Development and Characterization of Microencapsulation Systems for Bioactive Ingredients of Interest in the Development of Functional Foods

Tesis por compendioThe aim of the present work was to design and characterize novel encapsulation structures of interest in the development of functional foods. For this purpose, various biopolymer-based microencapsulation structures were obtained through different processing technologies, with an emphasis on electrospraying as an advantageous alternative to conventional microencapsulation techniques. Firstly, novel microencapsulation structures were produced from aqueous solutions and in mild conditions by electrospraying, using different biopolymers as encapsulation matrices. For this purpose, the processing conditions were optimized and the relationship between the feed solution properties and the morphology of the electrosprayed materials was studied. The developed microstructures were then used to microencapsulate model bioactive ingredients with different properties, including hydrophilic molecules, hydrophobic compounds and probiotic microorganisms. For the hydrophobic ingredients, different strategies were evaluated in order to disperse them within the aqueous biopolymer solutions, such as the preparation of emulsions and liposome dispersions prior to electrospraying. An in-line setup for the continuous mixing of liposomes with the biopolymer and their subsequent hybrid capsule formation was also developed by combining microfluidic and electrospraying technologies. For the probiotic microorganisms, the convenience of preparing the feed suspensions from fresh culture or freeze-dried bacteria, as well as the impact of adding a surfactant and a prebiotic carbohydrate to the formulation, were also evaluated. The performance of the proposed encapsulation systems was evaluated in terms of microencapsulation efficiency, stabilization of the bioactive ingredients against degradation under detrimental conditions and/or their impact on the bioaccessibility of the microencapsulated ingredients after in-vitro digestion. Spray-drying was also used to microencapsulate some of the bioactive ingredients, in order to compare the results obtained through electrospraying with a more conventional encapsulation technique and using different encapsulation matrices. Additionally, a novel concept of bio-inspired encapsulation was proposed in this thesis: the potential of intact plant cells isolated from potato tubers to bind phenolic compounds was explored, and the impact of starch gelatinization on the loading capacity of these proposed encapsulation vehicles was also assessed. Finally, the impact of microencapsulation in real food systems was also studied. Yoghurts and biscuits were enriched with a peptide hydrolysate and a green tea extract, respectively, and the stabilization effect of the protective matrices during food manufacturing was assessed. The consumers' acceptability of the enriched biscuits was also studied.El objetivo de este trabajo fue el diseño y caracterización de nuevas estructuras de encapsulación de interés en el desarrollo de alimentos funcionales. Para ello, se obtuvieron distintas microestructuras de encapsulación biopoliméricas utilizando diferentes tecnologías de procesado, con especial hincapié en el electroesprayado como alternativa ventajosa a las técnicas de microencapsulación convencionales. En primer lugar, se desarrollaron nuevas microestructuras de encapsulación a partir de disoluciones acuosas y en condiciones suaves mediante la técnica de electroesprayado, utilizando diferentes biopolímeros como matrices de encapsulación. Para ello, se optimizaron las condiciones de procesado y se estudió la relación existente entre las propiedades de las disoluciones de partida y la morfología de los materiales electroesprayados obtenidos a partir de ellas. Posteriormente, se utilizaron las estructuras desarrolladas para microencapsular ingredientes bioactivos modelo con diferentes propiedades, incluyendo moléculas hidrofílicas, compuestos hidrofóbicos y microorganismos probióticos. En el caso de los ingredientes hidrofóbicos, se evaluaron diferentes estrategias para dispersarlos en las disoluciones poliméricas acuosas, como la preparación de emulsiones y de liposomas para su posterior procesado mediante electroesprayado. También se desarrolló un proceso en línea para mezclar los liposomas con el biopolímero y electroesprayarlos en continuo, combinando las tecnologías de microfluídica y electroesprayado. En el caso de los microorganismos probióticos, se evaluó también la conveniencia de preparar las suspensiones bacterianas a partir de cultivo fresco o de liófilos, así como el impacto de añadir un surfactante y un carbohidrato prebiótico a la formulación. El comportamiento de los sistemas de encapsulación propuestos se evaluó en cuanto a eficiencia de encapsulación, estabilización de los ingredientes bioactivos frente a condiciones de estrés y/o impacto sobre la bioaccesibilidad de dichos ingredientes tras un proceso de digestión in-vitro. También se utilizó la técnica de secado por pulverización para microencapsular alguno de los ingredientes bioactivos, con el fin de comparar los resultados obtenidos mediante electroesprayado con los de una técnica de encapsulación convencional, y utilizando diferentes matrices de encapsulación. Además, en esta tesis se propone un nuevo concepto de encapsulación bio-inspirada, basada en el potencial de células vegetales de patata intactas para unirse a compuestos fenólicos. Asimismo, se evaluó el efecto de la gelatinización previa del almidón presente en las células en la capacidad de carga de estos potenciales vehículos de encapsulación. Por último, se estudió el impacto de la microencapsulación en alimentos reales. Se enriquecieron yogures y galletas con un hidrolizado de péptidos y un extracto de té, respectivamente, y se estudió el efecto estabilizador de las microcápsulas durante el procesado de estos alimentos. También se estudió la aceptabilidad por parte de los consumidores de estas galletas enriquecidas.L'objectiu d'aquest treball va ser el disseny i caracterització de noves estructures d'encapsulació d'interès en el desenvolupament d'aliments funcionals. Amb aquesta finalitat, es van obtenir diferents microestructures d'encapsulació biopolimèriques utilitzant diferents tecnologies de processat, amb especial èmfasi en el electrosprayat com a alternativa avantatjosa a les tècniques de microencapsulació convencionals. En primer lloc, es van desenvolupar noves microestructures d'encapsulació a partir de dissolucions aquoses i en condicions suaus mitjançant la tècnica de electrosprayat, utilitzant diferents biopolímers com a matrius d'encapsulació. Per a això, es van optimitzar les condicions de processament i es va estudiar la relació existent entre les propietats de les dissolucions de partida i la morfologia dels materials electrosprayats obtinguts a partir d'elles. Posteriorment, es van utilitzar les estructures desenvolupades per microencapsular ingredients bioactius model amb diferents propietats, incloent molècules hidròfiles, compostos hidrofòbics i microorganismes probiòtics. En el cas dels ingredients hidrofòbics, es van avaluar diferents estratègies per a dispersar-los en les dissolucions polimèriques aquoses, com la preparació d'emulsions i de liposomes per al seu posterior processat mitjançant electrosprayat. També es va desenvolupar un procés en línia per a mesclar els liposomes amb el biopolímer i electrosprayar-los en continu, combinant les tecnologies de microfluídica i electrosprayat. En el cas dels microorganismes probiòtics, es va avaluar també la conveniència de preparar les suspensions bacterianes a partir de cultiu fresc o de liòfils, així com l'impacte d'afegir un surfactant i un carbohidrat prebiòtic a la formulació. El comportament dels sistemes d'encapsulació proposats es va avaluar respecte a eficiència d'encapsulació, estabilització dels ingredients bioactius front a condicions d'estrès i / o l'impacte sobre la bioaccesibilitat d'aquests ingredients després de la seva digestió in-vitro. També es va utilitzar la tècnica d'assecat per polvorització per microencapsular algun dels ingredients bioactius, per tal de comparar els resultats obtinguts mitjançant electrosprayat amb els d'una tècnica d'encapsulació convencional, i utilitzant diferents matrius d'encapsulació. A més, en aquesta tesi es proposa un nou concepte d'encapsulació bio-inspirada, basada en el potencial de cèllules vegetals de creïlles intactes per unir-se a compostos fenòlics. Així mateix, es va avaluar l'efecte de la gelatinització prèvia del seu midó en la capacitat de càrrega d'aquests potencials vehicles d'encapsulació. Finalment, es va estudiar l'impacte de la microencapsulació en aliments reals. Es van enriquir iogurts i galetes amb un hidrolitzat de pèptids i un extracte de té, respectivament, i es va estudiar l'efecte estabilitzador de les microcàpsules durant el processat dels aliments. També es va estudiar l'acceptabilitat per part dels consumidors d'aquestes galetes enriquides.Gómez Gómez-Mascaraque, L. (2017). Development and Characterization of Microencapsulation Systems for Bioactive Ingredients of Interest in the Development of Functional Foods [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/93344TESISCompendi

Protein-based emulsion electrosprayed micro- and submicroparticles for the encapsulation and stabilization of thermosensitive hydrophobic bioactives

This work shows the potential of emulsion electrospraying of proteins using food-grade emulsions for the microencapsulation and enhanced protection of a model thermosensitive hydrophobic bioactive. Specifically, gelatin, a whey protein concentrate (WPC) and a soy protein isolate (SPI) were compared as emulsion stabilizers and wall matrices for encapsulation of α-linolenic acid. In a preliminary stage, soy bean oil was used as the hydrophobic component for the implementation of the emulsion electrospraying process, investigating the effect of protein type and emulsion protocol used (i.e. with or without ultrasound treatment) on colloidal stability. This oil was then substituted by the ω-3 fatty acid and the emulsions were processed by electrospraying and spray-drying, comparing both techniques. While the latter resulted in massive bioactive degradation, electrospraying proved to be a suitable alternative, achieving microencapsulation efficiencies (MEE) of up to ∼70%. Although gelatin yielded low MEEs due to the need of employing acetic acid for its processing by electrospraying, SPI and WPC achieved MEEs over 60% for the non-sonicated emulsions. Moreover, the degradation of α-linolenic acid at 80 °C was significantly delayed when encapsulated within both matrices. Whilst less than an 8% of its alkene groups were detected after 27 h of thermal treatment for free α-linolenic acid, up to 43% and 67% still remained intact within the electrosprayed SPI and WPC capsules, respectively.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO project AGL2012-30647 for financial support. Sara Díaz Cuesta is also acknowledged for experimental support.Peer reviewe

Stability and bioaccessibility of EGCG within edible micro-hydrogels. Chitosan vs. gelatin, a comparative study

Micro-hydrogels are very promising systems for the protection and controlled delivery of sensitive bioactives, but limited knowledge exists regarding the impact of this encapsulation on their bioaccessibility. In this work, two different hydrogel-forming biopolymers (gelatin and chitosan) were compared as wall materials for the microencapsulation of a model flavonoid, (−)-epigallocatechin gallate (EGCG). Results showed that gelatin was more adequate as wall material for the encapsulation of EGCG than chitosan, achieving higher encapsulation efficiencies (95% ± 6%), being more effective in delaying EGCG release and degradation in aqueous solution and exhibiting a 7 times higher bioaccessibility of the bioactive compound (in terms of antioxidant activity) after in-vitro gastrointestinal digestion. A very low bioaccessibility of EGCG in chitosan was observed, due to the neutralization of the carbohydrate in the basic simulating salivary conditions, thus precluding subsequent flavonoid release. Moreover, gelatin micro-hydrogels also hindered dimer formation during in-vitro digestion, thus suggesting greater bioavailability when compared with free EGCG.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO projectAGL2015-63855-C2-1 for financial support.Peer reviewe

Tannin-rich extracts improve the performance of amidated pectin as an alternative microencapsulation matrix to alginate

This work was funded by Teagasc, the Irish Agricultural and Food Development Authority (Fermoy, Ireland), by the “Plan propio de Investigaci ́on y Transferencia” of the University of Granada under both programs “Intensificaci ́on de la Investigaci ́on, modalidad B′′ and by the European Research Commission (Research Executive Agency) under the research project Stance4Health (Grant Contract N 816303) granted to Jos ́e A. Rufi ́an-Henares.Microencapsulation of tannin extracts through extrusion-gelation method was performed comparing two alter- native encapsulation matrices: alginate and amidated pectin. The microstructure of the generated microbeads was studied, as well as their microencapsulation efficiency and release properties. Overall, pectin-based beads performed better than their alginate-based counterparts. This, combined with a greater incorporation of tannins in the feed formulations led to a higher tannin load in the final beads. The best microencapsulation efficiency was given by pectin microbeads loaded with 10% tannin extract (w/w), but the final tannin content could be further increased by adding a 20% (w/w) concentration of the extracts. During a 14-days storage, only a marginal loss of tannins was recorded for pectin-based microbeads. The results reveal that great potential exists in producing pectin-based microbeads in presence of tannins, which allow better loading capacities and improving structural properties, thanks to the interactions between the tannins and the amidated polysaccharide.Teagasc, the Irish Agricultural and Food Development Authority (Fermoy, Ireland)Plan propio de Investigación y Transferencia” of the University of Granada under both programs “Intensificación de la Investigación, modalidad BEuropean Research Commission (Research Executive Agency) under the research project Stance4Health (Grant Contract N 816303

Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds

The molecular weight of chitosan is one of its most determinant characteristics, which affects its processability and its performance as a biomaterial. However, information about the effect of this parameter on the formation of electrosprayed chitosan microcapsules is scarce. In this work, the impact of chitosan molecular weight on its electrosprayability was studied and correlated with its effect on the viscosity, surface tension and electrical conductivity of solutions. A Discriminant Function Analysis revealed that the morphology of the electrosprayed chitosan materials could be correctly predicted using these three parameters for almost 85% of the samples. The suitability of using electrosprayed chitosan capsules as carriers for bioactive agents was also assessed by loading them with a model active compound, (−)-epigallocatechin gallate (EGCG). This encapsulation, with an estimated efficiency of around 80% in terms of preserved antioxidant activity, showed the potential to prolong the antiviral activity of EGCG against murine norovirus via gradual bioactive release combined with its protection against degradation in simulated physiological conditions.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Gloria Sanchez was supported by the “Ramón y Cajal” Young Investigator Program. The authors would like to thank the Spanish MINECO project AGL2015-63855-C2-1 and INIA grant RTA2014-00024-C04-03 for financial support.Peer reviewe

Electrosprayed gelatin submicroparticles as edible carriers for the encapsulation of polyphenols of interest in functional foods

In this work, the potential of the electrospraying technique to obtain food-grade gelatin capsules in the submicron range for sensitive bioactive protection was explored, studying the influence of the protein concentration on the size and morphology of the obtained particles. Gelatin was selected as encapsulating material because, being commonly used as a food ingredient, it possesses unique gelation properties and is commercially available at a low cost. The electrosprayed matrices were used to encapsulate a model antioxidant molecule, (−)-epigallocatechin gallate (EGCG). Very high encapsulation efficiencies, close to 100%, were achieved, and the antioxidant activity of the bioactive was fully retained upon encapsulation. The EGCG release profiles showed a delayed release of the encapsulated antioxidant in aqueous solutions. Furthermore, while free EGCG in PBS lost a 30% of their antioxidant activity being completely degraded in 100 h, encapsulated EGCG retained its whole antioxidant activity within this time period.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO project AGL2012-30647 for financial support.Peer reviewe

Microencapsulation of a whey protein hydrolysate within micro-hydrogels: Impact on gastrointestinal stability and potential for functional yoghurt development

Gelatin and chitosan micro-hydrogels containing a potentially bioactive whey protein hydrolysate were developed through spray drying and the impact of microencapsulation on protection during digestion and peptide stability against lactic acid fermentation during yoghurt manufacturing was assessed. The results showed that the protection exerted by the encapsulation structures during milk fermentation was sequence- and matrix-dependent, being chitosan more effective than gelatin in stabilising the peptides. However, only 5 out of the 21 fermentation-susceptible peptides identified could be protected through encapsulation within chitosan (1 of which was also protected by gelatin). Moreover, the encapsulation within chitosan microparticles did not substantially affect the peptide profile of the digested hydrolysate, and therefore, the peptide bioaccessibility was not expected to be compromised.Laura G. Gómez-Mascaraque is the recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. The authors would like to thank the Spanish MINECO projects AGL2015-63855-C2-1 and AGL2015-66886-R for financial support.Peer reviewe

Tannin-rich extracts improve the performance of amidated pectin as an alternative microencapsulation matrix to alginate

peer-reviewedMicroencapsulation of tannin extracts through extrusion-gelation method was performed comparing two alternative encapsulation matrices: alginate and amidated pectin. The microstructure of the generated microbeads was studied, as well as their microencapsulation efficiency and release properties. Overall, pectin-based beads performed better than their alginate-based counterparts. This, combined with a greater incorporation of tannins in the feed formulations led to a higher tannin load in the final beads. The best microencapsulation efficiency was given by pectin microbeads loaded with 10% tannin extract (w/w), but the final tannin content could be further increased by adding a 20% (w/w) concentration of the extracts. During a 14-days storage, only a marginal loss of tannins was recorded for pectin-based microbeads. The results reveal that great potential exists in producing pectin-based microbeads in presence of tannins, which allow better loading capacities and improving structural properties, thanks to the interactions between the tannins and the amidated polysaccharide

Optimization of electrospraying conditions for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion

Electrospraying has recently emerged as a novel microencapsulation technique with potential for the protection of probiotics. However, research efforts are still needed to minimize the viability loss observed during the processing of sensitive strains, and to maximize productivity. The aim of the present work was the optimization of the electrospraying conditions for the microencapsulation of a model probiotic microorganism, Lactobacillus plantarum, within a whey protein concentrate matrix. In a pre-optimization step, the convenience of encapsulating fresh culture instead of freeze-dried bacteria was established. Additionally, a surface response methodology was used to study the effect of the applied voltage, surfactant concentration, and addition of a prebiotic to the formulation on cell viability and productivity. Viability losses lower than 1 log10 CFU were achieved and the bacterial counts of the final products exceeded 8.5 log10 CFU/g. The protection ability of the developed structures during storage and in-vitro digestion was also evaluated.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Russell Cruz Morfin received a scholarship from the Mexican National Council for Science and Technology (CONACYT), Call 2014. Gloria Sánchez is supported by the “Ramón y Cajal” Young Investigator program of the MINECO. This work was financially supported by the Spanish MINECO project AGL2012-30647 and by the CSIC project 201470I002.Peer reviewe

Nano- and microstructural evolution of alginate beads in simulated gastrointestinal fluids. Impact of M/G ratio, molecular weight and pH

peer-reviewedAlginate microcapsules were prepared using three different alginate grades and incubated under simulated digestion conditions. Their micro- and nanostructural changes were studied using microscopy, laser diffraction and small angle X-ray scattering. Both the molecular weight and M/G ratio affected the size and nanostructural features of the capsules, but the changes in gastrointestinal conditions were mainly determined by the latter. All microcapsules swelled slightly in simulated gastric fluid (pH = 3) and swelled further in simulated intestinal fluid (pH = 7), particularly those with high mannuronic acid (M) contents. While high guluronic acid (G) beads maintained the nanostructural features characteristic of alginate gels (junction zones) in both media, these were rapidly disrupted in the M-rich capsules. Decreasing the pH of the gastric phase from 3 to 2 had dramatic structural impacts, resulting in a greater integrity of the microcapsules, thus highlighting the importance of the selected digestion protocol for rational microcapsule design