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ÖgeZein 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)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.
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ÖgeValorization 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)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.
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ÖgeEncapsulation of echium oil and saffron extract in electrospun nanofibers(Graduate School, 2022-11-15)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.
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ÖgeInvestigating the valorisation potential of hazelnut by-products: Transforming waste into functional food ingredients(Graduate School, 2022-12-09)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.
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ÖgeBioactive peptide encapsulation by electrospinning technique: Characterization of electrospun fibers and mathematical modelling of release kinetics(Graduate School, 2023-06-12)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.