts belonging to genera Hanseniaspora uvarum, Meyerozyma guilliermondii, and Metschnikowia aff. pulcherrima P01A016 were employed to inhibit P. digitatum in wounded and whole mandarins. All yeast cultures (73.85% - 80.64%) and their combinations (1:1, v/v; 1:1:1, v/v/v, 1 × 108 cells/mL) (78.40% - 83.18%) reduced the mycelial growth of green mould in vitro. M. aff. pulcherrima reduced the disease incidence and lesion diameters by 75.5% and 91.3%, respectively, demonstrating the highest biocontrolling activity alone. M. guilliermondii exhibited the highest biofilm formation (OD 0.93 ± 0.01) and antifungal activity (71.13%) via volatile organic compounds (VOCs), whereas H. uvarum exhibited cell-wall degrading β-1,3 glucanase activity in addition to mycocinogenic and VOCs activity. M. guilliermondii and M. aff. pulcherrima (M-1) were the most compatible, while M. aff. pulcherrima and H. uvarum (M-3) had the least compatibility. Combining these three yeasts resulted in synergistic cooperation, demonstrating the highest biocontrolling efficacy in vitro and in vivo. In Chapter 5, grape-isolated yeasts (Metschnikowia aff. fructicola, Metschnikowia pulcherrima, and Hansenispora uvarum) were proposed to control B. cinerea-caused grey mould disease in grapes, while also elucidating their potential inhibitory mechanisms. The antifungal mechanisms included iron depletion, secretion of hydrolytic enzymes, diffusible compounds, inhibition of conidial germination, biofilm formation, wound-site colonisation, and VOC emission. Metschnikowia yeasts yielded comparable in vitro results but in in vivo experiments M. aff. fructicola outperformed all other yeasts. Interestingly, VOCs from H. uvarum held considerable potential as an antifungal biofumigant. All yeasts are adapted well to their ecological niche to protect the grapes against grey mould. In Chapter 6, a novel EPS from R. glutinis was characterized by its monosaccharide composition by high-performance liquid chromatography (HPLC), molecular characterization by Fourier transform infrared (FT-IR) spectroscopy, thermal stability by differential scanning calorimetry (DSC), morphological characterization by scanning electron microscopy (SEM), crystallographic characteristics by X-ray diffraction (XRD) analysis, structural characterization by nuclear magnetic resonance (NMR) spectroscopy, antioxidant activity by •OH, DPPH•, CUPRAC, and ABTS radical scavenging activities, and rheological characterization by a rheometer. The findings revealed that EPS was a heteropolysaccharide composed of glucose and galactose. NMR and FT-IR analyses confirmed that the EPS-BMD26 structure had glucose and galactose without mannose. Differential scanning calorimetry (DSC) analysis revealed its thermal stability up to 326.16 °C. Scanning electron microscopy (SEM) revealed the porous microstructure with fissures. X-ray diffraction (XRD) analysis revealed that it was semi-crystalline. It demonstrated moderate and concentration-dependent antioxidant potential through •OH, DPPH•, CUPRAC, and ABTS radical scavenging methods. EPS-BMD26 exhibited high water holding capacity of 190 ± 0.22% with a water solubility index of 60.6 ± 8.3%. The EPS-BMD26 also inhibited biofilm formation by Staphylococcus aureus ATCC 25923 (79.5% inhibition at 1250 ppm). Rheological analysis revealed its shear thinning and pseudoplastic behaviour. Finally, in Chapter 7, based on the main outcomes of the previous chapters, the general discussion, conclusion, and future prospects in the biocontrol yeasts and yeast-derived exopolysaccharides are presented. From a biocontrol perspective, further research is required to examine the relationships between yeast, fungal pathogens, fruit, and microbiome. Integrating antagonistic yeasts with different microorganisms, natural agents, and physical processes can open new opportunities for designing effective microbial consortia for a tailored biofungicide. Further research is required to develop novel yeast-based biofungicides and understand their precise mode of action in large-scale applications.
DBUVC ve UVCLED ışınlamanın kiraz ve vişne yüzeyinde mikrobiyel inaktivasyona ve meyve kalite özelliklerine etkisi

(Lisansüstü Eğitim Enstitüsü, 2022-04-21) Bilgin, Ayşenur Betül ; Akocak Başaran, Pervin ; 506181502 ; Gıda Mühendisliği

Ultraviyole – C (UVC), fiziksel bir yöntem olan ve genellikle gıdalarda dezenfeksiyon amaçlı kullanılan mor ötesi ışıktır. Son yıllarda, geleneksel düşük basınçlı UVC (DBUVC) lambaların ve son yıllarda yeni UVC kaynağı olarak kullanılan UVC – ışık yayan diyotların (light-emitting diodes, LED) (UVCLED) gıdalarda meyve ve sebze dezenfeksiyon çalışmaları yoğunluk kazanmıştır. Bu projede, UVC kaynağı olarak UVCLED ve DBUVC ışınlanmasıyla kiraz ve vişne yüzeyinin dezenfeksiyonun sağlanması ve meyvelerin raf ömrünün arttırılması hedeflenmiştir. Kiraz ve vişne yüzeyine daldırma ve spreyleme yöntemi ile Pseudomonas syringae, meyveden izole edilen maya inoküle edilmiştir. Meyve yüzeyinde doğal olarak bulunan toplam aerobik mezofilik bakteri (TAMB), toplam küf ve maya (TKF) ve inoküle edilen mikroorganizmalar hedeflenerek meyve yüzeyi 30 saniye, 3 ve 10 dakika UVCLED (200 – 260 nm) ve DBUVC (253,7 nm) ile ışınlanmıştır. Her iki UVC lamba ile ışınlanmış meyveler, 25-28˚C (%40-60 nem)'de ve +4˚C'de depolanarak mikroorganizmalardaki inaktivasyon etkisi periyodik olarak değerlendirilmiş ve ürünlerin raf ömrü incelenmiştir. Işınlama öncesi ve sonrası meyvelerde meydana gelen fizikokimyasal (meyve ağırlığı, pH, toplam çözünebilir katı madde miktarı, su aktivitesi, toplam fenolik madde miktarı, antioksidan aktivite ve renk) değişimler gözlenmiştir. Tüm bu sonuçlar ile UVCLED ve geleneksel DBUVC lambaların inaktivasyon etkinliği ve kalite üzerinde etkisi karşılaştırılmıştır. 3 ve 10 dakika DBUVC ışınlamanın, UVCLED'e göre, 30 saniye UVCLED ışınlamanın ise DBUVC'ye göre daha etkili olduğu tespit edilmiştir. Toplamda tüm sürelerde ve mikroorganizmalarda en düşük ve en yüksek inaktivasyon UVCLED ışınlama ile 0,31 – 0,86 kob/g, DBUVC ile 0,11 – 1,56 kob/g olarak gerçekleşmiştir. Hem kiraz hem de vişnede ışınlama öncesi ve sonrası toplam ağırlık, su aktivitesi, pH, toplam çözünür katı madde miktarı ve renk değerlerinde (L*, a*, b* ve H˚) önemli bir farklılık bulunmamıştır (p > 0,05). Vişnede UVCLED ışınlama ile fark edilebilir düzeyde (∆EUVCLED* = 1,17 – 3,30) renk değişimi tespit edilmiştir. Toplam fenolik madde ve antioksidan madde miktarında çoğunlukla önemli bir artış gözlemlenmiştir (p < 0,05). Işınlanmış meyvelerin +4 ve 25˚C'de depolanmasıyla raf ömrünün ortalama %30 oranında arttığı tespit edilmiştir. Sonuç olarak; DBUVC, UVCLED ışınlamaya göre genel olarak mikroorganizma inaktivasyonunda daha etkili olduğu, antioksidan aktivitesi ve toplam fenolik madde miktarının artması dışında meyvelerin fizikokimyasal özelliklerinde önemli bir etkisinin bulunmadığı tespit edilmiştir.
Development of pea protein agar based biodegradable film with the incorporation of essential oils

(Graduate School, 2022) Ozhan, Selin ; Yeşilçubuk Şahin, Neşe ; Saygun, Ayşe ; 725922 ; Food Engineering Programme

In recent years, there is an increased awareness of sustainability in the world. People are looking for more sustainable solutions in packaging the industry since it is seen as one of the most polluting agents in the environment. Edible films and coatings are alternatives to reduce plastic packaging usage in the food industry. While it has an advantage for being sustainable, it also provides a good barrier, sensory, and quality properties in the foods. This study aimed to investigate the characterization of pea protein and agar edible film and then add essential oils to increase the antimicrobial effect of the edible film. For this purpose, 4 different essential oils are used; eucalyptus, thyme, lemon and niaouli in 1%, 2%, 3%, 4% and 5% (v/v) concentrations. Edible films are produced by using the solvent casting method. As the first step of this study, different film-forming materials were investigated and prepared in the laboratory to observe their edible film-forming capability. According to the results, pea protein and agar were chosen as the main edible film materials because they can form flexible and transparent films. Furthermore, essential oils were researched in the literature. The proper essential oil combination with edible films and the concentration were investigated. Thyme, eucalyptus, niaouli, and lemon oil were chosen because of their high antimicrobial activities and being capable to incorporate with edible films. After the decision of main materials, the pea protein and agar films were produced and thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA) was checked to understand the characterization of the main film. After the addition of essential oils, swelling analysis, antimicrobial activity, and color analysis were done. TGA indicates the weight decrease of the film due to the water, volatiles, and plasticizer losses during the analysis. In DSC analysis, the Tg value is observed as 73℃. Since the pea protein and agar film seems flexible structure at room temperature, having a higher Tg value than room temperature may show that it is not mechanically strong for processing and it will be in more rubbery and softer structure at the higher temperatures. Lastly, DMA analysis was unsuccessful due to the weak mechanical strength of the film. After essential oils addition to pea protein based film, antimicrobial activities of essential oil incorporated edible films were analyzed. Based on the results, swelling and color analysis were conducted for the films that showed antimicrobial effect and also 5% oil concentration. For antimicrobial analysis, the agar disc diffusion method was used and essential oil incorporated film's antimicrobial activity was investigated against Escherichia coli, Staphylococcus aureus, and Aspergillus niger. According to the results, 1% addition of essential oil to the edible film did not show any antimicrobial effect against any of the microorganisms. In 2% essential oil addition, thyme and niaouli oil started to show antimicrobial effects against gram (+) and Gram (-) bacteria. After 3% addition of thyme oil, antimicrobial effects were observed against both bacteria and fungus. In bacteria, there is no direct correlation between the concentration of essential oil and the antimicrobial zones in the Petri dishes. The largest zone which was 2.7 cm observed against E. coli is 3% thyme oil incorporated edible film, and the largest zone against S. aureus is from all 3%, 4%, and 5% thyme oil that all give 2.3 cm in one of the analysis. On the other hand, there was a direct correlation between the concentration of essential oil and the antimicrobial activity against fungus. In 5% thyme oil incorporated film, there was almost no growth in the Petri dishes however the growth of the fungus increases as the concentration of thyme oil decreases. Additionally, swelling analysis was conducted for 5% essential oil incorporated films and 2% niaouli, 2% thyme, 3% thyme, and 4% thyme incorporated films that show antimicrobial effects against bacteria. It is found that 5 ml thyme oil is the least absorbent one in the both water and saline water environment. In a saline water environment, it started to lose its weight from the first hour. Hence, it can be said that it is not suitable for salted food application or any salty environment. On the other hand, 2 ml niaouli oil incorporated edible film is seen as the most absorbent one in both environments. During the analysis, all edible films show some peaks in different time zones. The reason can depend on the oil concentrations and hydrophobicity rates. In terms of color analysis, the same samples were used for swelling analysis. According to the average of six repetitive measurements, 2 ml niaouli oil incorporated edible film gives the least yellowish color, and the 4 ml thyme oil incorporation of the film gives the most yellowish. The results are evaluated by using the b* parameter which provides the rate of yellow color on the materials. In conclusion, this study shows that pea protein and agar can yield an edible film, and when incorporates with the proper amount of thyme oil, it can show antimicrobial activity against gram-positive and gram-negative bacteria and fungus.
Effect of atmospheric non-thermal plasma against food-borne bacteria on food packaging film surfaces

(Graduate School, 2024-01-18) Doğanöz, Dilan ; Güler Karbancıoğlu, H. Funda ; 506211507 ; Food Engineering

Over the past decade, non-thermal plasma has become established as a potential technology for microbial inactivation. As commonly known, plasma treatment can produce highly specific surface modifications, so it has been extensively used in packaging. Methods such as heating, surface washing with hydrogen peroxide, irradiation, or their combinations, widely used for sterilization in the packaging industry, are known to have negative drawbacks. For instance, using chemicals or preservatives may cause residue problems, or high thermal processes may cause the loss of the desired structure of the food or package. Despite that, sterilization by non thermal plasma has several advantages: A highly energy-efficient system, eco environmental nature, low cost, and versatility. Vegetative and especially spore forming bacteria exhibit strong resistance to external factors such as environmental stress, chemicals, and thermal inactivation due to their intrinsic resistance, outer layers, and low water content. These characteristics make spores more difficult to kill than vegetative forms. In this study, the effect of atmospheric non-thermal plasma application on Gram positive Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 39327 and Clostridium sporogenes ATCC 3584 spores, which are among the important food pathogens, was compared in different packaging materials. Within the scope of the study, microbial inactivation at different exposure times and application types (wet or dry application) was investigated using dielectric barrier discharge (DBD) plasma and corona discharge plasma devices. Low-density polyethylene (LDPE), biaxially oriented polypropylene (bOPP), and polyethylene terephthalate (PET) were used as packaging materials. Each film (3×3 cm2) was initially treated with DBD with an electrode gap set to 3 mm for 0.4 seconds. The plasma power was high, with 100% voltage and minimum frequency set for all treatments. The test microorganisms were inoculated into the center of the 3x3cm2 DBD-treated film surface before corona NTP treatment. The corona NTP power was set at 25-27% voltage, low power, and minimum frequency. The inoculated films were exposed to cold plasma (with the setting of 1.5 cm electrode gap, ~17 kHz wet or dry application) at different application periods depending on microorganism. Exposure periods for S. aureus and P. aeruginosa were 60, 120, and 180s and for C. sporogenes was 0, 360, 540, and 720s. The results showed that non-thermal plasma had an antimicrobial effect for all microorganisms, and wet application, by adding 10 µL of sterile distilled water before exposure to plasma, enhanced the microbial inactivation effect. There was also a direct relationship between exposure time and microbial inhibition. A significant antimicrobial effect was observed only after longer exposures. Considering the results of dry corona plasma application of all microorganisms, the highest D-value belongs to C. sporogenes inoculated on the PET film surface with 94.81±31.56 min. In addition, it was statistically observed that C. sporogenes was the most resistant bacteria to corona treatment for all films (p <0.05). For S. aureus, after dry and wet corona plasma application, all films showed statistical similarity and wet corona application was more effective than dry application (p<0.05). Dry corona application were not performed for P. aeruginosa due to the lack of vitality on the surface after drying. Among vegetative bacteria, the Gram-negative bacterium P. aeruginosa showed higher microbial inactivation than the Gram-positive S. aureus. The most effective result for P. aeruginosa after wet corona plasma application is 1.99±0.03 min and 1.96±0.02 min, with no statistical difference between LDPE and PET films (p>0.05). The results obtained in this study have provided a new perspective on the surface sterilization of packaging materials used in the food industry with cold plasma application of DBD or corona discharge non-thermal plasma systems. In addition, compared to the currently used packaging surface sterilization methods, its disadvantages have been reduced, and an environmentally friendly, affordable system has emerged without the need for complex high/low-pressure systems or gas systems. Thanks to the atmospheric cold plasma devices used in the study, the effective and innovative plasma system design can be easily integrated into the industry without damaging the film surface by using O2 in the atmosphere without needing a pressurized environment or additional gas systems.
Effects of cold plasma treatment on the quality andantioxidant properties of mixed fruit juice

(Graduate School, 2023) Şahinoğlu, Ecre ; Gözükırmızı Kırkın, Celale ; 814088 ; Food Engineering Programme

In recent years, non-thermal processes have been used for food sterilization instead of thermal treatment methods. Cold plasma is an example of these new technologies. The fourth state of matter is called as plasma. Main plasma types are classified as cold and termal plasma. Cold plasma refers to plasma with a gas temperature below 1000 K. In this type of plasma, molecules, ions, and electrons are not thermodynamically in equilibrium due to its low temperature. The electron temperature ranges from 10^4-10^5K, while the ion temperature is approximately 25 °C. Cold plasma is applied at low pressure and low energy levels. On the other hand, thermal plasma refers to plasma with a gas temperature above 1000 K. It is used for the modification of heat-resistant inorganic materials due to its ability to reach high energy levels. Cold plasma can be applied at low energy levels, hence it is suitable for use in food applications. Food sterilization, packaging sterilization, surface disinfection of food, and enhancing seed germination speed obtained by plasma in different studies. The sterilization mechanism is achieved through reactive species released by plasma. It relies on the interaction between reactive components in the gas phase and microorganisms, leading to the disruption of their structure. Plasma is a mixture containing gases such as oxygen and nitrogen. When these gases are excited by plasma, reactive compounds generated such as free radicals and ions. These compounds can attach to the surfaces of microorganisms or react with their surroundings, causing damage to the cellular structure of microorganisms. Another inhibition mechanism is the destruction of microorganisms' genetic material by UV radiation and atomic erosion at the atomic level. This method can effectively play a role for inhibition of both vegetative cells and bacterial spores at ambient temperature and in a short time. Additionally, reactive compounds created by cold plasma can interact with microorganisms on the surface of food, leading to their inactivation. These interactions can disrupt the cellular membranes of microorganisms, denature their proteins, or render their enzymes inactive, as a result of this reactions product staling time can be longer. Various mechanisms are being used to generate cold plasma. One of the most commonly used mechanisms is Dielectric Barrier Discharge (DBD) which is having a dielectric material placed between two electrodes and applying an alternating electric voltage to these electrodes. The dielectric material interacts with the electric field, leading to the formation of plasma. Another system is microwave plasma, which generates plasma using microwave energy. Microwave energy is delivered to the plasma chamber through a resonator or waveguide. Microwave radiation stimulates gas molecules, resulting in plasma formation. Corona discharge is a mechanism in which an electrical discharge occurs between a high-voltage electrode and the ground, ionizing the electrical gap to create plasma. The electric field on the electrode ionizes the gas molecules, leading to plasma formation. High-frequency plasma involves applying high-frequency electric current between electrodes to trigger plasma formation. Radio frequency plasma is another mechanism that utilizes radio frequency energy to create plasma. A radio frequency electric voltage is applied between electrodes, resulting in plasma formation. In this study, DBD mechanism was used and plasma treatment was carried out using a pulsed DC power source at 40 kV (56 Hz, 10 mA) for 0 min (control) , 10 min, or 20 min. Its effects on mixed fruit juice was compared to pasteurized samples to observe differences in the quality properties of juice. The outputs of study submitted that cold plasma treatment has positive effects on the quality and antioxidant peculiarities of fruit juice. The color change in samples operated with plasma was barely noticeable compared to pasteurized samples. However, there was an impressive difference in color between pasteurized juice and the control sample. The Brix° values remained unchanged as 10.5 in both processes. The total acidity significantly decreased in the pasteurized sample but no significant difference was observed between the plasma-handled samples and the control sample (p>0.05).The total viable count was found to be extremely low (Log <2), indicating effective microbial reduction. Despite the inhibition observed, there was no significant decrease in mold and yeast counts during the plasma application (p>0.05). Nonetheless, a decrease in mold and yeast count was observed in the pasteurized sample compared to the control sample (p<0.05). It was concluded that cold plasma did not have an effect on the microbial count, but this can be improved by optimizing process parameters. The activity of antioxidant was evaluated using three different methods. No difference was observed according to the DPPH assay. The ABTS value decreased after the cold plasma treatment. The highest CUPRAC value was recorded after the 20 min treatment. The DPPH and CUPRAC values declined in the pasteurized samples compared to the cold plasma samples, but no significant difference was observed in the DPPH assay (p>0.05). The total phenolic content showed a slight decrease in the pasteurized sample compared to the cold plasma samples, but this distinction was not statistically important (p>0.05). The color properties of the fruit juice were recorded after both processes. In the pasteurized samples, a significant color change was observed compared to the plasma- samples. The total color difference was recorded as 20.86±1, while the ratio in the plasma-treated sample was 2.86±2.3, indicating that this color difference was too small to be noticeable. In conclusion, cold plasma process preserved the appearance, taste, and antioxidant properties of fruit juice compared to pasteurization. By optimizing process parameters for microbial inhibition, this method can be applied to fruit juice production.
Effects of dielectric barrier discharge cold plasma on the quality of dandelion root infusions

(Graduate School, 2023-06-20) Elçik, Berfin Ada ; Gözükırmızı Kırkın, Celale ; 506201526 ; Food Engineering

Since ancient times, humans have sought solutions to their illnesses from plants. In the past 30 years, consumers' dietary preferences and interest in healthy nutrition have significantly changed, leading to increased research on medicinal plants. The health effect of medicinal plants primarily stems from their rich sources of bioactive compounds and antioxidants. Taraxacum officinale, commonly known as dandelion, stands out due to its bioactive properties due to its bioactive compounds. The root of Taraxacum officinale, which often emerges as waste in the food industry, is an intriguing plant root due to its bioactive compounds. The compounds found in the root may exhibit antioxidant effects and help prevent cellular damage caused by oxidative stress. Before food products are commercialized, they need to undergo processing to minimize health risks, provide convenience and usability, enable long shelf life, reduce food waste, and offer variety. Traditional food processing methods that have been widely used since ancient times and they are temperature-dependent. However, heat-dependent conventional processing techniques can result in nutrient losses or physical demage in food. Cold plasma, charged and reactive gas molecules and these molecules inactivate harmful microorganisms which present in foods and food packaging materials, is a promising green and appropriate technique for heat-sensitive foods. This method can help preserv the bioactive compounds in foods. Currently, cold plasma (CP) technology is used for various purposes, including extraction of volatile oils, promotion of seed germination, modification of surface structures, inactivation of enzymes and microorganisms, and degradation of pesticides. Antioxidants are bioactive compounds that naturally found in foods and they can reduce the risk of various diseases. However, these most of the bioactive compounds are recognised as sensitive to thermal processing. DBD cold plasma processing will enable food processing without the loss of antioxidant capacity in food due to its operation at low temperatures. Understanding the interaction between plasma types and bioactive compounds is crucial to prevent nutrient degradation and other undesirable effects. Cold plasma is a form of plasma consisting of high-energy ions, electrons, free radicals, and other reactive molecules. This plasma can interact with bioactive compounds and alter their properties. While there are many studies in the literature on the antimicrobial effects of DBDCP application, research on improving the process efficiency is limited. This study focus to analyze the effects of subjecting dandelion roots to cold plasma treatment during tea brewing, specifically focusing on the changes of the exposure time on the antioxidant activity, color concentration and total phenolic content. For this study, dried dandelion roots were obtained from a local vendor in Izmir and ground using a coffee grinder, and samples to be used in the study were separated through the sieves of 212 micrometers and 450 micrometers and samples called ground. Subsequently, a dielectric barrier discharge cold plasma (DBDCP) was generated using a pulsed direct current power supply (400W, 40 kV , 56 kHz, and 1 mA). The process was conducted as follows: Both ground and unground samples underwent treatment with 40 kV DBDCP for 10 min and 20 min. Untreated samples were used as the control. All treatments were repeated three times. The electrode gap was set at 1.1 cm. The samples subjected to cold plasma were prepared for the brewing process. The prepared samples were brewed at 95±2 0C for 4 minutes. During the brewing process, 2 grams of sample and 200 milliliters of distilled water were used. For grounded samples, 2 grams of ground dandelion root was added to 200 mL of distilled water at 95±2 0C and allowed to steep for 4 minutes, followed by centrifugation through 4000 rpm, 5 minutes at a controlled temperature of 4°C. For unground samples: 2 grams of dandelion root was immersed in 200 mL of distilled water at 95±2 0C using a metal sieve and allowed to steep for 4 minutes. After the infusion process, the metal sieves were removed from the water and the samples were cooled to room temperature. Subsequently, the necessary analyses were performed. The first analysis conducted was color measurement to evaluate the brewing efficiency. Color intensity during brewing has positive effects on consumers. In the color measurement results, a statistically difference (p<0.05) was observed only for the b* value. The b* value represents the yellowness. The brewed dandelion roots transitioned from transparent to yellow throughout the brewing process. Thus, it is possible to say that DBDCP can increase the color intensity. Total phenolic content (TPC) analysis is a commonly used method to calculate the amount of phenolic compounds present in a products, reflecting their antioxidant capacity. In the analysis of samples ground into powder, a significant increase was observed only in the 20 min-treated samples (p<0.05). In unground samples, a decrease at the 10th minute and a slight increase at the 20th minute were observed. The observed increase in antioxidants after processing in ground samples can be attributed to an increase in surface area and enhanced interaction with reactive compounds. This phenomenon may be linked to the increased ability of antioxidants to neutralize free radicals through their interaction. However, in unground samples, a decrease followed by a slight increase through processing time can be observed. This may be due to the initial decrease in the amount of antioxidants on the outer surfaces of larger particles. With the application of DBDCP for a longer time, bioactive compounds in the cell can be reached by breaking down the cell wall in the samples. Accordingly, the total phenolic content of the all samples were as followed: 0.122 mg GAE/mL for ground samples without cold plasma treatment, 0.128 mg GAE/mL for ground samples which implied cold plasma for 10 min, 0.140 mg GAE/mL for ground samples treated with cold plasma for 20 min, 0.045 mg GAE/mL for unground samples without cold plasma treatment, 0.027 mg GAE/mL for unground samples treated with cold plasma for 10 min, and 0.036 mg GAE/mL for unground 20 min cold plasma treated samples for. 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a free radical compound that undergoes a change in color when interacts with substances exhibiting antioxidant properties. In the analysis of samples ground into powder, a significant DPPH radical scavenging activity increase was observed only in the 20 min-treated samples compared to the control (p<0.05), while no significant difference was observed in unground samples (p>0.05). Accordingly, the DPPH radical scavenging activity of the samples were as follows: 0.668 mg TE/mLfor ground samples without cold plasma treatment, 0.695 mg TE/mL for ground samples applied with cold plasma for 10 min, 0.774 mg TE/mL for ground samples applied with cold plasma for 20 min, 0.290 mg TE/mL for unground samples without cold plasma treatment, 0.218 mg TE/mL for10 min cold plasma applied ungrounded samples, and 0.241 mg TE/mL for unground samples which treated with cold plasma for 20 minutes. 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay is a commonly used method for evaluating the antioxidant activity. Accordingly, the ABTS radical scavenging activity of the samples were as follows: 0.119 mg TE/mL for ground samples without cold plasma treatment, 0.124 mg TE/mL for ground samples that applied cold plasma for 10 min, 0.135 mg TE/mL for ground samples treated with cold plasma for 20 minutes, 0.049 mg TE/mL for unground samples without cold plasma treatment, 0.036 mg TE/mL for unground samples which cold plasma applied for 10 minutes, and 0.044 mg TE/mL for unground samples which cold plasma applied for 20 minutes Ferric reducing antioxidant power (FRAP) assay is a rapid evaluation of antioxidant activity method. Accordingly, the antioxidant activity as assessed by FRAP assay were as follows: 0.358 mg TE/mL for ground samples without cold plasma treatment, 0.374 mg TE/mL for ground samples cold plasma applied for 10 minutes, 0.387 mg TE/mL for ground samples cold plasma applied for 20 minutes, 0.144 mg TE/mL for unground samples without cold plasma treatment, 0.090 mg TE/mL for unground samples cold plasma treated for 10 minutes, and 0.118 mg TE/mL for unground samples cold plasma treated for 20 minutes. The sensory analysis was conducted by a panel of 12 individuals from the Department of Food Engineering at ITU. The samples were placed in screw-capped test tubes, labeled with randomLy assigned three-digit numbers. Subsequently, the samples were presented to the panelists and evaluated using a 7-point hedonic scale. The scale was used to assess odor intensity, color intensity, clarity, and overall impression. All measurements were reported as means and standard deviations. Minitab software was used for statistical analysis, applying analysis of variance (ANOVA) and Tukey's test for comparisons. No significant differences were observed among any of the samples in the sensory analysis (p>0.05). This study investigates the effect of DBDCP treatment on the process efficiency and antioxidant activity during the brewing process of Taraxacum officinale roots as tea. The study shows that cold plasma treatment can enhance the process efficiency by preserving the antioxidant activity of Taraxacum officinale roots. It can be said that dielectric barrier discharge cold plasma treatment can be utilized in the brewing process of Taraxacum Officinale roots as tea, leading to an improvement in the process efficiency with the preservation or even enhancement of antioxidant activity.
Effects of novel food processing techniques on bioaccessibility and transepithelial transport of cranberrybush polyphenols

(Graduate School, 2021-08-06) Özkan, Gülay ; Çapanoğlu Güven, Esra ; 506142507 ; Food Engineering

Phenolic compounds, which are present in a wide variety of foods such as fruits, vegetables, flowers and leaf of plants, exhibit a variety of beneficial effects including antimicrobial, antioxidant, antidiabetic, diuretic, hypoglycemic, cough reliever, antiinflammatory and antiviral activities as well as prevention of cardiovascular, pancreas, liver and kidney diseases. However, most of the polyphenols have poor water solubility, chemical instability in gastrointestinal tract and, thus, a reduced bioavailability. Therefore, a wide variety of attempts have been investigated to improve the solubility, stability, bioaccessibility and bioavailability of phenolic compounds. Considering the above, a research framework to study the effects of novel processing techniques on the antioxidant capacity, bioaccessibility and bioavailability of cranberrybush polyphenols has been developed. The objectives of this Ph.D. thesis were (i) to determine the effects of novel non-thermal food processing on cranberrybush polyphenols and vitamin C; (ii) to investigate the effects of non-thermal food processing and food matrix on bioaccessibility and transepithelial transportation of bioactive compounds, in particular chlorogenic acid, from cranberrybush (Viburnum opulus) using combined in vitro gastrointestinal digestion/Caco-2 cell culture model; (iii) to obtain an effective Supercritical Anti-Solvent (SAS) coprecipitation of quercetin or rutin with polyvinylpyrrolidone (PVP), enhancing the dissolution rate, and, therefore, improving the bioavailability of these natural antioxidant compounds; (iv) to determine the effects of SAS processing and food models on the antioxidant capacity, bioaccessibility and transport dynamics of flavonol-loaded microparticles by using combined in vitro gastrointestinal digestion/Caco-2 cell culture model. To achieve these goals, four different experiments (Chapters 3-6) were conducted. Firstly, effects of high pressure processing (HPP) and pulsed electric field (PEF) treatments on physicochemical properties, bioactive compounds, antioxidant capacities and polyphenol oxidase activities of cranberrybush purée samples were evaluated (Chapter 3). Following that, non-thermal treated cranberrybush purée samples as well as cranberrybush juice/water, bovine or almond milk blends were subjected to combined in vitro gastrointestinal digestion/Caco-2 cell culture (Chapter 4). In line with the outcomes of previous chapter, in order to increase the bioavailability of some phenolic compounds that could not be absorbed across the gut epithelium after transport experiments with cranberrybush samples, the micronization of two flavonoids, quercetin and rutin, and their coprecipitation with PVP were studied by using SAS processing to increase their solubility and enhance their stability during gastrointestinal tract (Chapter 5). Finally, SAS-processed flavonoids in different simulated food models were exposed to combined in vitro gastrointestinal digestion/Caco-2 cell culture in order to investigate their transport dynamics (Chapter 6). In Chapter 1, research framework and objectives of this Ph.D. thesis are introduced. Following that, in Chapter 2, comprehensive reviews on the antioxidant properties, bioaccessibility and bioavailability of polyphenols are presented, with a specific focus on the application of novel processing techniques. Initially, a critical evaluation of the effects of novel non-thermal food processing technologies on the beverage antioxidants have been provided. Then, the studies about microencapsulation methods for food antioxidants regarding principles, advantages, drawbacks and applications have been reviewed. Afterwards, effects of encapsulation on the bioaccessibility and bioavailability of phenolic compounds were discussed. Lastly, in vitro and in vivo approaches on interactions of phenolics with food matrix were described. In Chapter 3, the effects of high pressure processing (HPP; 200-600 MPa for 5 or 15 min) and pulsed electric field treatment (PEF; 3 kV/cm, 5-15 kJ/kg) on physicochemical properties (conductivity, pH and total soluble solids content), bioactive compounds (vitamin C, total phenolic, total flavonoid, total anthocyanin and chlorogenic acid contents), antioxidant capacities (DPPH and CUPRAC assays) and polyphenol oxidase activity of cranberrybush purée samples were evaluated. Results showed that conductivity increased significantly after PEF (15 kJ/kg) treatment. PEF and HPP treatments resulted with a better retention of bioactive compounds (increase in the total phenolic content in the range of ~4 – 11% and ~10 – 14% and total flavonoid content in the range of ~1 – 5% and ~6 – 8% after HPP and PEF, respectively) and antioxidant capacity compared to untreated sample. HPP reduced residual enzyme activity of PPO comparatively better than PEF. Besides, cranberrybush polyphenols were identified along with their detected accurate mass, molecular formula, error in ppm (between the mass found and the accurate mass < 10 ppm) of each phytochemical, as well as the MS/MS fragment ions. UPLC–QTOF–MS/MS analysis of cranberrybush led to the identification of flavan-3-ols (catechin, epicatechin, epi(catechin) hexoside), proanthocyanidins (procyanidin dimer, procyanidin trimer, procyanidin dimer monoglycoside), flavonols (quercetin, quercetin-deoxyhexose, quercetin-3-O-glucoside, quercetin pentoside hexoside, rutin, isorhamnetin-3-O-rutinoside), flavone (diosmetin-rhamnosylglucoside), phenolic acids (caffeic acid, chlorogenic acid, coumaric acid, p-coumaroyl-quinic acid) as well as anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside and cyanidin-3-xylosyl-rutinoside). In conclusion, high retention of bioactive compounds was achieved, with a potential extraction of vitamin C, phenolics, flavonoids and anthocyanins in cranberrybush purées after HPP and PEF treatments at selected processing intensities. In Chapter 4, effects of food matrix and non-thermal food processing on bioaccessibility and transport dynamics of cranberrybush phenolics, in particular chlorogenic acid, in a combined in vitro gastrointestinal digestion/Caco-2 cell culture model were studied. Results showed that PEF treatment at 15 kJ/kg specific energy input resulted in a higher recovery of total flavonoid content (TFC; increase of 3.9% ± 1.1%, p < 0.0001), chlorogenic acid content (increase of 29.9% ± 5.9%, p < 0.001) and antioxidant capacity after gastrointestinal digestion. The present study also demonstrates that untreated and treated samples display comparable transport across the epithelial cell layer. Besides, addition of milk matrix have a positive effect on the stability and transportation of chlorogenic acid. JM increased the transport efficiency of chlorogenic acid by 3.5% ± 0.8% (p < 0.0001), while JA increased the transport of chlorogenic acid by 3.3% ± 0.5% (p < 0.001) in comparison with JW blend. The in vitro gastrointestinal digestion/Caco-2 cell culture method applied in this chapter was used in the succeeding chapter (Chapter 6). In Chapter 5, micronization of two flavonoids, quercetin and rutin, and their coprecipitation with polyvinylpyrrolidone were studied by using the SAS process. In particular, optimum conditions in terms of operating pressure, type of the solvent, total solute concentration and polymer/active ratio for the formation of spherical composite microparticles were determined. Morphology, mean size and size distribution of the particles were analyzed and discussed. The effectiveness of the process was also verified through entrapment efficiency and dissolution tests. Overall, amorphous microparticles were produced with total solute concentrations greater than 20 mg/mL. Furthermore, release studies confirmed the improvement of the flavonoids dissolution rates: 10 and 3.19 times faster dissolution rates were achieved with PVP/quercetin and PVP/rutin microparticles rather than those of unprocessed quercetin and rutin, respectively. Besides, the high entrapment efficiencies, up to 99.8%, were achieved for quercetin and rutin coprecipitates by using DMSO, which was the solvent chosen to coprecipitate the flavonoid compounds with PVP by the SAS process. Consequently, the characteristics of the powders could allow to use of these quercetin and rutin loaded microparticles in pharmaceutical and nutraceutical applications due to their high antioxidant and anticancer benefits for, in which the flavonoid compounds have high stability and bioavailability. In Chapter 6, effects of SAS processing on bioaccessibility and transepithelial transportation of quercetin and rutin were investigated by using a recognized combined gastrointestinal digestion/cell-based assay. Moreover, aqueous hydrophilic and acidic conditions were simulated to analyze food-related factors that could have an impact on the transport of these compounds across the gut epithelium. SAS processing improved the recovery of the quercetin (94 and 13 times in hydrophilic and acidic conditions, respectively) and rutin (7 and 2 times in hydrophilic and acidic conditions, respectively) after in vitro digestion. Besides, transepithelial transportation of PVP/quercetin and PVP/rutin microparticles were found to be much higher rather than unprocessed quercetin and rutin. Finally, in Chapter 7, based on the outcomes of the previous chapters, the general discussions and conclusions on the antioxidant properties, bioaccessibility and bioavailability of polyphenols were presented. The status and main outcomes of this thesis were discussed under the headings of fate of the polyphenols after application of novel non-thermal food processing techniques, effects of encapsulation on the food phenolics and interactions of phenolics and food matrix. During the discussion on the effects of encapsulation on the food phenolics, important factors to be considered during encapsulation, advantages and drawbacks of these techniques, their impacts on the antioxidant properties, bioaccessibility and bioavailability of phenolic substances were discussed. Besides, while referring to the interactions with food matrix, special attention has been paid to comparison of the different in vitro and in vivo digestion models.
Emerging approaches for non-thermal modification of proteins isolated from de-oiled sunflower cake

(Graduate School, 2021-09-10) Gültekin Subaşı, Büşra ; Çağanoğlu Güven, Esra ; 506172506 ; Food Engineering

When considered their biological accessibilities, animal-based proteins are known to be closer to the concept of "complete protein" rather than that of plant-based proteins with their higher bioavailabilities. However, increment of world population, damaging of natural sources due to false agricultural policies and climate crisis, high carbon release ratios during their production, high potential for extreme consumption and pollution of water sources, animal welfare issues, religion/ethnic concerns, expansion of diet styles and philosophies like veganism/vegetarianism are all some of the major reasons of community, industry, and food scientists' increased attention about sustainable plant-based proteins in order to replace animal proteins, day by day. Along with their nutritional importance, due to owning the techno-functional properties such as foaming, film/gel-forming, and emulsification, proteins had extensive application areas in the food industry. Recent studies concerning the sustainable plant-based protein sources mainly focused on protein extraction or recovery that have functional properties from plant wastes and/or by-products. Discovery of novel plant proteins with high or improvable techno-functionalities is of extreme importance due to being used as the replacers of animal-based proteins, urgently. For this purpose, sunflower is one of the most studied plants as a protein source coming after the soy, which is the heading plant for this area in the world. Due to having a high protein content, including no allergenic or toxicologic concerns enable the sunflower press cake, which is the oil extraction by-product, more attractive for the mentioned purposes. Under the lights of these explanations, research was planned to frame the characterization and investigating the techno-functional properties of proteins that will be extracted from industrially de-oiled sunflower cake. The objectives of this Ph.D. thesis was (i) isolation and characterization of proteins from de-oiled sunflower cake; (ii) developing a novel non-thermal treatment approach aiming to change the structural properties of protein isolate; (iii) proposing a novel non-thermal treatment perspective using a well-known thermal method aiming to change the structural properties with the purpose of improving its functionality (iv) and investigating the effect of this proposed method on sunflower protein emulsification property in detail. Four different experimental studies (Chapters 3-6) were conducted in order to fulfill the declared purposes. Initially, protein isolates were extracted from de-oiled sunflower cake, characterized and its functional properties were evaluated from a wide-angle perspective (Chapter 3). Then the protein was exposed to a developed nonthermal "moderate electric field treatment" against a reference protein and their structural differences were assessed (Chapter 4). Based on obtained data from the previous chapter, another novel approach, "non-thermal electromagnetic field treatment" was proposed and applied on dry powder protein thereafter the structural alterations of protein were discussed (Chapter 5). Emulsification properties of proteins that were treated with non-thermal electromagnetic field were examined extensively (Chapter 6). In Chapter 1, the main goals and the scope of this present Ph.D. thesis are defined. Right after in Chapter 2, a review study that comprehensively covering the techno-functional properties and potential modification methods of sunflower protein are presented. Initially, compositions of studied sunflower proteins with their quantitative content ranges were reported. Following that, the ways of how de-oiled press cake and isolated sunflower protein were applied in real food systems have been reviewed. Afterwards, the varying methods for protein extraction, isolation of phenolic compounds, and recovery for removed phenolics were assessed. As the last but is the focal point of this review was examining all defined and studied functional properties of sunflower protein up to date, and the modification methods that were used to improve them. In Chapter 3, both types of protein isolated were extracted from sunflower cake; as it is in natural form with phenolic compounds and as de-phenolized isolates. Natural phenolic compounds (dominantly chlorogenic and caffeic acids) that exist in the press cake make complexes with protein molecules and are isolated as adjoints to the structure. In this part, the effect of these natural phenolic compounds on protein content, color, amino acid and mineral compositions, protein surface structure, protein secondary structure, thermal properties, ζ-potential, foaming and the viscoelastic properties at the air/water interface were investigated. According to the proximate composition analysis, it was observed that the moisture and the crude protein ratios increased around 59 and 9%, respectively while the ash content decreased 53% when the phenolic compounds were achieved to be removed at 98%. The color of dephenolized protein was visibly changed from dark green to light brown, the protein surface was observed as roughened and porous rather than natural protein isolate. Isoelectric points were calculated as pH. 4.37 and 4.82 for natural and de-phenolized sunflower proteins, respectively. After the removal of phenolic compounds, significant decreases for all minerals were observed except for Se and Sr. No significant changes were obtained for protein secondary structure and thermal properties however, its hydrophobicity increased when de-phenolized. The most substantial differences were observed for foam stability and interfacial properties of de-phenolized protein at the air/water interface and it was reported that functional properties significantly improved after removal of phenolic compounds. Based on the results of this study, de-phenolized sunflower protein isolate was used as the only material for the following studies (Chapters 4-6). During industrial oil processing, using high treatment temperatures for high production efficiencies coupled with applied mechanical forces induce the globular sunflower protein, helianthinin (11S:2S with a ratio of 7:3) to build more compact globules and gain a kind of heat resistance. According to the literature, in order to unfold this "already denaturized" sunflower protein to improve its functional properties, a heat treatment over 90 °C should be applied. Due to the fact that, within the scope of this Ph.D. thesis, a novel "non-thermal moderate electric field treatment" was developed and applied on sunflower proteins. In Chapter 4, sunflower protein was exposed to an electric field with varying voltages for different times below the 45 °C. Since it was the first application of this proposed technique, sodium caseinate, as a widely used and known reference protein, was exposed to the same processing conditions aiming a better understanding for the effects of non-thermal moderate electric field. Proteins were examined in terms of both structural and functional properties after processing. Smaller average particle size, lower interfacial tension at the air/water interface as well as changed secondary and tertiary structures besides different thermal properties were observed. Sunflower protein was successfully unfolded with the proposed method, non-thermally. In Chapter 5, due to the very same reasons and motivations about the unfolding of heat-resistant sunflower protein, another novel, and non-thermal approach was proposed and applied. Microwave treatment as one of the most widely known electromagnetic radiation applications is a thermal processing method however it also has a simultaneous non-thermal effect on samples during processing, whose exact mechanism is still unclear. Due to allowing for processing the sample on "dry basis", it was assumed that the polar amino acids in the protein structure will absorb the electromagnetic energy, enables rotating around the central carbon atom and/or forming free radicals and consequently, inducing the structural changes such as partial unfolding and/or refolding. After processing it was observed that, the polar amino acid ratio of processed protein was changed, particle size decreased, protein's secondary and tertiary structures altered, thermal stability decreased and thermogravimetric losses were obtained. This second proposed non-thermal novel treatment succeeded to induce the sunflower protein for partial unfolding. Following the exposure of sunflower proteins to non-thermal electromagnetic field and observing promising structural alterations, a functional property was decided on and examined in detail in Chapter 6, instead of a general overview covering multiple functional properties. Electromagnetic field application increased the protein solubility and surface hydrophobicity besides more homogenous and stable (1.43 fold) emulsions with smaller droplet size were obtained. Similarly, in that of Chapter 4, sodium caseinate was used as a reference protein and exposed the same treatments to compare particularly emulsification properties. Despite the higher surface tensions at the oil/water interface were observed for sunflower protein samples rather than sodium caseinate, more elastic but less stretchable solid-like protein layers were determined at the interface. Consequently, the proposed application fulfilled the aim of altering the structure of sunflower protein and having the potential to improve its functional properties. Finally, in Chapter 7, based on the data obtained from the previous parts a comprehensive discussion and results about the changing the structure of sunflower protein using proposed novel treatment approaches and their potentials to improve protein's techno-functional properties are presented. Advises for future researches are also provided. The results observed from this Ph.D. thesis were examined under the titles of characterization of sunflower protein, the effect of natural phenolic compounds on protein structure and functional properties, the effect of non-thermal moderate electric field application on protein structure, the effect of non-thermal electromagnetic field application on protein structure and functional properties. Foaming and emulsification were chosen as the functional properties to investigate however, possibilities to change the protein structure were predominantly focused on throughout the whole study.
Encapsulation and release of amino acids in double emulsions

(Graduate School, 2021-03-18) Kocaman, Esra ; Van Der Meeren, Paul ; 506162503 ; Food Engineering ; Gıda Mühendisliği

Double emulsions have been studied for many years, given their potential as encapsulation systems. It is also possible to control the release of diverse bioactive components by means of double emulsions. As amino acids might be degraded to some extent due to environmental factors such as pH, temperature, light exposure as well as some reactions (i.e. oxidation, Maillard), their encapsulation may be advantageous to avoid these issues. Besides, encapsulation may enable to release of these compounds in a later stage of the gastro-intestinal tract. The main research question of our research project was to what extent the release of encapsulated components from double emulsions can be controlled by the emulsification method, emulsion composition and environmental factors. Moreover, it was evaluated whether the release kinetics were substantially influenced by the molecular properties of the encapsulated compounds. Hence, this thesis studies the influence of some parameters on double emulsion stability as well as amino acid encapsulation and release in double emulsions. The current study consist of the evaluation of these parameters: solute characteristics (i.e hydrophobicity, molar mass) and concentration, pH of the aqueous phases, hydrophobic and hydrophilic emulsifier, homogenization and thickener. For the investigation of the effect of these parameters, the emulsion droplet size, and the entrapped water volume fraction were evaluated to characterize the double emulsions. Moreover, the release of amino acids was observed during storage using spectrophotometric and Nuclear Magnetic Resonance (NMR) techniques. A modification of the original method was performed to enable the optimum conditions for amino acid quantification (section 4.1). Due to the high background absorbance of the reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) which was the case for many of the measured concentrations, different TNBS concentrations were evaluated in order to determine the optimum concentration. Hence, the solution containing 0.6 mM TNBS was choosen as it demonstrated the lowest absorbance among the studied concentrations as a blank and the TNBS solution reacted with leucine. As the absorbance was not substantially changed after 3 hours, it was used as the reaction time. In section 4.2, the effect of solute characteristics on double emulsion stability and release of encapsulated compounds were presented. Different amino acids (i.e. hydrophilic and hydrophobic) were used to investigate the hydrophobicity effect at different temperatures. Also, di-peptides were used as encapsulated compound in order to evaluate the influence of molecular mass. The results showed that an increase was observed from 50 up to 90 μm in the average droplet size for the samples homogenized with Ultra-turrax at 17500 rpm within the 32 days time frame. The double emulsions at 4 °C indicated a higher increase in average droplet size as compared to 37 °C. To investigate the main instability mechanism in the emulsion, double emulsions were diluted with sodium dodecyl sulfate (SDS) before laser diffraction measurement. The measurement of the droplet size in the presence of SDS showed that flocculation was the main instability mechanism, which caused an increase in droplet size. On the other hand, a constant enclosed water volume fraction was found in double emulsions during 16 days of storage, independent from the temperature and hydrophobicity studied in this thesis. The encapsulation efficiency of amino acids in the inner water droplets was found to be higher than 80% in all cases. From the release results, amino acid hydrophobicity and storage temperature were found to largely influence the release rate of the encapsulated amino acids. The amino acid release rates were fastest at 37 °C, which was the highest temperature examined in this section of the thesis. This can be explained by the higher solubility as well as increased diffusion rate of amino acids in the intermediate phase. Also, an increase was observed in the release rates of amino acids as a result of higher hydrophobicity. The significant effects of hydrophobicity and temperature, as well as the constant enclosed water volume revealed that the release of amino acids from the inner to the outer water phase was mainly governed by a direct diffusion mechanism. As the di-peptides released faster than the amino acids, it follows that the increased solubility overruled the effect from the decreased diffusion coefficient of the dissolved compound in the oil phase. In section 4.3, the influence of solute concentration (i.e. 5, 10, 20 and 40 mM) on the release and double emulsion stability was investigated. The varying concentrations of amino acid did not cause a significant difference in the increase of volume weighed droplet size during 16 days. The entrapped water volume was stable for double emulsions that contained varying solute concentrations except from the double emulsion which contained 40 mM where a decrease was observed through 16 days of storage. This can be a result of the faster diffusion velocity of the amino acid across the oil phase to the external water phase as compared to the diffusion of potassium chloride (KCl) through the oil phase to the internal water phase. Hence, a fraction of the internal phase was expelled to the external water phase to equalize the osmotic pressure which resulted in a decrease in yield of entrapped water volume. Regarding the average residence time (ta) values, the double emulsion that contained the highest solute concentration studied (i.e. 40 mM) in this thesis indicated a faster release as compared to the other samples at 37°C, whereas there was no significant difference among the samples at 4°C. The pH effect of the aqueous phases on the release of amino acids and di-peptides was evaluated in section 4.4. Regarding the average droplet size, there was no significant difference between samples as a function of pH of the aqueous phases. Considering the release, the transport of the amino acids and di-peptides was faster at neutral pH as compared to acidic and basic pH values, which was thought to be due to the increased solute solubility in the oil phase for the zwitterionic (rather than ionic) form of the more hydrophobic molecules at neutral pH. The oil type effect on amino acid release and double emulsion stability was demonstrated in section 4.5 comparing long chain and middle chain triglycerides. The average droplet size of the long chain triglyceride (LCT) containing double emulsions were larger than of the medium chain triglyceride (MCT) containing samples. This can be due to the stronger aggregation of LCT containing samples as a consequence of the higher viscosity of the LCT oil. From the release results, much faster transport of L-leucine was observed through MCT oil as compared to LCT oil due to its higher solubility. Also, the lower viscosity of MCT oil gives rise to a higher diffusivity of dissolved compounds, which may also fasten molecular transport. In section 4.6, the influence of the hydrophobic emulsifier concentration (from 1 to 5%) on the double emulsion stability and release of entrapped amino acids was demonstrated. The entrapped water volume fraction of the polyglycerol polyricinoleate (PGPR) stabilized samples remained around 100% during 32 days of storage, except from the one with only 1% PGPR which had a decreasing yield due to insufficient stabilisation of the internal water droplets. It follows that the use of higher concentrations of PGPR enabled the entrapped water volume to remain constant, whereas a PGPR concentration below the critical micelle concentration (CMC) caused a water flux from the internal to the external phase. The average residence time (ta) of enclosed L-leucine among the PGPR stabilized double emulsions was lowest at the highest PGPR concentration, which indicates the faster release of L-leucine in the presence of an excess of reverse PGPR micelles in the oil phase. The effect of partial replacement of PGPR by native and phosphatidylcholine (PC) depleted lecithin on double emulsion stability and amino acid release was shown in section 4.7. Although a droplet size increase was observed in the PGPR-stabilised double emulsions during storage, the use of 5% of a PGPR-native lecithin (1/1) mixture resulted in a constant droplet size during storage. The used PGPR and PC-depleted lecithin concentration influenced the droplet size of the double emulsions. The lowest droplet size was about 30 µm just after preparation and during storage in double emulsions containing 5% PC-depleted lecithin. This indicates that partial replacement of PGPR can be beneficial in terms of stability of the double emulsion droplet size. Considering the entrapped water volume, the inclusion of PC-depleted lecithin could not facilitate to overcome the instability at too low (i.e. less than 2% in this case) PGPR concentration. In fact, lecithin addition had a negative impact on the etrapped water volume fraction. The average residence time ta, on the other hand, was much lower in PC-depleted lecithin-containing double emulsions as compared to the emulsions with only PGPR. The effect of hydrophilic emulsifier concentration on amino acid release and double emulsion stability was investigated (section 4.8). It was found that the use of a higher Tween 80 concentration facilitated a less pronounced increase in average droplet size during storage. The use of less than 2% Tween 80 concentration seemed to be insufficient to cover the interface between oil and outer aqueous phase. A constant entrapped water volume fraction was obtained during storage regardless of the Tween 80 concentration. Differences in Tween 80 concentration, varying from 0.5 to 2.0%, did not change the release kinetics to a large extent. In section 4.9, the influence of microfluidization (at 0.75 and 1.00 bar of driving compressed air pressure) and rotor stator homogenization treatment (at 17500, 21500 and 24000 rpm of Ultra-turrax) and the presence of xanthan gum were investigated. Considering the particle size distribution, multimodal and monomodal particle size distributions were observed for microfluidized double emulsions and those prepared by rotor stator homogenization treatment, respectively. The inclusion of xanthan gum decreased the size of the oil droplets, which resulted from the decreased viscosity ratio between the oil and the aqueous phase. Also, an increased homogenization intensity induced a decreased droplet size, resulting from the higher shear stress applied to the fluid. The entrapped water volume fraction was about 90% for all double emulsions prepared with rotor stator homogenization treatment and without xanthan gum. As the cream and serum layers of the double emulsions stabilized with xanthan gum were not separated during 2 hours of analytical centrifugation, the reliable estimation of the enclosed water volume fraction was troublesome. The release rate of L-leucine in double emulsions prepared with rotor stator homogenization treatment was proportional with the homogenization level, which can be explained from the smaller droplet size: a faster release rate was observed at higher homogenization intensity as a result of a smaller droplet size. Xanthan gum addition remarkably increased the release rate of L-leucine, which was thought to be due to the smaller droplet size. Preliminary gastrointestinal tests indicated that double emulsion encapsulation provided a gradual release of amino acids in the gastrointestinal environment (section 4.10). The release of amino acids might be governed by diffusion in the gastric environment, whereas the oil digestion can change this mechanism as well as the relase rate. The smaller droplets obtained after intestinal digestion was likely due to the triglycerides hydrolysis which resulted in the disruption of the oil phase and hence release of encapsulated amino acid. In section 4.11, the release of L-phenylalanine was investigated by means of high resolution NMR diffusometry. As the first and last decay profile of water overlapped, it follows that the enclosed water volume fraction remained constant during incubation (at 30 and 50 °C). Moreover, a slower amino acid diffusion coefficient was obtained in the external water phase as compared to the internal water phase (i.e. before emulsification). This might be due to the presence of xanthan gum in the external (but not in the internal) water phase, which restricts the thermal motion of the amino acids, and hence the diffusion behaviour. The diffusion behaviour of L-phenylalanine in double emulsions exhibited a typical bi-exponential decay, which enabled to discriminate between encapsulated (slowly diffusing due to restriction in a spherical confinement) and released (fast diffusing due to the absence of confinements) amino acid. Whereas the main purpose of the experiment was to enable a more detailed investigation of the influence of the incubation temperature, a clear conclusion was hampered by the extensive release before the start of the NMR experiment. This research enables a better insight to understand the influence of molecular properties and double emulsion composition on the release kinetics. From a practical point of view, our results provide guidance in the design of colloidal systems for the encapsulation and controlled release for nutritional applications. In order to extend this study, the double emulsions containing amino acids can be incorporated in the food matrix or drugs.
Encapsulation of aqueous Hibiscus sabdariffa L. extract in food-grade high internal phase pickering emulsions stabilized by soy protein isolate

(Graduate School, 2023-06-15) Çavdar, Hümeyra ; Güven Çapanoğlu, Esra ; 506211516 ; Food Engineering

Hibiscus Sabdariffa L. is a highly versatile plant that finds applications in various industries due to its abundant nutrients, potent bioactive compounds such as phenolics and anthocyanins, and natural colour pigments. In addition to its use as a colouring agent, its positive health effects, such as antibacterial, antioxidant, anticholesterol and prevention of gastrointestinal problems, spread the consumption of the product for health purposes. However, the fact that bioactive ingredients are easily affected and damaged by many factors limits this product's use in many industries. Therefore, integrating Hibiscus Sabdariffa into food products and various formulations becomes more complex and limits the methods used. Encapsulation, a preferred method for much bioactive protection, appears at this point. Encapsulation comes to fields as a technique that allows us to apply in different areas, such as preserving these components and controlled release and integrating them into product formulations. Emulsification by encapsulation method has gained a solid place in industry and literature. High Internal Phase Emulsions are among the leading emulsions with high volume encapsulation and high stability against external factors. Similarly, double emulsion systems can enhance bioactive components' protection with their nested phase structure. The debate over the many adverse effects of preferred emulsifiers and stabilizers for emulsion stability, such as human health and environmental pollution, has accelerated scientific research into discovering alternative products. Plant proteins have gained tremendous interest in recent years as they have the potential to be an alternative to conventional emulsifiers and stabilizers. The various functional properties and amphiphilic characteristics indicate that these proteins are significantly effective in the stability of the emulsion system. Like many other types of plant proteins, soy protein is a preferred product due to its easy availability and processability. In this study, analyses were conducted to identify and evaluate the encapsulation of aqueous Hibiscus extract in the High Internal Phase Pickering Emulsions (HIPPE) and High Internal Phase Double Emulsions (HIPP-DE) systems stabilized by soy lecithin and soy protein isolate. The stability of the emulsions obtained, characteristics and the effects of in vitro gastrointestinal digestion on the phenolics and anthocyanins in the extract with the presence of soy protein and soy lecithin were investigated. The extraction of phenolics and anthocyanins from powdered Hibiscus calyces was realized by ultrasonic method, and water was used as a solvent. Emulsions with an internal volume of 80% have been created using soy protein isolate (SPI) gel and lecithin. A HIPPE resistant to phase separation could not be obtained using soya protein isolation gel alone, while emulsions (L4 and L6) containing 4% and 6% lecithin and phase separation resistance could be achieved. Combinations of different concentrations of lecithin and soy protein isolated gel stabilized other HIPPEs. After 24 hours of storage, HIPPEs stable against phase separation were obtained by homogenizing HIPP-DEs at a volume of 50% with a 6% SPI gel. When emulsions stored for 24 hours were observed, SPI gels and lecithin at varying concentrations acted as a synergistic mechanism that effectively prevented phase segregation and ensured stability in the emulsion system. All HIPP-DEs also showed superior resistance to phase separation. According to the CI results, an increase in the concentration of soy lecithin from 2% to 4% in HIPPE containing 6% SPI gel resulted, indicating increased stability, leading to a decrease in the CI value on day 1; however, no significant difference in CI was observed between HIPP-DEs. EAI and ESI values show a statistically significant increase when the SPI concentration increases from 2% to 4% in HIPPEs and HIPP-DEs with 4% and 6% lecithin concentrations. Still, surprisingly, no significant increase was observed with further increases in SPI. In contrast, the particle size of HIPPEs with 4% and 6% lecithin concentrations showed a significant decrease as the SPI gel concentration increased from 2% to 6%. In HIPPEs, the zeta potential was negative, and the absolute values increased by increasing the SPI gel concentration from 2% to 4%, while in L4S4, it was observed that the HIPPE had the highest absolute zeta potential (-41.21 ± 1.23 mV). However, an increase in SPI gel concentration from 4% to 6% decreased the absolute zeta potential value. The increase in the concentration of soy protein isolate (SPI) from 2% to 6% resulted in a significant decrease in the PDI values of HIPPE containing 4% and 6% lecithin. The lowest PDI values were obtained in L4S6 and L6S6 HIPPE with an SPI concentration of 6%. The same decline has been observed in HIPP-DEs. Different changes have been observed in the stability and characteristics of emulsions, as well as in the study of bioactive components and their properties. Encapsulation Efficiency (EE) determinations stated that HIPP-DEs produced higher EE than HIPPEs due to high SPI gel concentrations. The total anthocyanin content (TAC), total phenolic content (TPC), DPPH and ABTS of the resulting aqueous Hibiscus extract were analyzed. The TAC (mg Cy-3-GC equivalent/100 g), TPC (mg GAE/100 g), DPPH (mg TE/100g) and ABTS (mg TE/100g) values were found as 31.13±1.23, 2619.01±17.31, 335.12±1.21 and 223.21±2.56 respectively. Significant decreases in TPC in Hibiscus extract were observed with in vitro gastrointestinal digestion. In HIPPEs prepared with a concentration of 4 % lecithin (L4, L4S2, L4 S4, L4S6), the SPI gel concentration increased from 0 % to 4 %, while anthocyanins in the stomach environment showed more excellent stability compared to the intestinal environment. When the TPC of the digested phenolics of Hibiscus extract was compared to those of digestive HIPPEs and HIPP-DEs, higher TPC values were observed in all emulsions. Maximum TPC values in the stomach and intestinal digestion reached 709.82 ±2.06 mg/100g and 1160.71 ±21.01 mg/100g, respectively. In the stomach and intestinal phases of in vitro digestion, the rate of phenolic release has been significantly influenced by the pH of the environment. According to HPLC results, aqueous Hibiscus extract has detected anthocyanins of delphinidin-3-glucoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside. Four phenolic acids have also been detected in the extract and all emulsions, including gallic acid, syringic acid, ferulic acid and chlorogenic acid. After gastric digestion, a decrease in the content of chlorogenic acid, ferulic acid and gallic acid was observed, while syringic acid increased, showing a different tendency. The highest concentrations of each anthocyanins and phenolics concentrations were obtained in L6S6-D. As a result, changes in SPI gel and lecithin concentrations have been effective in many conditions, such as the stability of emulsions, their properties, and the effects of in vitro digestion on anthocyanins and phenolics. In addition, according to the results of encapsulation efficiency, it can be stated that HIPPEs and HIPP-DEs are effective emulsion systems in the encapsulation of Hibiscus extract.
Encapsulation of cumin seed oil in chickpea protein- maltodextrin matrix

(Graduate School, 2022-02-14) Atlı, Onur ; Karaça, Aslı Can ; 506181509 ; Food Engineering

This study aimed to investigate developing a plant-based protein matrix for microencapsulation of cumin oil, by determining optimum pH for solubility, emulsion stability of chickpea protein isolate (CPI) and investigating the effect of the matrix composition on encapsulation effiency and aromatic composition of cumin oil. Solubility, net surface charge, and emulsifying properties (emulsion activity/stability indices, and creaming stability) of CPI at ranging pH 3.0-9.0 were investigated. The highest protein solubility (94.4%) and emulsion activity index (61.8 m2/g), and the highest creaming stability (9.8% separation) was obtained at pH 9.0. CPI stabilized emulsions were optimized based on pH due to the higher emulsion stability index, which is 123.8 min at pH 7.0. CPI concentration of 0.5-2.31% (w/w) and oil concentration of 5-23.11% were adjusted in order to investigate the emulsion stabilization capacity of CPI using response surface methodology. It was observed that obtained CPI has the lowest effect on emulsion formation. Optimum conditions for minimum creaming were identified as: 0.19% CPI concentration and 6.83% oil concentration. CPI was found to be thermally stable with high denaturation temperature (161.4°C) and required 142.2 J/g enthalpy for denaturation. Cumin (Cuminum cyminum L.) seed essential oil was microencapsulated employing the complex matrix formation of CPI and maltodextrin DE 12-16 using spray drying. Effects of cumin oil concentration (10-20%), CPI concentration (1-3%) and maltodextrin (MD) concentration (25-35%) on the physicochemical characteristics and aromatic composition of the microcapsuled cumin oil were investigated. It was observed that, CPI-MD complex matrices had positive effects on microencapsule properties such as relatively lower surface oil, and higher encapsulation efficiency and oil retention, when they were formulated properly. Oil retention of 86.6–96.4%, encapsulation efficiency of 90.9–98.4%, and surface oil of 1.4–7.9% were obtained in samples. Correspondingly, it was observed that MD–CPI interaction was an effective parameter for emulsion stability. Optimum conditions for maximized oil retention (92.9%) and encapsulation efficiency (98.60%) were identified as: CPI concentration of 2.1%, cumin oil concentration of 14.8% and MD concentration of 35%. Moreover, GC-MS analysis of obtained microencapsulates was also carried out to determine the changes in essential oil composition during spray drying. It was observed that cymene, α-pinene, β-pinene, sabinene, terpinene, terpineol, phellandrene, and cumin aldehyde were the major volatile components in cumin oil. It was observed that optimized design not only had the highest encapsulation yield, but also provided better protection against the degradation of volatile composition of cumin oil.
Encapsulation of echium oil and saffron extract in electrospun nanofibers

(Graduate School, 2022-11-15) Najafi, Zahra ; Yeşilçubuk Şahin, Neşe ; Altay, Filiz ; 506152510 ; Food Engineering

In this doctoral thesis, it was aimed to investigate the production of nanofibers containing Echium seed oil and bioactive compounds of saffron using biopolymers, the characterization of nanofibers and the in vitro release and kinetic studies of obtained nanocarriers. In addition, the different applications of nanofibers (carrier system or food coating material) were studied. Bioactive compounds possess many health promoting properties, therefore there is a growing interest in development of functional foods fortified with them. Echium seed oil is an important plant-origin source of long chain polyunsaturated fatty acids (LC-PUFAs), especially stearidonic acid (SDA). The importance of SDA, is due to its function as a precursor in biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and its conversion efficiency to EPA (30 %) is higher than ALA (around 7%). Moreover, they have anti-cancer activity, and they probably reduce coronary diseases and immune disorders. Saffron is also attracting consumers' attention due to including valuable bioactive compounds exert important health-promoting effects. Crocins, picrocrocin, and safranal are the three main bioactive ingredients present in saffron stigmas and they exhibited antioxidant, antitumor and neuroprotective activities. Therefore, in this thesis study, electrospinning as an emerging electrohydrodynamic method, has been applied for encapsulation of PUFAs and saffron extract (SE), in addition the potential of SE as a natural antioxidant to enhance oxidative stability of encapsulated oil in nanofibers was evaluated. First, saffron bioactive compounds were extracted by conventional and novel approaches using different solvents, then the extract with the highest antioxidative activity was freeze-dried and incorporated into several edible oils to retard their lipid oxidation measured by Rancimat test (Chapter 3). Then, in Chapter 4, it was aimed to produce nanofibers from SE and EO by electrospinning using different coating materials. The electrospun Pul-Pec and Pul-PPI-Pec nanofibers (NFs) loaded with SE, SE loaded nanoliposome (SENL) and EO emulsion were produced using water as a solvent. Morphological studies by scanning electron microscopy (SEM) showed that uniform Pul-Pec and Pul-PPI-Pec NFs with average diameters of 112 nm and 115 nm were fabricated, by the addition of EO, the diameters of fibers increased to 163 and 125 nm. Moreover, thicker fibers were formed by incorporation of both bioactive compounds (EO and SENL) into electrospinning blends. SE and EO embedded into the blend NFs had encapsulation efficiencies (EE) greater than 70% and 65%, respectively. The FTIR spectra of all NFs were recorded at various storage days (50°C), and the A 3010 cm-1/A 2925 cm-1 ratio were calculated for each sample. This ratio indicates the unsaturation degree of the encapsulated oil. The values of this ratio which was calculated for samples revealed an upward trend, and the largest values belonged to EO-loaded PPI-Pul-Pec NFs with SELN. Therefore, this encapsulant provided the best protection for EO against oxidation. Beside FTIR method, isothermal differential scanning calorimetry (DSC) method was used to determine the oxidative stability of EO and EO embedded in NF matrix. The onset oxidation times (Ot) were obtained from DSC exotherms of NF samples. Four different temperatures were used to calculate activation energy values (Ea) and to predict the shelf-life of EO loaded NF samples. The DSC outcomes were in consistent with FTIR results. Incorporating SENL in EO loaded Pul-PPI-Pec NFs caused up to a three-fold increase in Ot at 20°C compared to control samples (EO loaded Pul-PPI-Pec NFs without SE). In addition, the greatest Ea (100.8 Kj.mol-1) and longest shelf-life was observed for this sample. The release behavior of both bioactive compounds and the kinetics involved were evaluated by fitting the release profile data to different kinetic models such as Rigter-Peppas, Zero-order, First-order, and Higuchi. The crocin-4 release rate from SELN loaded NF blends (58–62% over 7 hours) was noticeably slower than that of unencapsulated SE (80% over 3 hours). Crocin-4 transfer from unencapsulated SE followed zero-order kinetics, although its release from NF samples followed Ritger-Peppas model involved Fick-diffusion mechanism. EO release from Pul-PPI-Pec NFs governed by a Fickian diffusion mechanism according to the best fitted model (Ritger-Peppas). However, for cross-linked Pul-Pec loaded EO NFs under simulated intestinal fluid, the release mechanism was non-Fickian which governed by combinations of diffusion and erosion. The release rate of EO was slower in cross-linked Pul-Pec NF blend due to their greater resistance against degradation. In Chapter 5, zein nanofibers (ZNs) loaded with SE were produced by electrospinning method, which were subsequently used as a nanocoating material. The influences of concentration and voltage are investigated on the electrospinning process. The zein polymer was prepared in three different concentrations (20, 25 and 30 wt%) through dissolving in ethanol-water (80:20) and then exposed to high voltages (6 and 14 kV). In addition, the solution properties including viscosity, surface tension and electrical conductivity of polymers were determined and correlated with the morphology of resulted fibers. SEM images showed that smooth and bead-free NFs were obtained via electrospinning of zein at 30% w/v concentration, while zein particles and mixtures of nanofibers and beads was generated from zein solutions at 15 and 20 wt% concentrations. Moreover, fibers obtained at applied voltage of 6 kV resulted in narrower fibers. Consequently, zein nanofibers (30 wt%) was selected as a carrier to encapsulate SE (5 and 10 wt% respect to zein weight). The resulted ZNs loaded with SE were characterized in terms of morphology, thermal and molecular properties, encapsulation efficiency and antioxidant activity. Addition of SE (10%) into ZNs caused a significant increase in mean fiber diameter from 369 to 440 nm at 6 kV. The encapsulation efficiency (EE) of SE components within ZNs was assessed by HPLC method. EE of total crocin and picrocrocin, in ZNs loaded with SE (ZNLSE10%), were 64% and 47%, respectively. Picrocrocin and four glycosyl esters of crocetin, namely trans-crocin-4, trans-crocin-3, cis-crocin-3, and cis-crocin-4, were detected in SE by LC-MS. The alteration in the crystal structure of SE was validated by DSC profiles, demonstrated that SE molecules were successfully embedded into the zein proteins. The FTIR spectra of ZNLSE, indicated the disappearance of several peaks because of shifting in signals and in plane-bending of hydroxyl groups, it can be proof for formation of secondary interactions between hydroxyl functional groups of crocins and amino groups (NH2) of zein. The ZNLSE (10 wt%) exhibited the greatest antioxidant activity compared to SE and ZN as controls. In final step, with the aim of exploring the efficiency of ZNLSE on shelf-life and quality of fish fillets, skinless fish fillets were nanocoated with ZNLSE (10%). Deterioration of the fish samples at 2 ± 1 °C during the 8-days-storage period was investigated through several physicochemical tests including volatile basic nitrogen (TVBN), thiobarbituric acid reactive substances (TBARS), peroxide value (PV), free fatty acid (FFA) and pH. The TVBN values of the coated samples were 30% lower than those of the control group on the 8th day of cold storage. Lipid oxidation in coated samples was also retarded according to the results of PV and TBARs analysis. In contrast to coated samples, PV of uncoated samples increased gradually from 1.3 to 4.4 meq O2/kg until the 4th day of storage, and then decreased until 8th day whereas PV of coated samples showed an increasing trend and reached to 3.27 meq O2/kg on 8th day, and their PV were lower than control. The FFA values of control and treated samples slowly increased throughout storage, however the rate of increase for FFA values remained slower than control. It has been concluded that zein based nanofibers loaded with SE have the potential as an active food packaging layer to extend the shelf life of fish fillets. In Chapter 6, the fabrication, characteristics, and release behaviors of SE (10 wt%) loaded zein, Pul-Pec, and Pul-PPI-Pec NFs were investigated. The morphology of three different NFs was investigated by SEM. The resulted NFs were smooth and homogenous without bead structure, and they had fiber diameters ranging from 103 to 115 nm. To observe the interactions of the bioactive compounds in saffron with various polymers as well as changes in the secondary structure of proteins, FTIR tests were also carried out. The in vitro release of crocin from NFs were kinetically studied under gastrointestinal media, with and without the digestive enzymes. Furthermore, in vitro release studies were performed using Franz diffusion cells in PBS solution. The fitting of in vitro release data into Ritger-Peppas model, indicated that crocin transfer followed Fickian diffusion mechanism for Pul-Pec and Pul-PPI-Pec NFs samples and non-Fickian for zein NFs. The release data belongs to in vitro release studies by Franz-diffusion cells best fitted with Ritgar-Peppas and Higuchi models, in addition the crocin release was governed by Fickian controlled diffusion transport. According to the results, it can be concluded that SE-loaded NFs have the potential to be used as a carrier to provide prolonged release of SE and maybe for transdermal applications as a food supplement. In the final part of the study, the general discussions and concluding remarks are given in Chapter 7 along with prospects and challenges.
Encapsulation of oil-based cheese aroma by using spray-drying and efficience of microcapsules in model foods

(Graduate School, 2022) Ertan Tütüncü, Sena ; Özçelik, Beraat ; Özgüven, Mine ; 737832 ; Food Engineering Programme

Since the world has existed, food consumption has been critical for the continuation of life. Appearance, flavor, and taste play an important role in the preference of foods in nature. In modern times, these parameters have been controlled and improved. Fermentation is invented to increase the shelf-life of the food; in addition, new volatile compounds are formed during the fermentation process. Cheese is the most consuming fermented product, and different types of cheese are made by changing fermentation and process parameters. These parameters also affect the odor and taste, providing the uniqueness of cheese. Each volatile compound has a special odor and taste, therefore, containing various volatile compounds allows different cheese varieties to have their unique odor and taste. Various extraction techniques are distillation, static headspace, dynamic headspace, solid-phase microextraction (SPME), stir bar sorption extraction, and vacuum distillation applies for the extraction of the cheese volatile compounds. Depending on the volatile compounds' qualifications such as the sensitivity of heat, light, and temperature; appropriate extractions should be chosen. The encapsulation process prevents flavors from being affected by adverse environmental conditions. Various techniques are used to encapsulate the active material. Selecting the suitable encapsulation method depends on the core material release features, wall material properties, project cost, and aim. Preparation of emulsion is the first and fundamental step of encapsulation. Wall materials that cover the core material may be consists of carbohydrates, protein, or other materials. Starch, gum, and maltodextrin carbohydrate-based derivatives to use in encapsulation; however, for getting more encapsulation efficiency carbohydrate and protein-based materials are preferred together. Both animal and plant-based derivatives are used as protein wall materials. High pressure, micrtechniquesing technique, and ultrasonication are used to homogenize and provide emulsion to good dispersion. The spray drying technique is one of the most common encapsulation techniques in the food industry. The spray drying process is based on the principle of atomizing the material to be dried under high pressure and then drying it in a very short time with hot dry air. In this experiment, commercial cheese aroma is encapsulated by the spray drying technique. Using maltodextrin and protein are used as wall material at different concentrations. 30 % of the emulsion mixture consists of solid ingredients. This solid content is prepared with 16 % aroma and 84 % mixture of maltodextrin with dextrose equivalent 11-16 and whey protein concentrate, 50 %. Protein content in the solid mixtures is 8 % and 4%. These emulsions are kept in a shaking bath at 40 C° for 20 hours at 80 rpm to improve hydration. 16 % aroma of total emulsion is added to both emulsions. After the hydration and adding aroma, the emulsion is mixed first at 450 rpm, for 1 hour; then homogenized by Ultra-turrax at 17000 rpm for 5 minutes. Laboratory scale Buchi B-290 is used at an 8 mL/min feed rate, 180 ± 2 C° inlets, and 80 ± 2 C° outlet temperature. Encapsulation duration keeps 40 minutes. Encapsulated and commercial aroma is added, a total of 1 % flavor, in cracker dough samples that include 60 % flour, 15 % oil, and other ingredients. Then the crackers are cooked in the oven at 220 C°, for 10 minutes. The aroma added cracker is oiled at % 10 without aroma. Cracker dough without aroma is prepared and cooked; however, the encapsulated aroma and liquid aroma are added in the oil process at 0.2 %. GC & MS analysis results are compared with the results of commercial liquid flavoring as a reference. Encapsulated A and B aroma volatile compounds concentrations show similar results. Butyric acid is lost after the encapsulation process; however, 9,12-Octadecadienoic acid (Z, Z) concentration dramatically increases after the encapsulation in both encapsulated aromas. 2-Decenal and 2-Nonenal concentrations show a slight rise and these compounds are also derivatives of the oxidation. Heptanoic and Undecanal concentrations in the encapsulated aroma are higher than in the liquid flavor. Other compound concentrations are similar between A & B aromas and commercial products. Added the encapsulated aromas and commercial aroma in cracker doughs, volatile compounds concentrations do not show dramatically difference; however, 2- Decenal concentration in three samples slightly increases with comparing the reference liquid aroma. 9,12-Octadecadienoic acid concentration is differentiated between encapsulated aroma and liquid aroma, higher concentration in the encapsulated aroma. Heptanoic acid concentration could not be maintained in doughs when using liquid flavoring and showed a decrease. After the cooking, adding the aroma to the top oil process demonstrates the different volatile compounds concentrations. Heptanoic acid, 2-Nonenal, 2-Decenal, dihydro-5-pentyl-2(3H)-Furanone, and Tetradecanal compounds concentrations are higher than reference liquid aroma and cracker that include aromas in the dough. Other compounds' concentrations are similar to the commercial aroma. The change in the concentrations of the volatile components in the aroma may be caused by oxidation and the combination of the aroma with other components and creating different reactions. The panelists do not realize dramatically difference between using encapsulated B aroma and liquid aroma in dough with cheese odor, cheese flavor, aftertaste of cheese flavor, and general taste. Significant differences are observed between crackers that use oil including aroma and added commercial aroma in the oil process. The samples using the encapsulated B flavor in the oil are more like in terms of cheese odor and flavor, aftertaste cheese flavor, and general taste than the crackers using the reference aroma. In line with these results, industrial cheese flavor managed to preserve most of its volatile components with its wall material consisting of maltodextrin and whey protein concentration. The encapsulated aromas are used in the dough of cracker samples, which are applied at high temperatures during cooking, and in the oil applied to them after cooking. Although some volatile compounds of crackers, such as butyric acid and 2-nonenal, are lost when high temperature is applied; a high amount of volatile components are retained. Added aroma in oil shows greater volatile compound concentrations, also added encapsulated aroma in oil is more preferred than others.
Endüstriyel havuç suyu atıklarından enzim ve ultrason destekli ekstraksiyon ile gıda bileşeni üretilmesi

(Lisansüstü Eğitim Enstitüsü, 2024-06-28) Demir, Yasemin ; Karıncaoğlu Kahveci, Derya ; 506201523 ; Gıda Mühendisliği

Küresel olarak, insan tüketimi için üretilen gıdaların yaklaşık üçte birinin kaybolması veya israf edilmesi en önemli gıda sistemi sorunlarından biridir. Nüfus ve tüketim sürekli artarken, gıda talebinin en azından bir sonraki 40 yıl boyunca artması beklenmektedir, bu da doğal kaynaklar olan toprak, su ve enerji üzerinde baskı oluşturmaktadır. Gıda israfının önlenmesi bir önceliktir, ancak bazen bu ekonomik veya teknolojik olarak mümkün olmamaktadır. Bu nedenle, gıda atıklarının yem ve enerji üretimi gibi çeşitli süreçler için kaynak olarak kullanılması için çaba gösterilmektedir. Tarım ve gıda sektörlerinden gelen atıklar besin maddeleri bakımından zengin olmasına rağmen, bunların bertarafı çevresel ve sağlık sorunlarına yol açabilmektedir. Araştırmacılar, bu sorunları en aza indirmek ve değerli biyoaktif bileşikleri çıkarmak için ultrasonik ve enzimatik destekli ekstraksiyon gibi çevre dostu teknolojileri araştırmaktadır. Çevre dostu metotlar arasında yer alan enzim ve ultrason destekli ekstraksiyon; çözgen kullanımını azaltarak, daha az enerji sarfiyatı sağlayarak ve verimi artırarak fayda sağlamayı amaçlamaktadır. Havuç sebzesi meyve suyu fabrikalarında işlendikten sonra genellikle endüstriyel atık olarak atılır veya hayvan yemi olarak kullanılırken, ortaya çıkan bu yan ürün karotenoidler ve fenolik bileşikler gibi yüksek oranda yararlı bileşikler içermektedir. Dolayısıyla bu yan ürünün farklı endüstrilerde (gıda, sağlık, kozmetik vb.) kullanılmasının hem katma değerli ürün elde edilmesinde hem de çevresel konularda faydalı olabileceği düşünülmektedir. Bu çalışmanın amacı, endüstriyel havuç suyu atıklarına çevre dostu ön işlemler (enzimatik ve ultrasonik destekli ekstraksiyon) uygulanarak gıda endüstrisinde önemli kullanım alanı olan bileşenlerin elde edilmesi için Sürdürülebilir Kalkınma Hedefleri'ne uygun bir yaklaşım geliştirilmesi amaçlanmakla birlikte çalışmada bahsedilen yöntemler üzerinde optimizasyon koşullarının belirlenmesidir. Öncelikle, geleneksel, enzimatik ve ultrasonik yöntemlerin her biri için solvent türü, katı-sıvı oranı, enzim türü, enzim miktarı, enzimatik ve ultrasonik destekli ön işlem süresi parametreleri beta karoten, likopen, toplam fenolik madde konsantrasyonlarına ve DPPH % inhibisyonuna göre optimize edilmiştir. Analizler spektrofotometre ile belirlenen dalga boylarında ölçümlenmiştir. Yapılan çalışmalar sonucunda solvent türü ve katı-sıvı oranı sırası ile etanol ve 1:40 (w/v) olmuştur. Enzimatik ön işlem parametreleri ise enzim türü için Pectinex® XXL, enzim miktarı için 300 μL ve ekstraksiyon süresi için 120 dk optimum koşulları vermiştir. Ultrasonik ön işlemin süresi 20 dk olarak belirlenmiştir. Sonuçlara göre, belirlenen bu optimum koşulların geleneksel, enzimatik ve ultrasonik ön işlem destekli ekstraksiyon yöntemlerine uygulanması ile istatistiksel olarak enzim ve ultrason ile ön işlem uygulamasının bu gıda bileşenlerinin geri kazanımını artırabileceğini göstermiştir (p<0.05). Optimum koşullar belirlendikten sonra havuç posaları geleneksel, enzimatik destekli, ultrasonik destekli, enzimatik-ultrasonik destekli ve ultrasonik-enzimatik destekli 5 farklı ekstraksiyon yöntemi ve kombinasyonu ile ekstrakte edilmiştir. Elde edilen bütün sıvı ekstraktların önce dönel buharlaştırıcı ile içerdiği etanol uçurulmuş daha sonra ise dondurarak kurutulmuştur. Bütün analizler katı madde üzerine uygulanmıştır. Ekstraksiyon yöntemlerine göre elde edilen bileşenlerin konsantrasyon sonuçlarına baktığımızda enzimatik destekli ekstraksiyonun sonuçları diğer yöntemlere göre daha etkili bulunmuştur. Enzim destekli ekstraksiyonun sonuçları şu şekildedir: 26,37 ± 0,26 mg/g beta-karoten konsantrasyonu, 20,93 ± 0,005 mg/g likopen konsantrasyonu, 9,97 ± 1,38 mg GAE/g toplam fenolik madde konsantrasyonu, 209,44 ± 7,98 mg rutin/g toplam flavonoid madde konsantrasyonu ve 25,57 ± 4,15 DPPH % inhibisyonudur. Ayrıca hem E.coli hem de Salmonella Typhimurium için en etkili antimikrobiyal aktivite gösteren yöntemin enzim destekli ekstraksiyon yöntemi olduğu gözlemlenmiştir. Bulgulara bakıldığında enzim uygulamasının hücre duvarını parçalaması özelliğinden dolayı hedef bileşenlerin salınımını büyük ölçüde etkilediği söylenebilmektedir. Ek olarak Enzim - Ultrason Destekli Ekstraksiyon uygulamasının ise ikinci etkili yöntem olduğu tespit edilmiştir. Bu uygulamada ise ek olarak ultrason kullanımının kavitasyon etkisi ile hücre duvarını parçalayarak verimi artırmada etkili rol oynadığı gözlemlenmiştir.

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