Ενθυλάκωση βιοδραστικών ενώσεων σε φυσικά πολυμερή με εφαρμογή καινοτόμων μεθόδων νανοεγκλεισμού με σκοπό την ενσωμάτωσή τους σε συστήματα τροφίμων
Authors: Δρόσου Χριστίνα 
Published: November 2021

The development of functional foods, which are enriched with bioactive ingredients providing beneficial properties to human health, is an area of growing interest for the food industry. Among the bioactive ingredients, which are widely used in the food industry improving the nutritional characteristics and organoleptic properties of existing food products, carotenoids play an important role. The most commonly used carotenoids in the food industry are β-carotene, lycopene, astaxanthin, lutein and zeaxanthin, which are incorporated into foods due to their antioxidant activity and coloring properties. β-carotene, which is a precursor of vitamin A, is the most common of all carotenoids and is a lipophilic micronutrient that is important for maintaining human health. However, the use of β-carotene in functional foods and supplements is currently limited due to its low solubility in water and its chemical instability when exposed to environmental factors. Therefore, a challenge for the food industry is to develop stable food products by maintaining the bioavailability of the active ingredients during their processing and storage.In this context, various techniques have been developed in order to enhance the stability and bioavailability of bioactive ingredients. Among them, encapsulation techniques have found wide application in the food industry and are based on trapping an active ingredient in a food-suitable carrier in order to protect it against environmental conditions and enhance its targeted delivery (release). There are many encapsulation techniques that can be used to develop carrier systems, and depending on the choice of method and its operating conditions, the functional properties of the structures are affected, such as morphology, micro- and macrostructure, encapsulation efficiency, stability and prolonged release of the active ingredient. Therefore, the selection of the appropriate encapsulation method is necessary for the development of encapsulation products with desired properties depending on the characteristics of the carrier food to be incorporated and the physical and chemical properties of the core and coating materials.The aim of this PhD dissertation was to study the encapsulation of β-carotene in natural polymers and to test its ability to be incorporated into food systems. The present PhD dissertation focuses on the study of the effect of the encapsulation technique on the properties of the encapsulation products and on the 'shelf life' of β-carotene during storage under various conditions. The encapsulation techniques studied were the two conventional encapsulation methods, spray drying and freeze-drying, as well as the innovative electrospinning process. Biopolymers such as pullulan (PUL) and whey protein isolate (WPI), which are widely used as matrices for the encapsulation of bioactive substances in food products due to their functional and nutritional properties, have been selected as encapsulation carriers.Initially, the development of innovative electrospun nanofibers from blends of WPI and PUL through electrospinning process was investigated in order to be used as β-carotene encapsulation matrices for functional food applications. More specifically, the effect of different blend proportions of the two biopolymer systems (WPI: PUL, 20:80, 30:70, 50:50, 70:30 and 80:20 w/w, respectively) on the morphology and diameter of the developed structures was investigated and the results were correlated with the properties of the solutions of the various blends, such as viscosity, surface tension and electrical conductivity. In addition, the effect of the process operating parameters on the fiber morphology, such as solution flow rate (0.4-0.6 mL/h), applied voltage (18-22 kV) and the tip-to-collector distance (8-12 cm), was studied using a central composite design (CCD). The morphology and diameter of the structures were determined by scanning electron microscopy (SEM) and the molecular interactions between biopolymers was examined by attenuated total reflectance infrared spectroscopy (ATR-FTIR). Finally, the thermal properties of the structures, such as glass transition temperature (Tg) and thermal stability, were determined through Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The addition of pullulan in the blends, at a concentration greater than 50% wt., resulted in increased viscosity and lower conductivity of the solutions which were beneficial to form more uniform nanofibers with diameters of around 231 nm. In addition, increasing the concentration of PUL in the solution led to the formation of larger fiber diameters. In terms of process operating parameters, the fiber diameter increased as the flow rate augmented, while the effect of this parameter diminished when the solution viscosity was low. On the other hand, applied voltage and tip-to-collector distance did not have excessive influence on the size of the nanofibers. Therefore, solution properties are the most significant parameter for the electrospinning process using such hydrocolloid mixtures. Infrared spectroscopy indicated that the composite fibers exhibited spectral features of both polymeric components, besides pointing to intermolecular interactions between the polymeric species. Regarding the thermal properties of the structures, Tg ranged from 32.2°C to 50.6°C and its value decreased with increasing concentration of PUL in nanofibers. Finally, WPI:PUL fibers showed slightly better thermal stability than that of the pure WPI which seems to concur with the interactions noted between the protein components and the polysaccharide chains by the FTIR spectroscopy.Subsequently, the development of b-carotene loaded structures was performed by electrospinning process (EL), spray drying (SP) and freeze-drying (FZ) using WPI and PUL as wall materials. For each encapsulation method, the effect and optimization of the operating parameters in terms of β-carotene encapsulation efficiency was studied using the response surface methodology (RSM) and central composite design (CCD). Specifically, regarding the electrospinning process, the operating parameters studied were the solution flow rate of the wall material (X1: 2.0-2.4 mL/h), the solution flow rate of β-carotene solution (X2: 0.1-0, 2 mL/h), the applied voltage (X3: 19-23 kV) and the tip-to-collector distance (X4: 17-19 cm) using a mixture of WPI: PUL (30:70) and PUL as wall materials. As regards spray drying, the emulsion concentration (X1: 4-8% w/w), the emulsion flow rate (X2: 200-800 mL/h) and the inlet air temperature (X3: 160-180 °C) were studied, while regarding freeze-drying, the effect of the emulsion concentration (4-8% w/w) on the encapsulation efficiency of β-carotene was examined using a mixture of WPI: PUL (80:20) as carrier. Encapsulation efficiency was determined using a High Performance Liquid Chromatography (HPLC) system and the presence of b-carotene in the different structures was confirmed using the ATR-FTIR technique. The morphology of the obtained fibers was studied using SEM. The adsorption isotherms as well as the Tg values in various storage humidities (11%-95%) were determined for the encapsulated β-carotene structures resulting from the optimal process conditions. In addition, the stability of the encapsulated β-carotene was studied during storage under various conditions of temperature (25°C, 35°C, 45°C), relative humidity (aw: 0.11, 0.33, 0.53, 0.75, 0.95) and exposure to ultraviolet radiation. To evaluate the results of the application of the various encapsulation techniques studied, the encapsulation efficiency of the process, estimated as the percentage of retention of the active substance in relation to its initial quantity, and the retention of β-carotene during storage were used as criteria. The results of the study of the kinetic degradation of β-carotene at different temperatures and water activities were correlated with the physical state (through the study of sorption isotherms) and the Tg of the encapsulation structures.Regarding electrospinning process, β-carotene was successfully encapsulated in WPI:PUL (30:70) and PUL fibers with an average encapsulation efficiency of 80.52% and 88.98%, respectively. Therefore, PUL led to the development of structures with higher encapsulation efficiency compared to WPI:PUL blend due to the higher viscosity (3.67 ± 0.04 Pa s) and the lower conductivity (0.35 ± 0.02 mS / cm) of the PUL solution compared to WPI: PUL blend (0.59 ± 0.01 Pa s and 0.60 ± 0.02 mS / cm) resulting in a more stable Taylor cone and charged jet. Based on the results of the response surface methodology, the maximum β-carotene encapsulation efficiency was found in the following experimental conditions: X1 = 2.0 mL/h, X2 = 0.1 mL/h, X3 = 19 kV and X4 = 17 cm, as well as X1 = 2.4 mL/h, X2 = 0.1 mL/h, X3 = 23 kV and X4 = 19 cm using WPI: PUL and PUL, respectively, as encapsulation carriers. In addition, the flow rate of the β-carotene solution was the parameter with the most significant effect on the encapsulation efficiency for both of the wall materials used and its increase led to a decrease in the encapsulation efficiency. Regarding the study of the morphology of the encapsulation structures, it is observed that the use of PUL as wall material led to the development of uniform fibers, while the presence of WPI in the shell solution led to the development of fibers with the presence of elongated beads with diameters of 530,36 and 444,84 nm, respectively. Based on the study of adsorption isotherms, WPI:PUL (30:70) blend fibers showed higher percentages of moisture content than PUL fibers for each water activity and storage temperature. Also, PUL fibers with encapsulated β-carotene showed lower Tg values than WPI:PUL blend fibers at all levels of humidity-temperature studied, ranging from 34.14°C to 79.88°C and from 43.18°C to 83.44°C, respectively. For spray drying, β-carotene was effectively encapsulated in WPI:PUL (80:20) blend encapsulation structures with an average encapsulation efficiency of 71.92%. The maximum β-carotene encapsulation efficiency was found in the following experimental conditions: X1 = 6.0% w/w, X2 =500 mL/h, X3 = 170°C. The variables with the greatest effect on β-carotene encapsulation efficiency were emulsion concentration and emulsion flow rate, while inlet air temperature did not contribute significantly, at least in the range of values studied (160-180°C). The encapsulation products obtained by spray drying showed a spherical shape with irregular surfaces, without visible cracks or fractures in the whole range of concentrations examined (4-8% w/w), with an average diameter of 6.53 μm. The Tg of the WPI:PUL particulate carriers ranged from 61.01°C to 87.17°C at the three storage temperatures examined (25, 35, 45°C) and it was observed that an increase in the moisture content of the samples led to a decrease in Tg while an increase in the storage temperature of the samples led to higher Tg values. For freeze-drying, successful development of encapsulated β-carotene structures was performed using a mixture of WPI:PUL (80:20) as an encapsulation carrier, with an average encapsulation efficiency of 66.36%. The maximum β-carotene encapsulation efficiency was found for emulsion concentration equal to 8% w/w. The encapsulation products showed an amorphous structure with a leafy appearance, which is a characteristic morphological structure of the lyophilized products. The Tg of the encapsulation structures also decreased with increasing storage humidity, with its value ranging from 59.01°C to 93.25°C, for the three storage temperatures of the product. Based on the above, it is concluded that, depending on the encapsulation technique applied, significant differences were observed in the morphology, the encapsulation efficiency of β-carotene and the thermal properties of the structures produced. More specifically, each method led to the development of structures with different morphology so that it is possible to choose the appropriate structure to be used depending on the desired final application of the encapsulation products. Regarding the encapsulation efficiency of the three methods tested, the EL encapsulation structures showed the highest encapsulation efficiency, followed by SP and FZ. In any case, the encapsulation efficiencies observed were very high for all three methods tested (> 65%), which proves the suitability of these techniques for the development of β-carotene encapsulation structures.Regarding the degradation study of the encapsulated β-carotene under exposure to ultraviolet radiation, the degradation reaction rate constant (k) showed the lowest value for SP structures, followed by EL-WPI:PUL, FZ and EL-PUL ones. Compared to the half-life value of free β-carotene, its encapsulation in WPI and PUL carriers led to an increase in half-life by 11.01, 4.50, 3.36, 1.16 times for the SP, EL-WPI:PUL, FZ and EL-PUL structures, respectively. Regarding the degradation rate study of encapsulated β-carotene during storage under various humidity and temperature conditions, this process was found to be satisfactorily described by the first-order kinetic reaction model for all encapsulation structures developed. In addition, in all the encapsulation structures studied an obvious increase in the rate constant (k) and a corresponding decline in the half-life values of β-carotene was observed with increasing humidity and storage temperature. Specifically, the k values increased with the increase of aw, noting a maximum value in the intermediate water activity (aw= 0.53), and then decreased for aw= 0.75 and 0.95 due to the structural collapse of the samples. In contrast, the storage of structures in environments with low water activity (aw= 0.11 and 0.33) led to increased protection of β-carotene. Comparing the encapsulation structures developed in terms of β-carotene retention, it is concluded that the SP structures showed the highest β-carotene stability, followed by the EL and FZ structures in all water activities and temperatures studied. Specifically, at water activity aw= 0.11 and storage temperature of 25°C, where the maximum stability of the encapsulated β-carotene was noted, its half-life was 130.27, 22.16, 18.68 weeks for SP, EL-WPI:PUL and FZ structures, respectively. Finally, the determination of Tg was not found to be a reliable approach to be used effectively in predicting β-carotene stability. More specifically, it has been found that significant loss of β-carotene occurs even at temperatures below the Tg temperature of the amorphous materials used as encapsulation carriers.Finally, the encapsulated structures were incorporated into white chocolate and the effect of the incorporation of β-carotene in free (CF) or encapsulated form (CEL, CS, CFZ through EL, SP, FZ, respectively) on their quality characteristics (such as color, melting temperature, viscosity and hardness), the shelf life of the quality characteristics of the products and the stability of the β-carotene during storage of the products for a period of 4 months at room temperature was investigated. Based on the results of the effect of the incorporation of the β-carotene encapsulation structures on the quality characteristics of the white chocolate products, differences are observed depending on the encapsulation technique applied for the development of the β-carotene structures. In particular, in terms of the thermal properties of the products, there were no significant differences between the peak temperature and the completion of the melting temperature of the samples with encapsulated β-carotene in relation to the control sample. However, the CF and CS samples showed lower melting energy (apparent enthalpy) than the control sample, in contrast to the CFZ and CEL samples which showed higher. Therefore, the CFZ and CEL samples show a more compact structure than the other samples tested. Regarding the rheological properties, the plastic viscosity of the CF and CS samples did not differ significantly from the CC control sample (p<0.05), while the viscosity of the CFZ and CEL samples was higher than the CC, with a maximum for the CFZ sample. Regarding the yield stress, all the samples examined showed significant differences compared to the control sample (p<0.05) and especially in the case of the CEL sample where the yield stress value was much higher than the other samples. The hardness of the CF and CS samples did not differ significantly from the CC sample, in contrast to the CFZ and CEL samples. The CFZ sample noted slightly increased hardness compared to the control sample while the CEL sample showed lower. Regarding color changes from the addition of encapsulated and non- encapsulated β-carotene, the incorporation of encapsulated β-carotene structures has been an effective method of masking the intense color of β-carotene. Compared to the encapsulation structures incorporated in white chocolate, the structures obtained through electrospinning process led to the development of white chocolate products that approach the values of parameters a* and b* of the reference sample. This is followed by the CS sample, while the CFZ sample approaches the values of the CF sample with free β-carotene. Based on the results of the shelf life of the quality characteristics of white chocolate products, an increase in melting point, values of rheological parameters and hardness was observed with increasing storage time while the values of color indicators a* and b* decreased for all samples examined. Among the encapsulation carriers studied, CS white chocolate products provided the best protection in terms of color stability, followed by CEL and CFZ samples. Also, the incorporation of β-carotene in encapsulated form into white chocolate products has led to better color stability than non-encapsulated form. Finally, the degradation rate constant showed the lowest value and respectively the longest half-life for CS samples, followed by CEL and CFZ samples. Compared to the half-life of β-carotene in CF samples, its incorporation into encapsulation carriers increased the half-life by 12.39, 2.27, 4.22 times for CS, CFZ and CEL products, respectively.The present study therefore concludes that encapsulation is an effective technique for enhancing the shelf life of β-carotene and its subsequent incorporation into food systems thus leading to the production of new/ innovative functional products of high standards in terms of their bio-functional characteristics.