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ÖgeBioprospection of antagonistic yeasts for biocontrolling postharvest pathogenic fungi and physicochemical characterization of a yeast exopolysaccharide(Graduate School, 2023-05-04)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.
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ÖgeProteins and bioactive peptides extraction from food wastes and their modification by different processes to produce functional food products(Graduate School, 2024-05-15)Watermelon seeds (WMS) have valuable protein content and possess health beneficial effects regarding the functional foods. They generally comprise approximately 35-40% protein per weight, rendering them a significant protein reservoir for individuals adhering to vegetarian and vegan diets. Additionally, watermelon seeds encompass healthy fats, including monounsaturated and polyunsaturated fats, which contribute to heart health. Moreover, these seeds boast dietary fiber, facilitating digestion and promoting digestive well-being, alongside serving as a rich source of minerals like magnesium, iron, zinc, and potassium, essential for diverse bodily processes. In terms of health benefits, watermelon seed protein is a nutritious and sustainable plant-based protein source that offers various health benefits and a valuable addition to a post-workout or muscle-building diet. Protein and fiber in watermelon seeds can help promote satiety, which may aid in weight management by reducing overall calorie intake. While watermelon seed protein offers numerous health benefits, it's essential to consume them as a protein source for a balanced. Incorporating watermelon seed protein into the diet of people preferring vegan, vegetarian or healthy diet can be a delicious and convenient way to boost the protein intake and support overall health and well-being. Even though WMS are a potential protein source owing to their high protein content and the balanced amino acid composition, they are often discarded when eating the fruit and actually packed with nutrients, including protein. In terms of valuable contents of bioactive peptides, protein, amino acids and, WMS protein come close to soy protein, being the most broadly used plant protein source so far.
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ÖgeUV-C ışık uygulamasının taze kesilmiş deveci armutunun dekontaminasyonu ve kalite parametreleri üzerine etkisi(Lisansüstü Eğitim Enstitüsü, 2024-09-04)Meyve ve sebzeler açısından zengin bir beslenmenin, insanları kanser ve kronik hastalıklara karşı koruduğu ve hastalık riskini azalttığı bilinmektedir. Taze kesilmiş meyveler tüketicilere tüketim kolaylığı, taze benzeri kalite ve çeşitli sağlık yararları ile yüksek duyusal ve besinsel nitelikler sunar. Sağlık yararlarının yanı sıra taze halde tüketim imkanı ve kolaylık sağlaması nedeniyle de son yıllarda popülerleşmektedir. Taze kesilmiş meyvelere uygulanan soyma, çekirdek çıkarma ve kesme gibi mekanik işlemler enzimatik esmerleşme, lezzet bileşenlerinin kaybı, yumuşama ve mikrobiyolojik bozulma gibi biyokimyasal bozulmalara neden olur. Tazeliği korurken, mikrobiyal riski de en aza indirecek yeni teknolojilerin geliştirilmesi, artan talebi karşılamak için önemlidir. Taze kesilmiş ürünlerin kalitesini korumak ve raf ömrünü uzatmak için genellikle modifiye atmosferde ambalajlamayı takip eden bazı kimyasal ön işlemler uygulanır. Kimyasal ön işlemler arasında askorbik asit, sitrik asit ve kalsiyum tuzları kahverengileşme ve yumuşama önleyici maddeler olarak en yaygın olanlarıdır. Modifiye atmosferde ambalajlama uygulaması taze kesilmiş meyvelerin hava bileşimine kıyasla azaltılmış O2 ve yükseltilmiş CO2 altında paketlenmesini içerir. MAA, taze kesilmiş ürünlerde aerobik solunum oranlarını, enzimatik esmerleşmeyi, yumuşamayı ve mikrobiyal büyümeyi azaltır ve böylece raf ömrünü uzatır. Optimum modifiye atmosferler farklı taze ürünler için farklılık gösterir ve genellikle aerobik solunum oranının en aza indirildiği MA olarak alınır. Bu nedenle, taze ürünler için uygun bir MAA tasarımı, optimum MA'nın belirlenebilmesi için O2 ve CO2'den etkilenen solunum hızının ölçülmesini gerektirir. Taze ürünler için optimum MA'ya sahip MAA, ürün tipi, ürün ağırlığı, ürün solunum hızı, gaz konsantrasyonu, depolama sıcaklığı, yüzey alanı ve ambalaj malzemelerinin gaz geçirgenliği gibi çeşitli faktörler göz önünde bulundurularak tasarlanır. Son zamanlarda, taze meyve ve sebzelerde ultraviyole (UV-C) ışınların kullanımı üzerine yapılan araştırmalar, bu teknolojinin, taze ürünlerin raf ömrünü uzatmak için geleneksel ısıl işlem ve kimyasal yöntemlere alternatif olarak önemli bir umut vaat ettiğini göstermiştir. UV-C işleminin etkinliği, UV dozu (J/m2), UV yoğunluğu (W/m2), yüzey özellikleri, başlangıçtaki mikroorganizma yükü ve mikroorganizma türü gibi çeşitli faktörlere bağlıdır. Yüksek verimlilikte üretime olanak sağlayacak koşulların optimize edilmesi, UV-C teknolojisinin ticarileştirilmesi için önemlidir. Bu çalışmanın amacı, taze kesilmiş Deveci armut dilimlerine uygulanan ön işlemler olan esmerleşme/yumuşama önleyici çözeltilere daldırma ve MAA uygulamalarına, UV-C ışınlama uygulamasının optimize edilerek entegre edilmesi ve bu uygulamaların taze kesilmiş armut dilimlerinin raf ömrü ve kalite üzerine etkisinin belirlenmesidir. Bu tez kapsamında, ilk olarak UV-C ışığın armut diliminde penetrasyon derinliği hesaplanmış ve farklı kesitlerdeki dilimler üzerinde ışığın hem enerji penetrasyonunun hem de dokularda etki penetrasyonunun dikkate alınarak şiddete bağlı olarak belirlenmiştir. Daha sonra, meyve dokusundaki esmerleşmenin ve yumuşamanın kontrol edilebilmesi amacıyla taze kesilmiş armuta uygulanacak ön işlem solüsyonu seçilmiştir. Armut meyvesinin en düşük aerobik solunum yaptığı gaz kompozisyonunun tespiti ile optimum MAA koşulları oluşturulmuş ve bu koşullara uygun ambalaj tasarlanarak dilimler ambalajlanmıştır. MA ambalajlama uygulamasının dilimlerin kalite parametreleri üzerine etkileri değerlendirilmiştir. Sonraki aşamada önemli duyusal ve kimyasal kalite kriterlerini olumsuz etkilemeyen, uygulanabilecek maksimum UV-C dozu belirlenmiştir. Yüksek ve düşük doz hızında farklı dozlarda UV-C uygulamasının, petri kutularına ve meyve yüzeyine inoküle edilen patojen mikroorganizmalar Escherichia coli, Staphylococcus aureus, Penicillium expansum ve Zygosaccharomyces bailii üzerindeki inaktivasyon etkinliğinin belirlenmiş ve kinetik olarak modellenmiştir. Son iş paketinde, taze kesilmiş armutlara ön işlemler, UV-C uygulaması ve MAA sistemi entegre bir şekilde uygulanarak depolama sırasında mikrobiyal, duyusal ve kimyasal kalite üzerine etkileri belirlenmiştir. Armut dilimlerinin ışık geçirgenliğinin ürünün çok ince bir yüzey tabakası ile sınırlı olduğu ve UV-C ışığın belirli derinliklerdeki armut hücrelerinin hem fonksiyonel aktivitesini hem de mikrobiyal hücrelerin canlılığını etkileyebileceği belirlenmiştir. Daha az esmerleşmeye ve sertlik kaybına neden olan %1 sitrik asit + %1 kalsiyum klorür solüsyonu armut dokusundaki enzimatik esmerleşmenin ve yumuşamanın kontrol edilebilmesinde daha etkilidir. Solunum hızını gaz konsantrasyonunun bir fonksiyonu olarak açıklayan en iyi model 'inhibisyonsuz Michaelis-Menten modeli' olmuştur. MAA, %2,1 O2 ve %8,7 CO2'den oluşan denge atmosferi altında solunum hızını azaltmış ve kısa süreli depolama süresi boyunca taze kesilmiş 'Deveci' armut dilimlerinin renk, pH, TSS, TA ve sertlik gibi kalite özelliklerini korumuştur. 5,15 kJ/m2 dk yüksek doz hızı ve 1,30 kJ/m2 dk düşük doz hızında ve 50, 100 ve 150 kJ/m2 UV-C dozları uygulandığında; yüksek doz hızında 50 kJ/m2 UV-C uygulaması, dilimlerde daha az sertlik kaybına ve toplam renk değişimine, daha yüksek L* ve hue açısı değerlerine ve daha düşük a* ve Chroma değerlerine neden olarak dilimlerin kalite parametrelerini daha iyi korumuştur. Besiyeri ortamında iki farklı doz hızında ve 4 farklı mikroorganizma türünün hepsinde mikrobiyal inaktivasyon kinetiğinin modellenmesi sonucu uyumlu bulunan tek model Weibull iken, armut dokusunda tüm mikroorganizmaların Weibull ve Bifazik modellere daha iyi uyum sağladığı görülmüştür. Genel olarak, buzdolabında depolama süresince mikrobiyal yükün artmaması, UV-C uygulaması ve soğuk depolama birlikte uygulandığında gıda güvenliği ve kalitesini korumada etkili bir strateji olduğunu göstermektedir. Meyve dokularının aynı doz uygulansa bile daha uzun süre UV-C ışığına maruz kalması ciddi hücre hasarıyla ilişkilendirilir ve bu da ürünün taze görünümünün hızla azalmasına neden olur. Bu nedenle bu teknolojinin raf ömrünü uzatma etkisi uygulamanın dozuna bağlı olduğu gibi uygulanan dozun şiddetine de bağlıdır. UV-C ve MAA uygulanmasının ayrı ayrı değil, entegre bir şekilde kullanılması armut meyvesinin kalitesinin korunmasını iyileştirmektedir. Ancak bu durum, optimize edilmiş O2/CO2 geçirgenliğine sahip uygun bir MAA filminin gaz konsantrasyonunu ve dolayısıyla ambalaj içindeki meyve solunumunu etkin bir şekilde kontrol etmesi halinde geçerlidir. Taze kesilmiş armutlara ön işlemler, MAA sistemi ve UV-C uygulaması entegre bir şekilde uygulanarak 21 günlük depolama süresi sonunda, 50 kJ/m² UV-C uygulanıp modifiye atmosferde ambalajlamanın (UV50+MAA) mikrobiyal kaliteyi, renk ve sertlik değerlerini, suda çözünen katı içeriğini, titrasyon asitliği miktarını ve pH değerlerini daha iyi korumuştur. UV-C uygulanan dilimlerin PAL enzim aktivitesi, toplam antioksidan kapasite, toplam fenolik madde miktarı ve toplam flavonoid madde miktarı daha yüksektir. Depolama süresi uzaması ile beraber UV-C ışını ve modifiye atmosfer ambalajının birlikte kullanımı dilimlerde gözlenebilecek olumsuz etkileri yavaşlatmakta, mikrobiyal kaliteyi korumakta ve armutların daha uzun süre taze kalmasına yardımcı olarak depolama süresi boyunca kalitenin korunmasına katkıda bulunmaktadır. Bu çalışmalarla, ülkemize özgü olan 'Deveci' armut türünün, yeni bir teknoloji olan UV-C uygulamasının ön işlemler ve MAA sistemine entegre edilip işleme koşulları optimize edilerek literatüre kazandırılmıştır. Böylece 'Deveci' armudundan taze kesilmiş katma değeri yüksek yeni ürünler geliştirilmesine katkı sağlanacaktır. Endüstriyel ölçekte kullanılan sistemlerin etkinliği; tasarımında yapılacak farklı modifikasyonlar ile arttırılabilecek, endüstriyel çaplı ve bilgisayar temelli yeni UV-C reaktörlerinin geliştirilmesine katkı sağlanabilecektir. Taze kesilmiş meyvelerin üretim verimi arttırılacak, depolanması sırasındaki çeşitli problemler giderilecek, ürün kayıpları önlenecek, ürün farklı gıda formülasyonlarına daha kolay entegre edilebilecek ve ürünün kullanımı kolaylaştırılacaktır. Böylece ülkemizde yeni yeni gelişmekte olan taze kesilmiş meyve ve sebze sektöründe, bu ürünlerin endüstriyel olarak işlenebilirliği artacak, israf önlenerek kaliteli alternatif ürünlere işlenebilecektir. Proje kapsamında elde edilen veriler, uluslararası hakemli dergilerde yayınlanacak veya kongrelerde sunulacaktır. Bu çalışmanın benzer çalışmaları tetikleyebileceği ve bilimsel ve endüstriyel kazanımlara katkı sağlayacağı öngörülmektedir.
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ÖgePotential of lactic acid bacteria fermentation as a strategy for valorisation and biotransformation of mushrooms(Graduate School, 2024-08-15)Consumers increasingly recognise the importance of healthy eating and the potential benefits of incorporating mushrooms into their diets. Known for their rich nutritional profile, mushrooms provide essential vitamins, minerals, fibre, and antioxidants, making them valuable plant-based food sources. They are also rich in bioactive compounds with anti-inflammatory, antioxidant, antitumor, antiviral, and antimicrobial properties, promoting health and reducing disease risks in humans. Among various bioactive components in mushrooms, phenolic compounds are particularly noteworthy. These compounds are considered significant secondary metabolites in mushrooms and are found in free and bound forms within food matrices. However, the bioaccessibility and bioavailability of bound phenolic compounds are lower than that of free phenolic compounds due to their covalent bonds to cell wall matrices, which prevent absorption in the small intestine. Additionally, mushroom production generates various by-products, which pose environmental and financial challenges due to their disposal. Innovative processing techniques are required to enhance the bioavailability of phenolic compounds and add value to mushroom by-products. Fermentation with lactic acid bacteria (LAB) is effective in this context. LAB fermentation not only extends the shelf life and improves sensory properties but also breaks down macronutrients such as carbohydrates and proteins. This alters the nutritional composition of the food and facilitates the transformation of bound phenolic compounds into more bioavailable forms, similar to free phenolic compounds. Through LAB fermentation, macronutrients are transformed, antioxidative peptides are released, and phenolic compounds are modified. This process enhances the health benefits of mushrooms by increasing the bioavailability of mushroom phenolics, making them more accessible for absorption and use by the body. Therefore, fermentation techniques can significantly improve mushrooms' use, health benefits, and by-products. Given this information, a research framework for this doctoral thesis explores modifying industrial mushroom wastes and specific extracted components, as well as mushrooms like L. edodes and La. deliciosus, which may become waste due to their short shelf life. The research plan is based on processing mushroom waste and mushrooms through LAB fermentation. The objectives of this doctoral thesis are: (i) to valorise the mushroom waste generated from bioactive substance extraction, modifying its structure and nutritional composition through fermentation with lactic acid bacteria; (ii) to ferment the mushrooms with lactic acid bacteria to facilitate the transition of phenolic compounds from bound to free form, altering the structure of L. edodes and La. deliciosus; (iii) to determine biotransformation of these phenolic compounds use analytical identification; (iv) to investigate the effects of LAB fermentation on the bioaccessibility and intestinal transport of mushroom phenolics using an in vitro gastrointestinal digestion model. To achieve these objectives, three different experimental studies (Chapters 3-5) were conducted within the scope of this thesis. The first study focused on the fermentation of L. edodes mushroom waste, L. edodes residue (LER), with lactic acid bacteria compared to L. edodes itself (LE), examining changes in its structure and nutritional composition for functional properties (Chapter 3). The second study was based on the changes in phenolic components, interactions with other metabolites, and the profiling of phenolic substances in fermented L. edodes and La. deliciosus (Chapter 4). Based on the previous chapter's findings, the third study examined phenolic components' bioaccessibility and antioxidant activity trends in an in vitro gastrointestinal digestion model.
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ÖgeGreen extraction and encapsulation of black rosehip polyphenols: İn vitro bioaccessibility, bioavailability, and biological activities(Graduate School, 2022-11-22)Due to the increased prevalence of degenerative diseases, there has been a high consumer demand for functional foods with added health benefits. The term "functional food" generally refers to products providing an additional health benefit, beyond basic nutrition. Consuming foods fortified with functional ingredients (e.g., vitamins, probiotics, omega n-3 fatty acids, minerals, and antioxidants) could help to mitigate the risk of chronic diseases and contribute to the physical and mental well being. In that sense, (poly)phenols, phenolic compounds containing at least one phenolic hydroxyl group, are the most pronounced secondary metabolites found in plants possessing strong antioxidant power. However, to exploit the use of underutilized wild edible plant resources, convenient extraction methods need to be developed to separate and concentrate the (poly)phenols without using toxic hazardous chemical solvents. Hence, 'green' isolation of value-added ingredients for enrichment purposes from underutilized plant sources have importance for food industry. Moreover, (poly)phenols are susceptible to against harsh processing and environmental factors such as heat, oxygen, light, alkaline pH, or enzymes and therefore mostly have poor solubility and bioavailability. Moreover, their biological functions are significantly dependent on their bioavailability. In this regard, encapsulation applications enable increasing their stability by protecting from stresses mentioned above and/or improve their solubility, bioaccessibility and intestinal uptake which is especially desirable for manufacturing of functional foods/beverages and dietary supplements. Taking these facts into consideration, this thesis consist of the following objectives: (i) to characterize the (poly)phenols composition of black rosehip (Rosa pimpinellifolia L.) and its antioxidant as well as antiproliferative activity in comparison to common rosehip (Rosa canina L.); (ii) to optimize the extraction conditions of black rosehip (poly)phenols by means of green methods, in particular pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction; (iii) to evaluate the effects of extraction methods on bioaccessibility and bioavailability of (poly)phenols using combined in vitro gastrointestinal digestion/Caco-2 cell culture model; (iv) to improve processing and digestive stability of hydrophilic and lipophilic (poly)phenols with high antioxidant acitivity by converting them into edible and stable form for functional foods by means of encapsulation and to investigate the effects of liposomal encapsulation and spray drying processing on bioaccessibility and bioavailability of (poly)phenols using combined in vitro gastrointestinal digestion/Caco-2 cell culture model. The first chapter was designed to characterize Rosa pimpinellifolia fruits collected from Gümüşhane Province in the Black Sea region of Turkey by means of (poly)phenol composition, in vitro antioxidant activity and other biological activities in comparison to commonly consumed rosehip specie. The angiotensin-converting enzyme inhibition potential of black rosehip was also assessed by spectrophotometric assay. Since rosehip (Rosa canina) fruit has traditionally been widely consumed in the form of herbal tea, tea-type infusions of both Rosa species were prepared to reveal the consumption potential of black rosehip. In this context, tea-type infusions and aqueous methanol (80%, v/v) extracts were analyzed by LC-MS/MS in both rosehip species studying different parts of the fruits; whole fruit, flesh, and seed. Apart from other rosehip species, Rosa pimpinellifolia has its dark purplish-black color due to its rich anthocyanin content. Unsurprisingly, any anthocyanin was absent in Rosa canina fruit. Chromatographic analysis showed that (poly)phenol contents varied between rosehip species and drying method applied (freeze drying and air drying). Gallic acid, catechin, protocatechuic acid, procyanidin-B2, procyanidin oligomers and quercetin acid derivatives were principal in both rosehip species. For black rosehip, high level of anthocyanins, (poly)phenols content and high antiradical scavenging capacity were characteristic. Reactive oxygen species (ROS)-induced-DNA damage has been linked to the onset of many degenerative and cardiovascular diseases. Polyphenolic compounds protect cells against the harmful effects of ROS in various mechanisms. Flavonoids and phenolic acids fractions of Rosa canina have been reported to inhibit cell proliferation, however no study exist regarding the antiproliferative properties of black rosehip to the best of our knowlege. In that sense, cytotoxicity by MTT assay, ROS generation, and apoptotic effects by Acridine orange assay in breast cancer cells were evaluated in tea-type infusions and hydroalcoholic extracts from both species. Response surface methodology was adopted to achieve an effective extraction procedure of (poly)phenols from black rosehip (Rosa pimpinellifolia) fruits. Pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction techniques were investigated by screening parameters including temperature, pressure, solvent composition, and solvent-to-solid ratio. Ultrasound assisted solvent extraction was also assessed. Simultaneous maximization in terms of extraction yield, total antioxidant capacity, total (poly)phenol content, catechin content, total monomeric anthocyanin content, and cyanidin-3-O-glucoside content was performed. Antioxidant activity was evaluated using 2,2-diphenyl-1- picrylhydrazyl radical (DPPH) radical scavenging activity and cupric ion reducing antioxidant capacity (CUPRAC) assays. The experimental data was subjected to the regression analysis to obtain second-order polynomial equations and the fitted polynomial equations are presented as contour plots also showing the linear and quadratic effects of the tested dependent variables. The optimum conditions for extraction was compared to the predicted values of RSM using an independent sample t-test. The optimized conditions were as follows: 75 °C with 10 mL g-1 solvent-to solid ratio under 100 bar during 60 mins, and 60 °C with 25 % aqueous ethanol under 280 bar during 60 mins for pressurized hot water extraction and ethanol modified supercritical carbon dioxide extraction, respectively. When these extracts were examined in terms of bioaccessibility and epithelial cells (Caco-2) uptake upon gastric and intestinal digestion in vitro, all extracts exhibited lower levels of total phenolic content compared to their undigested counterparts (p < 0.05). Chapter 4 aimed to achieve the encapsulation of black rosehip extracts obtained via green techniques applied in previous chapter and to evaluate their antioxidant properties as well as processing and digestive stability. The black rosehip extract (BRE) which was obtained at optimized pressurized hot water extraction conditions, was further encapsulated in biopolymer-coated liposomes and spray dried using maltodextrin as carrier. Being readily available natural biopolymers, chitosan and whey protein were used in coating of liposomes via electrostatic deposition method. In the literature, these biopolymers have been widely used for encapsulation purposes of various polyphenolic extracts. However, in the present thesis, for the first time a comparison between these materials for their efficiency to fabricate stable antioxidant powders. Nanosized particle diameters were achieved by 5 cycle microfluidization of the liposomal dispersions. During spray drying process, total phenolic content (TPC) in extracts decreased due to oxygen and heat exposure. However, the retention efficiency of TPC in biopolymer coated liposomal powders was found significantly higher than spray dried BRE. In a similar pattern as observed with retention of phenolics, retention of antioxidant capacity of the powders was confirmed with CUPRAC assay. In addition, encapsulation provided remarkable protection of the phenolics under in vitro gastrointestinal digestion conditions, resulting in up to a 5.6- fold more phenolics in the bioaccessible fraction, which also had 2.9–8.6-fold higher antioxidant activity compared to the non-encapsulated BRE. Similar results were obtained for encapsulated RPFE using ethanol injection method in preparation of liposomes. Besides, as an alternative colloidal delivery system to liposomes, solid lipid nanoparticles (SLN) were prepared using plant saponin glycyrrhizin to ensure stabilization at low concentrations which has great importance for the food, nutraceutical and pharmaceutical industries. For the first time a series of glycyrrhizin emulsified tristearin SLNs were successfully fabricated and physical stability was determined during 21 days of storage. The influence of glycyrrhizin on the physical stability and crystallization behavior of SLNs were evaluated by dynamic and static lights scattering, electrophoretic light scattering, optical microscopy, visual observations, and differential scanning calorimetry. The SLN formulations containing lower amount of surfactant revealed poor stability against aggregation as clearly observed by optical microscopy. Beyond bioaccessibility, the absorption of (poly)phenols through the intestinal epithelial layer is an important factor in functional food development. Thus, the behavior of individual phenolic compounds loaded in dried liposomes should be investigated to gain insight regarding their bioavailability and resulting bioactivity. In the last chapter, the co-encapsulation of phenolic compounds into nanoliposomes in combination was explored in terms of particle characterization and bioavailability compared to their native (non-encapsulated) form. Phenolic compounds representing berry matrix (catechin, epicatechin, ferulic acid, and resveratrol) were loaded into chitosan- and whey protein-coated liposomal powders. For control purposes, the phenolic compounds were also spray dried without liposomes to observe the effect of liposomal encapsulation on protection of bioactive molecules against processing or digestive conditions. The digestive fate of the samples was determined using in vitro digestion coupled with Caco-2 cell monolayer model. The phenolic compounds, both in encapsulated or free form, decreased upon simulated gastrointestinal digestion, except cis-resveratrol possibly due to trans-to-cis isomerization that occurs during in vitro digestion. Significantly higher digestive stability, solubility, micellization efficiency, and bioaccessibility were found in encapsulated phenolic compounds (p < 0.05). On the contrary, whey protein coated liposomal formulation resulted in substantial increase in the cellular uptake of trans-resveratrol in comparison to other encapsulated formulations or in native form (p < 0.05). The epithelial permeation of the native and encapsulated micellar phenolics were further evaluated in Caco-2 transwell model system. Moreover, all digested formulations was analyzed in terms of fatty acid induced lipid accumulation in human hepatic cancer (HepG2) cells as an indicator of the effect of encapsulation on the potential biological activity.