LEE- Gıda Mühendisliği-Doktora

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Phenolic and carotenoid profiles of tomatoes collected from different parts of Turkey and antioxidant properties of dried tomatoes

(Graduate School, 2021-02-18) Bakır, Sena ; Çapanoğlu, Güven, Esra ; 506132509 ; Food Engineering ; Gıda Mühendisliği

Tomatoes, which are the indispensable part of the Mediterranean diet, have attracted the attention of several researchers due to being one of the most consumed fruit all around the world. Tomato may be consumed as fresh and also in forms of processed products including paste, sauce, juice, etc. Tomato consumption is associated with reducing the risk of cardiovascular diseases, blood sugar, obesity, and also decreasing the carcinogenic cells in the human body which has been attributed to their high phenolic and carotenoid contents. Considering the health effects of tomato bioactives, phenolic and carotenoid profiling of tomatoes using diverse methods have taken the attraction of scientists. The objectives of this Ph.D. thesis were (i) to evaluate the phenolic and carotenoid profiles of some selected Turkish tomato varieties; (ii) to determine the effect of different drying techniques on the phenolic profile of tomatoes and monitoring the bioaccessibility of key phenolic components after simulated in vitro digestion; and (iii) to investigate the phenolic and carotenoid contents of a new functional food by enriching with tomato powder and to elucidate the interactions between these bioactives with proteins. Regarding these aims, first a comprehensive review was prepared on the functional properties of tomato and tomato by-products and their possible applications in foods (Chapter 2). In this part, phenolic profiling methods and their applications have been summarized. On the other hand, use of tomatoes and tomato by-products in different foods as functional ingredients was discussed. In the experimental part, firstly tomato landraces of Turkey were investigated, and 77 Turkish labelled tomato seed samples were collected. The obtained seed samples were planted in open fields for two harvest years, but only 50 of them were used for further analyzes. Harvested samples of both years were analyzed for their phenolic profile (Chapter 3), as well as carotenoids and other health-related compounds (Chapter 4). In order to evaluate the effect of drying on tomato bioactives, various drying treatments were applied and also some commercial dried samples were provided to evaluate the changes in their phenolic and carotenoid contents; moreover, bioaccessibility of phenolic compounds after drying was investigated by using a simulated in vitro bioaccessibility protocol (Chapter 5). Finally, a new functional food was produced using tomato powder, and investigated in terms of their phenolics, carotenoids, their interactions with proteins, and in vitro bioaccessibility (Chapter 6). In Chapter 3, the semi-polar metabolite profiling was aimed for Turkish tomato accessions. Tomato seeds were planted in an open field to accomplish this goal. Harvested samples were firstly evaluated for their colour and pH values. Subsequently, samples were ground under liquid nitrogen and dried with a lyophilizator which was followed by storing at -20⁰C for further analysis. Methanol extracts were prepared were phenolic profile determination. LC-MS equipment was used for the research, and outcomes of this analysis were evaluated with PCA and HCA diagrams. Results indicated that the phenolic content of landraces diverse mostly based on the fruit size on the PCA diagram. On the other hand, geographical area of seed samples where they were collected, did not directly affect the semi-polar metabolite content of tomato fruits. In Chapter 4, the biodiversity of potential health-beneficial compounds within a 50 tomato fruit accessions which were collected throughout Turkey, was assessed. The contents of phenolics, carotenoids, ascorbic acid and tocopherols, as well as their antioxidant capacities were investigated for each sample. By using complementary spectrophotometric assays, the antioxidant capacity of both hydrophilic and lipophilic extracts were determined after individual antioxidants were detected by HPLC using an on-line antioxidant detection method. Using HPLC with a photodiode array and fluorescence detection, phenolic acids, flavonoids, carotenoids and vitamins C and E were quantified. The results showed that concerning their hydrophilic and lipophilic antioxidants, there is a large variety within this set of samples. In Chapter 5, sun-dried, freeze-dried, semi oven-dried and oven-dried (at 60, 80, 100 and 120⁰C) tomato samples were compared with each other to monitor the influence of drying on phenolic and carotenoid contents, and also on some vitamins. Semi-polar metabolite profile of fruits was determined with LC-MS analysis. Sugar profile of samples was evaluated with RI-HPLC analysis. Individual phenolics and carotenoids were determined with HPLC coupled with PDA and fluorescence detectors, respectively. Despite of these, ergosterol content of smaples were measured with HPLC system. Moreover, in vitro bioaccessibility protocol was applied to understand the changes in phenolics during digestion. Metabolite diversity analysis indicated that semi-polar components in freeze-dried and semi-dried samples were close to each other, while the sun-dried samples were all located together on the PCA diagram as well as the oven-dried ones. These results were in accordance with the data obtained after HCA analysis. In Chapter 6, sun-dried tomato powder was added to simit dough at different concentrations to produce functional simit samples. Phenolic-protein and carotenoid-protein interactions as well as the content of lipophilic and hydrophilic antioxidants were investigated by using flours with different protein contents. For this purpose, 10.4, 11.5 and 13.1% protein containing flours were used to prepare simit doughs and flours in samples were replaced with 2, 4 and 8% tomato powder for the preparation of functional simit samples. The semi-polar metabolite contents of samples were analyzed by LC-MS and lipophilic compounds were determined with HPLC-PDA coupled with fluorescence detector. Metabolite profiles of samples prepared with 11.5% and 13.1% protein containing flour were found to be close to each other while 10.4% protein containing flour showed a different trend. The difference in t-lycopene content between samples were found to be statistically significant, having the highest levels in samples containing the highest levels of protein flour, whereas this difference was not significant in the case of β-carotene. According to the results of hydrophilic compounds, chlorogenic acid content in 13.1% protein flour and 8% tomato powder containing simit samples were found to be 4.7 times higher compared to its counterpart prepared with 2% tomato powder. According to the in vitro bioaccessibility results, the highest recovery value for chlorogenic acid was obtained with 13.1% protein flour and 4% tomato powder containing simit sample (44%) covered with sesame. In Chapter 7, the final part, all results obtained within this work were evaluated together, conclusions and recommendations for future research are provided. The main conclusions derived in each section were summarized including the changes in phenolic and carotenoid profiles of tomato samples, effect of simulated gastrointestinal digestion on the bioactives of tomatoes, changes in tomato bioactives during drying with different methods, and interaction of phenolics and carotenoids with proteins observed in a traditional bakery product. Finally, suggestions on the potential future work were provided.
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.
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.
Formation of anthocyanin-rich black carrot extract loaded potato protein particles by ternary compressed CO2-ethanol-water mixture extraction and PGSS-drying

(Graduate School, 2021-08-16) Düzgün Yavuz, Merve ; Özçelik, Beraat ; 506142501 ; Food Engineering

Black carrot anthocyanins have importance for food industry as it is a natural colorant and possesses health beneficial effects regarding the functional food and beverages. Anthocyanins are conventionally extracted by using organic solvents such as methanol, acetone or water with small amount of hydrochloric acid or formic acid. These methods are problematic due to the residues of organic solvents remaining in extracts that are associated with food safety or due to the degradation of anthocyanins at high temperatures which required for acidified water extraction. Due to these problems, it is crucial to develop novel methods which are environmentally sustainable and efficient, resulting in high yields. The extraction using sub- and supercritical carbon dioxide (sc-CO2) has been growing as an alternative to conventional extraction, as it can potentially fulfill these demands. The anthocyanin- rich extracts are still very labile to different environmental conditions. Encapsulation or complexation with different biopolymers of bioactive compounds provides a good solution before the incorporation of these valuable compounds in food and beverages. Complex coacervation is one of the encapsulation methods which finds a widespread relevance in functional biomaterials consisting the food and beverage area. It is a physicochemical process that be conducted at mild temperatures without high pressures. The other process applied in this thesis for the purpose of complexation was Particles from Gas saturated Solutions–Drying.
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.
Mercimek proteini ile soğan kabuğu fenolikleri interaksiyonunun fonksiyonel özellikler ve biyoaktivite üzerine etkisi

(Lisansüstü Eğitim Enstitüsü, 2022-04-24) Köroğlu, Deniz Günal ; Güven Çapanoğlu, Esra ; Gıda Mühendisliği

Fenolik bileşikler bitkilerde bulunan antioksidan özelliğe sahip bileşiklerdir. Tüketicilerin sağlıklı tercihlere yönelmesi ile birlikte, bitkilerden saflaştırılan fenolik bileşikler özellikle sentetik antioksidanlar yerine veya fonksiyonel gıdalarda bileşen olarak kullanılmaktadır. Bunun yanında, her geçen gün büyümekte olan gıda sektöründe büyük oranlarda gıda atıkları oluşmakta ve çevre kirliliği gibi birçok soruna neden olmaktadır. Doğal bir kaynak olan gıda atıklarından fenolik bileşiklerin geri kazanılması, gıda atıklarının tekrar kullanılması için olanak sağlamaktadır. Özellikle işlenmiş soğana talebin son yıllarda artması, soğan atıklarının çoğalmasına sebep olmuştur. Soğanın iç kısmının fenolik bileşikler yönünden zengin olmasına karşın benzer fenolik profiline sahip olan kabuk kısmının fenolik madde miktarı daha fazladır; fakat bu kısım gıda hazırlama sürecinde atılmaktadır. Bu çalışmanın amacı (1) floresan sönümleme ile mercimek proteini~soğan kabuğu fenolik ekstraktı kompleks oluşum mekanizmasını analiz etmek, in-vitro gastrointestinal sindirilebilirlikleri ile birlikte (2) model sistemlerde protein fonksiyonel özellikleri üzerine etkisini ve (3) fonksiyonel bir gıda ürününde kompleks oluşumunun etkilerini tanımlamaktır. İlk aşama olarak, mercimek proteinleri ile fenolik ekstraktların kompleks oluşum mekanizması hakkında bilgi almak amacıyla floresan sönümle analizi yapılmıştır. Her iki ekstraktın, mercimek proteinlerinin floresan yoğunluğunda konsantrasyonlarına bağlı olarak düşmeye sebep olduğu belirlenmiştir. Ayrıca, kırmızı soğan kabuğu fenolik ekstraktının sarı soğan kabuğu fenolik ekstraktına kıyasla daha düşük konsantrasyonlarda sönümleme etkisi olduğu belirlenmiştir. Mercimek proteini~sarı soğan kabuğu fenolik ekstraktı kompleksi için mercimek proteininin maksimum emisyonunda maviye kayma olmuştur. Diğer bir deyişle, interaksiyona giren fenolik grubunun proteinin içinde gömülü olduğunu göstermiştir. Ayrıca, kompleks oluşumu doğrusal bir Stern-Volmer grafiği ile statik söndürmeyi ifade etmektedir. Mercimek proteini~sarı soğan kabuğu fenolik ekstraktı kompleksinin termodinamik parametreleri hesaplanmıştır. Etkileşimin baskın olarak hidrofobik, entalpi güdümlü olduğu ve kendiliğinden gerçekleşmediği bulgusuna ulaşılmıştır. Mercimek proteini~kırmızı soğan kabuğu fenolik ekstraktı kompleksi için kırmızıya kayma, yani proteinlerin yapısal açılımını ifade etmektedir. Diğer bir yandan, Stern-Volmer grafiği doğrusal değildir. Dinamik ve statik olmak üzere iki sönümleme mekanizmasının mercimek proteini~kırmızı soğan kabuğu fenolik ekstraktı kompleksi için aynı anda meydana geldiği "eylem alanı modeli" (sphere of action) söz konusudur. Çalışmanın ikinci aşamasında, protein-fenolik interaksiyonunun mercimek proteinlerinin fonksiyonel özellikleri üzerine ve farklı sistemlerin (fenolik ekstrakt çözeltileri, protein-fenolik çözeltileri, emülsiyonlar) in-vitro gastrointestinal sindirilebilirliği üzerine etkileri araştırılmıştır. Fenolik bileşiklerin varlığı konsantrasyona bağlı olarak mercimek proteinlerinin çözünürlüğü, köpürme ve emülsiyon özelliklerini olumsuz etkilemiştir ve Pearson korelasyon katsayısı analizi ile fenolik konsantrasyonunun proteinlerin bu fonksiyonel özellikleri ile ilişkisinin negatif olduğu da belirlenmiştir. Farklı model sistemlerde (fenolik ekstrakt çözeltileri, protein-fenolik çözeltileri, emülsiyonlar) in-vitro gastrointestinal sindirim öncesi, toplam fenolik madde miktarları büyükten küçüğe sırasıyla ekstrakt çözeltisi, emülsiyon ve protein-fenolik çözeltisi şeklindedir. Mercimek proteini varlığında toplam fenolik madde miktarında düşme vardır. Toplam fenolik madde miktarı mide fazından sonra ekstrakt çözeltileri ve emülsiyonlarda düşmüştür ve protein-fenolik çözeltilerinde ise sindirim öncesi değerlere benzerdir. Bütün çözelti sistemlerinde, aynı çözelti için toplam fenolik madde miktarı bağırsak fazında en yüksektir. Antioksidan aktivite değerleri toplam fenolik madde sonuçları ile benzer değişmiştir. Protokateşik asit ve fenolik asit türevleri çözelti içerisindeki diğer fenolik bileşiklerin parçalanmasından dolayı sindirim öncesi değerlerinin neredeyse iki katı olmuştur. Kırmızı soğan kabuğu fenolik ekstraktında belirlenmiş her bir antosiyaninin miktarı sindirim öncesinde emülsiyon~ekstrakt çözeltisi>protein-fenolik çözeltisi şeklinde iken mide fazından sonra emülsiyon>ekstrakt çözeltisi~protein-fenolik çözeltisi şeklindedir. Bağırsak fazından sonra ise ekstrakt çözeltilerindeki antosiyanin miktarı diğerlerine kıyasla daha fazladır. Antosiyaninler protein içeren sistemlerde sindirim sonunda daha az tanımlanabilmiştir. Mercimek proteinlerinin varlığıyla sindirim öncesi her bir fenolik bileşiğin miktarında azalma gözlenmiştir. Bu azalma kuersetin ve türevleri için daha fazla olmuştur. Mide fazından sonra kuersetin ve türevlerinin miktarı düşmüş, bağırsak fazından sonra mide fazına benzer olmuştur. Bu durum, toplam fenolik madde miktarında ve antioksidan kapasitesinde gözlemlenen belirgin düşmenin sebebi olarak düşünülmüştür. Tüm sistemlerde bağırsak fazından sonra kuersetin tespit edilmezken, kuersetin türevleri emülsiyonlarda en yüksektir. Protein varlığında, model bir sistemde, her bir fenolik bileşiğin in-vitro gastrointestinal sindirimden farklı etkilendiği gözlenmiştir. Üçüncü aşamada, iki farklı oranda fenolik (soğan kabuğu tozu, soğan kabuğu fenolik ekstraktı ve kuersetin) içeren fonksiyonel krakerlerde fenolik içerikte/antioksidan aktivitede değişim protein-fenolik interaksiyonları dikkate alınarak değerlendirilmiştir. Fonksiyonel krakerler benzer besin değerlerine (p>0.05), daha düşük L* ve daha yüksek a* değerlerine sahiptir. b* değeri daha yüksek oranda soğan kabuğu tozu veya soğan kabuğu fenolik esktraktı ile düşmüş, kuersetin katkısı ile artmıştır. Fonksiyonel krakerlerde fenolik/antioksidan geri kazanımı fenolik oranı arttıkça azalmıştır ve kuersetin 7,4-diglikozit miktarı teorik değerden düşük bulunmuşken kuersetin miktarı yüksektir. İn-vitro gastrointestinal sindirim krakerlerde uygulanmıştır ve fenolik/antioksidan/protein ulaşılabilirliği belirlenmiştir. Fenolik (BIF) ve antioksidan (BIA) biyoerişilebilirlik indeksi in-vitro gastrointestinal sindirim öncesi ve sonrası toplam fenolik madde miktarı ve antioksidan aktiviteye göre hesaplanmıştır. Fonksiyonel krakerlerde BIF değeri küçükten büyüğe soğan kabuğu tozu, soğan kabuğu fenolik ekstraktı ve kuersetin eklenen krakerler şeklinde iken, BIA değerleri kuersetin eklenen krakerlerde en düşüktür. Kuersetin sadece soğan kabuğu fenolik ekstraktı içeren buğday veya mercimek krakerinde tanımlanabilmiştir. Ağız ve mide fazında tüm fonksiyonel buğday krakerlerinde TCA'da çöken peptid miktarı kontrolden küçüktür. Sindirim sonunda buğday krakerlerinin TCA'da çöken peptitleri tanımlanamamıştır. Ağız fazından sonra fonksiyonel mercimek krakerlerinin TCA'da çöken peptid miktarı birbirine yakın (p<0,05) kontrolden küçüktür, mide fazından sonra ise fazladır. Bağırsak fazından sonra TCA'da çöken protein miktarı kuersetin içeren mercimek krakerinde en düşüktür. Gastrointestinal sindirim boyunca genel olarak TCA'da çöken peptid miktarı azalırken, serbest amino grupları miktarında artış gözlenmiştir. Ağız fazından sonra serbest amino grupları miktarı kontrol krakerlere istatistiksel olarak benzer olmuştur (p<0.05). Mide ve bağırsak fazından sonra ise buğday krakerlerinde kontrole kıyasla daha düşük miktarda serbest amino grupları miktarı saptanmıştır. Mide fazından sonra fonksiyonel mercimek krakerlerinin serbest amino miktarı kontrolden yüksektir, bağırsak fazından sonra ise düşüktür. Sonuç olarak, sarı ve kırmızı soğan kabuğu fenoliklerinin mercimek proteinleri ile interaksiyon mekanizması bu çalışma ile ilk defa araştırılmıştır ve her iki ekstraktın farklı yollarla interaksiyona girdiği belirlenmiştir. Bu doktora çalışması, soğan kabuğu fenoliklerin mercimek proteinlerinin fonksiyonel özellikleri üzerine belirgin etkisi olduğunu ve özellikle protein varlığında fenoliklerin biyoerişilebilirliğinin hem model sistemlerde hem de bir gıda matriksinde önemli ölçüde etkilendiğini gösteren özgün bir çalışmadır.
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.
Green extraction and encapsulation of black rosehip polyphenols: İn vitro bioaccessibility, bioavailability, and biological activities

(Graduate School, 2022-11-22) Kasapoğlu, Kadriye Nur ; Özçelik Günşar, Beraat ; 506152507 ; Food Engineering

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.
Valorization of black chokeberry waste as a potential source of bioactive compounds: Their identification, microencapsulation and impact on the human gut microbiota

(Graduate School, 2022-12-07) Çatalkaya, Gizem ; Çapanoğlu Güven, Esra ; 506152502 ; Food Engineering

Epidemiological studies have suggested that adopting a diet rich in fruits and vegetables has been associated with a reduced risk of noncommunicable diseases, such as cardiovascular diseases, neurodegenerative diseases, type II diabetes and cancer. The presence of bioactive substances such as polyphenols has been linked to these potentially health promoting benefits. Polyphenols are secondary metabolites that determine the sensory and nutritional qualities of fruits and vegetables. However, polyphenols are processed as xenobiotics by the human body after consumption, hence the bioavailability of native substances is rather low. Only 5-10% of total dietary polyphenols, mostly those with monomeric and dimeric structures, are estimated to be directly absorbed in the small intestine. The remaining polyphenols pass to the colon, where they are further metabolized by the enzymatic activity of colonic bacteria to molecules with varied physiological significance. These phenolic compounds generated by the microbial catabolism are more absorbable than the original molecules present in foods and may have higher health benefits. In addition to this, dietary polyphenols reaching to the colon can act as prebiotics and they may modulate the gut microbiota by promoting the growth of beneficial bacteria and/or hindering the proliferation of harmful bacteria. Black chokeberry (Aronia melanocarpa) is one of the richest sources of phenolic compounds, especially anthocyanins, among the other berry types. In addition to the anthocyanins, they are a rich source of proanthocyanidins with a high degree of polymerisation. However, despite their health beneficial properties, they are seldomly ingested as fresh due to their distinct astringent flavor, which is perceived as undesirable by the consumers. For this reason, they are processed into juices, jams, etc. Juice processing generates by-products, such as pulp, that might be used in the production of natural colorant and the isolation of the natural nutraceuticals. Although anthocyanins possess potential health-promoting properties and are regarded as promising natural food colorants, unfortunately their unstable nature acts as an obstacle in their practical applications due to their poor bioavailability and susceptibility against environmental factors such as temperature, light, oxygen, pH change, etc. Therefore, encapsulation of these substances might be a suitable method to increase concentrations of bioactive anthocyanins in the gastrointestinal tract and thus boost their beneficial effects. In this useful system, anthocyanins are protected from degradation and prevented from premature color development. Taking all the above-mentioned information into account, this thesis was organized to (i) characterize the polyphenol content of the black chokeberry pulp, (ii) determine the most effective conditions and materials for the encapsulation of the anthocyanin-rich extract obtained from black chokeberry pulp, (iii) determine the effect of black chokeberry polyphenols in different matrices on the human gut microbiota under in vitro conditions. For this purpose, firstly the state of the art on the polyphenol bioaccessibility, bioavailability, interaction with the gut microbiota and analysis through omics approach was comprehensively reviewed and discussed in Chapter 2. In Chapter 3 the extract obtained from the black chokeberry pulp was characterised by both spectrophotometric methods and chromatographic methods. Total polyphenol and total anthocyanin contents of the extract were determined by Folin-Ciocalteu and pH differential methods, respectively. Also, the individual polyphenol composition of the extract was identified by using UPLC-ESI-QqQ-MS/MS method. Dry matter content of pulp was 35.6±0.2%, brix value of the extract was 20% and total anthocyanin content and total phenolic content of extract were determined as 4.91±0.297 mg cyanidin-3-glucoside/mL, 11.5±0.14 mg gallic acid equivalent/mL, respectively. According to LC-MS/MS analysis, ~72% of the total quantified polyphenols consisted of anthocyanins. It is widely known that black chokeberries contain four major anthocyanins, namely cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-xyloside. In this study, cyanidin-3-O-glucoside was identified and quantified. However, apart from the major anthocyanins some other anthocyanins were also detected (cyanidin-3,5-diglucoside, cyanidin-3-O-rutinoside, and pelargonidin-3-O-glucoside). In fact, to the best of our knowledge pelargonidin-3-O-glucoside was identified in black chokeberries for the first time. After characterizing the extract, the second goal of this study was to encapsulate the black chokeberry extract with different coating materials by using spray drying technique which was also detailed in Chapter 3. Among the encapsulation techniques, the spray drying method has been largely utilized for drying heat-labile nutraceuticals since it is precise, efficient, simple and cost-efficient in the processes. The selection of coating material to entrap the active material by spray drying is crucial to achieve an efficient encapsulation. Therefore, five different coating materials have been tested for the microencapsulation of black chokeberry extract (maltodextrin with dextrose equivalent of 6, maltodextrin with dextrose equivalent of 20, its blends with gum Arabic, xanthan gum or whey protein isolate). Spray drying conditions were chosen as follows: inlet temperature of 150 °C, the outlet temperature of 90 °C, 4.5 mL/min feed flow rate, 0.357 m3/h air flow rate, and an aspirator capacity of 100%. For the determination of the most effective system, physicochemical characteristics of the powders such as moisture content, particle size, capsule morphology, color, spray drying yield, encapsulation efficiency, total anthocyanin content, total and individual phenolic content, and total antioxidant activity were investigated. Within the five different wall materials, maltodextrin:gum Arabic provided the maximum encapsulation efficiency (71.5%) while MD6 resulted in the lowest encapsulation efficiency (38.3%). The spray-dried powders presented low moisture content in an acceptable range from 2.57 to 3.27%. Also, spray drying yield varied between 51.4 to 78.1%. The addition of gums or protein significantly enhanced both total phenolic content and total antioxidant capacity. The highest increase in total phenolic content was observed when gum Arabic was used along with maltodextrin as a coating material. Although significantly different results were obtained for most of the parameters tested for each wall material, all of them resulted in successful microencapsulation of black chokeberry pomace extract. However, within the tested wall materials, the maltodextrin:gum Arabic combination had better results compared to the other wall materials. For this reason, the next step of the study was continued with the spray-dried powders obtained by using maltodextrin: gum Arabic as a wall material. The effect of black chokeberry phenolics on the human gut microbiota in a sophisticated, computer-controlled dynamic colonic fermentation model (TIM-2) was investigated in Chapter 4. For this purpose, black chokeberry pomace as juice processing by-product, anthocyanin rich extract from black chokeberry pomace, and microencapsulated extract in maltodextrin-gum Arabic system were examined in terms of the changes in microbial composition, short-chain fatty acid (SCFA) and branched-chain fatty acid (BCFA) contents. Stool samples were collected from 5 healthy donors to prepare a standardized microbiota cocktail. The experiments in TIM-2 were last for 40h where the first 16h was adaptation period of the human fecal microbiota and the last 24h was the test period. Samples were collected from lumen and dial compartments at time 0h and 24h. Genomic DNA from the luminal samples was extracted and sequencing by polymerase chain reaction (PCR) amplification of the 16S rRNA gene V3-V4 region was carried out by using Illumina MiSeq and BCL2FASTQ pipeline. The QIIME2 (Quantitative Insights Into Microbial Ecology) software package was employed for taking sequencing data from raw sequences to interpretation for the microbiota analyses. The statistical analyses were done in RStudio. The abundances of microbial species in the total microbial community were calculated and shown as relative abundance (RA). According to the results, the fermentation of black chokeberry polyphenols in the in vitro colon model (TIM-2) resulted in shifts in the standardized microbiota and differentiation in the extent of the production of SCFA and BCFAs. Synergy between maltodextrin+gum Arabic+polyphenols resulted in an increase in the relative abundances of some health-promoting taxa (Anaerostipes, Blautia, Christensenellaceae R7 group, Prevotella 9) and decrease in the disease related taxa Alistipes. Encapsulation increased the SCFA production and decreased the BCFA production in the lumen. Nevertheless, none of the metabolites could be correlated with the identified operational taxonomic units. In the final chapter (Chapter 5), overall evaluation of the results obtained throughout this study, conclusions, and recommendations for the future research were presented. The main outcomes of this study revealed that pulp obtained from Turkish black chokeberries has a unique polyphenol profile with bioactive properties. The successful microencapsulation of polyphenols extracted from black chokeberry pulp can be used as a value-added natural colorant in powder form with bioactive properties in food, pharmaceutics or cosmetic product formulations. Also, clack chokeberry polyphenols present in the extract, pulp or encapsulate have the potential to be used for establishing a healthier gut as it caused a shift in the gut microbial composition and SCFA levels in a good way.
Investigating the valorisation potential of hazelnut by-products: Transforming waste into functional food ingredients

(Graduate School, 2022-12-09) Ceylan, Fatma Duygu ; Güven Çapanoğlu, Esra ; 506152501 ; Food Engineering

Hazelnuts are one of the most widely consumed nuts around the world. Considering the nutritional value of hazelnuts, a wide range of hazelnut-based food products are available in the market. Nevertheless, the processing of hazelnuts generates a large number of by-products and waste. The most valuable by-products of the hazelnut industry are shell, skin, and meal. These by-products are rich in bioactive compounds, protein, dietary fibre, mono- and polyunsaturated fatty acids, vitamins, minerals, phytosterols, and squalene. The current utilisation of hazelnut by-products is mostly limited to animal feed supplementation of hazelnut meal and skin and use as a low-value heat source for the shells. However, disposing of these by-products or using them as a low-value heat source or animal feed supplementation results in significant waste of a natural resource rich in nutritional components. Consequently, valorising hazelnut by-products as bioactive ingredients in diverse fields such as food, pharmaceutics and cosmetics has stimulated interest among scientists, producers, and consumers. In light of the above, a research strategy to investigate the valorisation potential of hazelnut by-products has been developed. The objectives of this Ph.D. dissertation were (i) to valorise hazelnut meal and explore the potential anti-obesity and antioxidant activities of its protein hydrolysates; (ii) to investigate the effect of the hydrolysis strategy (single or sequential hydrolysis) using Alcalase and Neutrase, as well as the application of microfluidization pretreatment on these activities and the functional properties of the protein isolates and hydrolysates; (iii) to examine the formation of the protein-polyphenol complex from dephenolised hazelnut meal protein isolates (dHPI) and hazelnut skin phenolic extracts (HSE); (iv) to monitor the bioaccessibility of hazelnut proteins and polyphenols after protein-polyphenol complexation. Two different research studies (Chapters 3-4) were conducted in line with these purposes. Firstly, hydrolysates of hazelnut proteins obtained with Alcalase and Neutrase were mainly examined for their physicochemical properties, potential anti-obesity effects, antioxidant capacities, and emulsifying properties (Chapter 3). Later on, protein-polyphenol complexes formed from dephenolised hazelnut meal protein isolates (dHPI) and hazelnut skin phenolic extracts (HSE) were investigated as well as their effects on bioaccessibility (Chapter 4). The background and objectives of this Ph.D. dissertation are introduced in the first chapter. Following that, an overview of current scientific knowledge about the main and most valuable hazelnut by-products and their actual valorisation, focusing on their chemical composition to inspire new applications of these valuable resources and fully exploit their potential, has been reviewed in the second part. In the third part, hazelnut meal protein hydrolysates obtained by a single or combined hydrolysis by Alcalase and Neutrase were mainly characterised for their physicochemical properties (SDS‒PAGE, particle size distribution, Fourier transform infrared, molecular weight distribution, etc.) and potential anti-obesity effect (FFA release inhibition), antioxidant activity (DPPH and ABTS methods), and emulsifying properties. The impact of microfluidization pretreatment was also investigated. The combination of Alcalase with Neutrase permitted the highest degree of hydrolysis (DH) (15.57%) of hazelnut protein isolate, which resulted in hydrolysates with the highest amount of low molecular weight peptides, as indicated by size exclusion chromatography (SEC) and SDS‒PAGE. There was a positive correlation between the degree of hydrolysis and the inhibition of FFA release by pancreatic lipase, with a significant positive effect of microfluidization when followed by Alcalase hydrolysis. Microfluidization enhanced the emulsifying activity index (EAI) of protein isolates and hydrolysates. Low hydrolysis by Neutrase had the best effect on the EAI (84.32 (NH) and 88.04 m2/g (MFNH)), while a negative correlation between the emulsifying stability index (ESI) and the DH was observed. Again, the combined Alcalase-Neutrase hydrolysates displayed the highest radical scavenging activities (96.63% DPPH and 98.31% ABTS). FTIR results showed that the application of microfluidization caused the unfolding of the protein structure. The individual or combined application of the Alcalase and Neutrase enzymes caused a switch from the β-sheet organization of the proteins to α-helix structures. In conclusion, hazelnut meal may be a good source of bioactive and functional peptides. The control of its enzymatic hydrolysis, together with an appropriate pretreatment such as microfluidization, may be crucial to achieve the best suitable activity. In the fourth part, the formation of the protein-polyphenol complex from dephenolised hazelnut meal protein isolates (dHPI) and hazelnut skin phenolic extracts (HSE), as well as its effect on the bioaccessibility of both hazelnut proteins and polyphenols, were investigated. The dHPI+HSE complexes were of considerable size and dependent on HSE concentration due to the occurrence of aggregation. Although catechin was the main component of HSE, it did not cause aggregation, except for a slight rise in particle size. According to fluorescence quenching, the hazelnut protein-phenolic extract complex had a linear Stern-Volmer plot expressing static quenching between 0-0.5 mM concentrations, and the interaction was mainly dependent on hydrogen bonding and van der Waals forces (ΔH<0 and ΔS<0) and the reaction was spontaneous (ΔG<0). According to Fourier Transform Infrared (FTIR) Spectroscopy results, higher phenolic extract concentration caused an increase in irregular structures in hazelnut protein, while the lowest phenolic concentration and catechin altered the regular structure. Skin extracts did not alter the digestibility of dephenolised proteins, but dephenolisation reduced the degree of hydrolysis by pancreatin. The formation of the protein-polyphenol complex had a beneficial effect on the bioaccessibility of hazelnut skin polyphenols predominantly on the gallolated form of the catechins such as gallocatechin gallate and epigallocatechin gallate. The final part presents the general discussions and conclusions, as well as future perspectives on the valorisation of hazelnut by-products, based on the findings of the previous chapters. A by-product of the hazelnut oil industry, hazelnut meal, was valorised as a source of bioactive peptides with an emphasis on their potential anti-obesity and antioxidant properties. First, hazelnut meal protein isolates were treated with microfluidization to improve their hydrolysis and functional properties. Then, sequential or individual hydrolysis by Neutrase and Alcalase was performed to prepare the protein hydrolysates. Finally, by combining Alcalase and Neutrase hydrolysis, we achieved the highest degrees of hydrolysis (DH), inhibiting FFA release by pancreatic lipase and scavenging free radicals. Regarding anti-obesity and antioxidant properties, hazelnut protein hydrolysates have the potential as functional food ingredients. Therefore, it is important to understand the process for protein processing (pretreatment, degree of hydrolysis, etc.) based on its intended application. Protein-polyphenol interactions improved the bioavailability of hazelnut skin polyphenols, particularly the gallolated form of catechins like gallocatechin gallate (GCG) and epigallocatechin gallate (EGCG). In recent years, researchers have focused on polyphenolic compounds and plant-based proteins isolated from natural sources. It is essential in vegan formulations such as foam-like products (mousse) or emulsions (mayonnaise), where the functional properties of proteins are crucial. Additionally, there is no consensus on whether protein-phenolic interactions affect polyphenol bioavailability positively or negatively. Consequently, more research needs to be conducted on this subject and reported in the literature.
Zein or gelatin nanofibers loaded with au nanospheres, SnO2 or black elderberry extract used as active and smart packaging layers for various fish fillets

(Graduate School, 2022-12-23) Çetinkaya, Turgay ; Altay, Filiz ; Ceylan, Zafer ; 506162508 ; Food Engineering

Fresh fish products spoil in a shorter time than other meat products. Consumers would prefer the freshest fish products that have higher initial quality. Therefore, it is important to investigate alternative methods to preserve the quality of fresh fish products and increase their shelf life. Furthermore, estimating the shelf life of fresh fish products in fast and easy methods has started to gain importance in recent years. These developments increase researchers' interest in active and smart packaging layers that are produced by nanotechnological methods. In this dissertation preparation, characterization, and practical application of biopolymer-based electrospun nanofibers as active and smart packaging layers are presented. First, the purpose and objectives are explained and recent studies are presented in Chapter l. Then, preliminary microbiological analyzes were applied to smoked fish products after their packages being opened (Chapter 2), suggesting that the smoking process inhibit microbial growth only if the package is not being opened. Then, the quality of three different fish meat samples (local salmon: YS, bream: Ç, sea bass: L) was determined in terms of the electrical conductivity value, surface tension values, and the ε″. Sensory evaluation results were also evaluated by photos and point scales. The electrical conductivity value of the YS, Ç, and L samples on the initial day (0.21, 0.24, and 0.233 mS cm-1) increased on the 4th day of storage (0.35, 0.39, and 0.47 mS cm-1), respectively (~65%, 63%, and 101% change). Dielectric loss factor values calculated at a frequency of 30 MHz were also increased with the highest change of L samples (29.68%) on the 4th day. On the contrary, surface tension values decreased with storage time. The surface tension of YS, Ç, L, declined from 38.22, 42.56, 37.57 mN m-1 to 16.2, 29.45, 25.94 mN m-1 on the 2nd day of storage. Results revealed these analyzes can be accepted as an important and rapid quality techniques in fish meat samples. Chapter 2 results revealed that active coating layers could be used to preserve shelf life and inhibit microbial growth. Therefore, in Chapter 3, skinless fish fillets nanocoated with fabricated AuZ-Nm (530 ± 377 nm) and TMAB growth at 4 ± 1 °C compared with the uncoated group during the 8-days storage. Microbiological results indicated that the use of zein nanofibers with gold nanospheres delayed the TMAB growth up to ~1 log CFU/g (p<0.05) and sensory deterioration. The monitoring dielectric properties of fresh fish fillets for evaluating their quality (Chapter 2), was also used in Chapter 3. In this sense, dielectric properties (ε′ and ε′′) of the fish fillets were treated with AuZ-Nm and were investigated. ε′ values of the uncoated fish samples were more variable (34.53%) compared to the nanocoated group (<30%) on the 7th day of storage. Similarly, a 40.55% decrease was recorded for the uncoated group according to the initial ε′′ value, while this decrease was 22.75% for nanocoated samples (p<0.05 between groups). It has been concluded that zein based nanofiber swith Au nanospheres have the potential as an active food packaging layer to extend the shelf life of fish fillets. In Chapter 4, the effect of electrical conductivity, ε′, ε′′, and loss tangent (ε′′/ε′) values (at 300 and 3000 MHz), on feed solution electospinnability was investigated. For the first time, the aggregation behavior of nanofibers was determined by ZetaSizer equipment. Electrospun samples dispersed in the ethanol had lower translational diffusion coefficient (water:2.03 μm2/s; ethanol: 1.85 μm2/s) and higher hydrodynamic radius (water: 242 nm; ethanol: 221 nm). Zein-gold nanofiber stability was also studied by zeta potential measurements (ethanol: +41.73 mV; water: +5.1 mV). In addition, the antimicrobial effects of AuZ-Nm were investigated and physicochemical characteristics were compared without Au. Specific shoulders in Au zein nanofiber spectrum indicated C=O carbonyl stretch vibrations in the amide I and amide II region, which does not appear for pure zein, but is observed also in the pure Au spectrum. After addition of Au, bands in the pure zein spectrum converted to stretching peaks, indicating the vibration frequency of Au–O ionic bond groups. These molecular observations and other signs (narrowing band, shifting wavenumber, transmittance changes) confirmed the successful integration of Au molecules. All these results indicated that although the process of smoking decreased the initial TMAB load, after opening the package, the TMAB values of the smoked fish increased more (Chapter 2). Using Au in nanofiber coating layers inhibited microbial growth more than the smoking process as explained in Chapter 3 and Chapter 4. However, since disruption is inevitable, it is also important to use these nanofiber materials as indicator layers to predict shelf life. In this context, gelatin-based nanofibers were developed to evaluate their color changing functions (Chapter 5). At first, the production of liquid gold NPs and dried gold nanopowders from gold salt (HAuCl4) was explained as stated in supplementary material. Then liquid gold added to feed solutions. SEM results of gelatin nanofibers the average diameter of pure gelatin nanofibers (GL) was 81.4 nm without any beads. The addition of 10% BE extract to the solution increased diameter to 277 nm (GLE). EDS elemental mapping peaks and Figures indicated that after 5% SnO2 incorporation into the feed solution, SnO2 NPs both encapsulated inside the nanofiber and attached to the surfaces of gelatin nanofibers (GLES). Small spots on nanofibers were observed with the addition of produced Au nanopowders at 2%, and the average diameter increased to 554 nm (GLESA). Nanofibers were deeply characterized by FTIR before and after exposed to fish meat for 30 hours (no direct contact). New methods were proposed for the first time to evaluate nanofiber stability by transmittance ratio values and produced degradation metabolites confirmed with the band differences in spectrums. Specific spectral changes indicated absorption/attachment of volatile amines to the nanofibers during the deterioration of fresh fish samples. Thermal stability between samples was compared by DSC and TGA. Thermal characteristic results of gelatin nanofiber samples also proved the new ionic bond complexes (initial) and thermal decomposition of absorbed volatiles (30th hour). Furthermore, L a b values of the nanofiber showed that interaction between flavonoids/phenolic acids and metal ions modified the color produced by the anthocyanins. Therefore, the lowest brightness values (L=64.23) with the highest redness (a=10.37) for the gelatin nanofiber that contains gold nanopowders (p<0.05) were obtained in the first measurement before the spoilage (GLESA). During 30 hour storage period the absorption of volatile amines influenced L a b in different directions. On the 24th hour, the color of the gold nanopowder added gelatin sample (GLESA) became more intense and turned to dark purple with the lowest b value (-3.11) and the lowest L value (24.14) (p<0.05; between nanofiber samples and between initial, 3rd, 6th hours). When all chapters are evaluated together, the results of this study enable a better insight into a) understanding of the determination of fish quality by rarely studied parameters such as electrical conductivity, surface tension, and dielectric loss factor, b) evaluation of various properties of prepared feed solutions for electospinnability, c) molecular, morphological, elemental characterization of nanofibers and NPs, d) searching aggregation behavior and stability of nanofibers by novel techniques such as DLS data calculations e) practical application of fabricated nanofibers containing Au nanospheres, SnO2, and BE extract on fish samples for gas sensing performance. These chapters guide possible nanoapplications of the nanofibers incorporated with antimicrobial agents, plant extracts, and metal-based NPs for active and intelligent packaging functions on meat products.
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.
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.
Production of functional food ingredient by enzyme and ultrasound assisted extraction from lemon waste

(Graduate School, 2024-03-15) Durmuş, Nihal ; Akyılmaz Kılıç, Meral ; 506152512 ; Food Engineering

Lemon (Citrus limon) belongs to the family of Citrus fruits which are commonly consumed in daily diet due to their high nutritional value, sensory properties and low price. A large portion of lemon is discarded after processing by the food industry as waste up to 50-70% of fruit weight depending on the cultivar. Citrus wastes are currently discarded to the environment or used for animal feed and biofuel production and these practices cause loss of valuable natural resources. Lemon peel constitutes a large portion of industrial lemon wastes contain several bioactive components such as phenolic acids and flavanones which exhibit several health benefits including antioxidant, antiobesity, antiinflammatory, antihypertensive, antihypercholesterolemic, antimicrobial, antidiabetic and anticarcinogenic activity. Most of the studies in the literature have focused on the extractable phenolics; however, significant amounts of phenolics in fruit peels are also present in non-extractable form as entrapped or bound in the cell matrix. Non-extractable phenolics are protected from harsh environmental conditions in the fruit matrix. They represent a potential uncovered resource for bioactive phenolics that could be used in food, nutraceutical, pharmaceutical and cosmetic industries. Determination of the distribution and amounts of phenolic compounds in extractable and non-extractable fractions can help in upcycling of lemon wastes. Recovery of non-extractable phenolics require use of an additional hydrolysis method to that used for extractable phenolics that needs to be optimized for an efficient production. Lemon peel constitutes a cheap potential resource for bioactive phenolics with antioxidant, antihypertensive and antidiabetic activities. Recovery of these bioactive compounds from lemon waste with green technologies will contribute to the efforts towards sustainable food systems and circular bioeconomy. The objectives of this study were (i) measurement of concentration and antioxidant activity of extractable phenolic compounds present in local lemon varieties, (ii) application of different extraction methods including conventional heat-, acid-, base-, ultrasound-, enzyme-, and ultrasound-enzyme-assisted extractions for maximum recovery of non-extractable phenolics found in lemon peel, (iii) determination of the antioxidant, antihypertensive, and antidiabetic activities of extractable and non-extractable fractions of phenolics in lemon peel (iv) production of an encapsulated functional food ingredient from lemon peel extract. The first part of the thesis is focused on determination of phenolic compounds and their antioxidant activity in different local lemon varieties. In addition, the solvent type and solid:solvent ratio for an efficient extraction were determined. Interdonato, Lamas, Kara lemon and Kütdiken lemon varieties used in the fruit juice industry in Türkiye were chosen for the study. Lemon peels were cut into small pieces and dried with a freeze dryer. Dried peels were ground into a powder and then sieved. Lemon powder was extracted with ethanol/water (50:50, v/v) or distilled water with a solid:solvent ratio of 1:10 or 1:20 (g/mL). The resulting extracts contained extractable phenolics. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity by 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cupric reducing antioxidant capacity (CUPRAC) assays were carried out. As a result of the analyses, TPC in lemon peels was found between 12.06±0.22-23.63±0.11 mg GAE/g dry matter, while the TFC was found between 10.02±0.99-30.10±0.33 mg RE/g dry matter. TPC of samples extracted with 50% aqueous ethanol was higher than that of the samples extracted with distilled water. Antioxidant activities of lemon peels were found in the range of 30.54±0.19-60.64±2.78 mg TE/g dry matter and 38.20±1.09-72.63±0.73 mg TE/g dry matter by ABTS and CUPRAC methods, respectively. It was revealed that Kara lemon had the maximum antioxidant activity, followed by Lamas, Interdonato, and Kütdiken variety. Considering all the results, 50% aqueous ethanol as a solvent, a sample:solvent ratio of 1:20 (g/mL), and the Lamas cultivar were chosen for further studies. The choice of the Lamas species was motivated by the fact that it is a local species unique to Mersin Province, Erdemli District, and is readily available. In the second part, effect of different extraction methods for recovery of non-extractable phenolics (NEP) in Lamas lemon peel was investigated. For this purpose, conventional heat-, acid-, base-, ultrasound-, enzyme-, and ultrasound-enzyme-assisted extractions were applied to the residue obtained after the extraction of extractable phenolics. To determine the maximum amount of non-extractable phenolics in the residues, acid and alkaline hydrolyses were applied. Alkali and then acid hydrolysis allowed the highest recovery. Preliminary experiments were performed to find the optimal parameters in all extraction methods. Preliminary trials were carried out for the conventional heat-assisted extraction method, with temperatures of 23, 40 and 60°C, duration of 40 min, 60 min, and 6 h. The best result was obtained by extraction at 40°C for 1 h. Ultrasound-assisted extraction (UAE) was applied at the amplitude level of 20 and 50%, temperature of 23 and 50°C, and duration of 5, 10, and 15 min. The optimum parameters obtained for maximum TPC, TFC and antioxidant activity were 50% ultrasound amplitude, the sample inlet temperature of 23°C and the extraction time of 15 min. When the effects of process parameters on bioactive compounds in ultrasound-assisted extraction were investigated, it was determined that the effect of ultrasound amplitude was statistically significant on TPC, TFC, and antioxidant activity with both ABTS and CUPRAC methods. Effect of extraction time had a significant effect TFC and antioxidant activity. In enzyme-assisted extraction, a lysing enzyme from Aspergillus sp. containing cellulase and pectinase, β-glucosidase, and a mixture of lysing enzyme and β-glucosidase were used. The residue was incubated at 40°C for 15 min, 60 min and 6 h. Hydrolysis with lysing enzyme for 60 min was found sufficient to release all phenolics. Ultrasound-enzyme-assisted extraction was carried out to determine if ultrasound in combination with enzyme would improve the extraction of phenolics. While in the first approach, enzyme and ultrasound were applied simultaneously, in the second way, ultrasound was applied to the residue first and then incubated with the enzyme. Application of ultrasound first and then enzyme hydrolysis provided higher TFC and antioxidant activity by ABTS and CUPRAC assays. Bioactivities of non-extractable phenolics from lemon peel obtained by conventional heat-, enzyme-, ultrasound-, and ultrasound-enzyme-assisted extractions were evaluated in comparison with those of the extractable phenolics. The antioxidant, angiotensin-I-converting enzyme (ACE), and α-amylase inhibitory activities and phenolic profile of the phenolic fractions were analyzed. While the extractable fraction had higher TPC, ascorbic acid content, and antioxidant activity, phenolic profile analysis indicated that the non-extractable fraction contained higher concentrations of phenolics, especially hesperidin and hesperetin. The concentrations of hesperidin and hesperetin in the non-extractable fraction were 270.9 mg/100 g dry weight and 415.9 mg/100 g dry weight, respectively, which were about two-fold higher than those present in the extractable fraction. Moreover, ACE and α-amylase inhibitory activities of non-extractable fraction were stronger than those of the extractable fraction. Total phenolic content, total flavonoid content, and antioxidant activity were increased by enzyme and ultrasound treatments compared to those by conventional heat treatment. However, ACE inhibitory activities of all the non-extractable fractions were similar while α-amylase inhibitory activity was higher in ultrasound- and ultrasound-enzyme-treated fractions. While ultrasound-assisted extraction slightly improved the yield of non-extractable phenolics, enzyme-assisted extraction yielded two-to four-fold increases in the amounts of individual phenolic compounds compared to heat-assisted extraction. Non-extractable phenolic fraction obtained by enzyme-assisted extraction from lemon peel was found to have a significant potential as an antihypertensive and antidiabetic agent. In the third part, encapsulation of the phenolic extract obtained by enzyme-assisted extraction with maltodextrin and whey protein concentrate was studied. Two maltodextrins with 6 and 19 dextrose equivalent (DE) were used. Two different drying methods including foam mat drying and foam mat freeze drying were applied. Encapsulated extract was analysed for encapsulation efficiency, moisture content, water activity, and glass transition temperature. In addition, encapsulated extract was stored at 40°C and 75% relative humidity for 5 d to determine storage stability. TPC, antioxidant activity, hesperidin content, ACE inhibitory activity, and α-amylase inhibitory activity were measured at the beginning and end of storage. Maltodextrin with 6 DE along with whey protein concentrate was found to be the best wall material. Foam mat-dried sample with maltodextrin 6DE showed the highest encapsulation efficiency, hesperidin content, storage stability and lowest hygroscopicity, while foam mat-freeze-dried sample with maltodextrin 6DE exhibited the highest TPC and antioxidant activity. There was no difference among ACE inhibitory activity of the samples before and after storage. ACE inhibitory activity was reduced after storage in all the samples. On the other hand, -amylase activity of the samples did not show a clear trend as a result of interference from the wall materials. Based on these findings, both foam mat-drying and foam mat freeze-drying can be chosen as the drying method in encapsulation. However, foam mat drying can be preferred as a practical method with low equipment and energy costs and short processing time. In this study, an encapsulated functional ingredient including both extractable and non-extractable phenolic compounds from lemon peel with significant antioxidant, ACE and α-amylase inhibitory activity was developed. The ingredient can be utilized for development of antihypertensive and antidiabetic functional foods or food supplements. Further in vitro and in vivo studies on stability, bioavailability, toxicity and health effects of the ingredient are recommended before its use.
Proteins and bioactive peptides extraction from food wastes and their modification by different processes to produce functional food products

(Graduate School, 2024-05-15) Şenol, Ezgi ; Özçelik, Beraat ; 506182503 ; Food Engineering

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.
Potential of lactic acid bacteria fermentation as a strategy for valorisation and biotransformation of mushrooms

(Graduate School, 2024-08-15) Sümer Ayar, Eda Nur ; Özçelik, Beraat ; 506172502 ; Food Engineering

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.
UV-C ışık uygulamasının taze kesilmiş deveci armutunun dekontaminasyonu ve kalite parametreleri üzerine etkisi

(Lisansüstü Eğitim Enstitüsü, 2024-09-04) Korkut Oğuz, Gözde ; Güneş, Gürbüz ; 506162505 ; Gıda Mühendisliği

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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