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ÖgeEmerging approaches for non-thermal modification of proteins isolated from de-oiled sunflower cake(Graduate School, 2021-09-10)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.
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ÖgeEffects of novel food processing techniques on bioaccessibility and transepithelial transport of cranberrybush polyphenols(Graduate School, 2021-08-06)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.
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ÖgeEncapsulation and release of amino acids in double emulsions(Graduate School, 2021-03-18)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.
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ÖgePhenolic and carotenoid profiles of tomatoes collected from different parts of Turkey and antioxidant properties of dried tomatoes(Graduate School, 2021-02-18)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.