This Diploma thesis talks about the production of concentrates and lyophilisate from the extract of elderberry moldlings (elderberry, Sambuvus nigra L.) and by determination by chosen chemical characteristics of these products. The theoretical part includes the botanical characteristics of the plant species Sambucus nigra L. Next is the part that is dedicated to biologicaly active substances found in elderberry and it‘s possible usage in the food industry. Next is the part where the extraction, purification and indentification of anthocyanic dyes are described. In the next part is the analyrical method HPLC described, including the description of instrumentation. The last part talks about the description of the used methods: methods for the concentration of the extract, UV-VIS spectrophotometry, determination of phenolic compounds, determination of dry matter, determination of active acidity and the overall titrating acidity. The experimental part contained the optimalization of the process of producing the extract from elderberry moldings. The extract obtained was then concentrated for preservation and characterization purposes based on selected chemical properties. The total content of anthocyanin dyes was chosen as a quality parameter. The most suitable process for the preparation of the extract was chosen to be the ratio of moldings and solvent 1:2, 4 (m/V). The most suitable solvent was chosen to be a mixture of water and ethanol in a ratio of 1:1 (V/V). According to the needs of the submitter, an extract with the use of water as the extraction agent is also provided. The prepared extract was then concentrated on a vacuum evaporator or lyophilized for storage purposes. The dye degradation while using the evaporator was the lowest in a 40 ° C water bath. The final products were characterized by the determination of soluble solids, pH, titration acidity, total phenolic compounds and total anthocyanin dyes. The anthocyanin pigments contained identified by liquid chromatography are: cyanidine-3-sambubioside, cyanidine-3-glucoside, cyanidine-3-sambubioside-5-glucoside, and cyanidine-3,5-diglucoside The total content of anthocyanins in the concentrate was determined to be equivalent to cyanidine-3-glucoside at 2.8 gl-1 and in the lyophilisate 2.5 gl-1.

<p>  </p> <p>With the recent growth in demand for biologics across the globe, it remains critical to manufacture these biologics in solid-state to improve stability as well as to increase the ease of transportation across the world. To meet these increased demands, it is of paramount importance to use various processing methods that have shorter processing times. It is also important to understand the impact of the processing methods and various formulation components on the stability of the proteins.  In Chapter 1, a review of the various processing methods that are used in the industry along with additional processing methods that are being investigated will be discussed. The common drying methods such as lyophilization and spray drying along with the novel techniques as well as specific examples of processing parameters to improve the processing conditions that better suit the protein formulations will be mentioned. </p> <p>The studies in Chapter 2 examined the effects of processing methods (freeze drying and spray freeze drying) and the excipients on the protein structure and physical stability. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements, and solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in the structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40oC for lysozyme.  The higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements, and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.</p> <p>Based on this work, it was identified that spray freeze drying can be a viable alternative to produce solid-state protein formulations with similar stability as the freeze drying process. However, due to the long processing times and scale-up issues involved in the spray freeze drying process, there is a necessity to explore additional drying processes. Chapter 3 focuses on using another novel technique known as electrostatic spray drying (ESD) to produce solid-state protein formulations at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70oC failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature at 5kV enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40oC. Low-temperature (70oC inlet temperature) drying with an electrostatic charge (5kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130oC inlet temperature) without charge.</p> <p>This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at a lower inlet temperature than traditional spray drying with better physical stability. While ESD can be a viable option for thermal-sensitive formulations, it is important to understand the impact of various formulation components on the stability of the proteins while using spray drying. Based on our previous studies, a good understanding of the effect of different sugars and the presence of surfactants on the spray-dried proteins has been established. However, the impact of the selection of buffer on protein stability has not been studied. In Chapter 4, the effect of buffer salts on the physical stability of spray dried and lyophilized formulations of a model protein, bovine serum albumin (BSA) were examined. BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40oC. PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. In summary, this study demonstrated that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.</p> <p>The study in Chapter 5 looked at examining the physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, with an excipient-free formulation as control. These formulations were produced using traditional spray freeze drying (SFD) or RF-assisted spray freeze drying (RFSFD). The dried formulations were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Traditional characterization tools such as ssFTIR and moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as the maximum deuterium uptake (Dmax) (R2 = 0.791) and deconvoluted peak areas of the deuterated samples (R2 = 0.914) showed a satisfactory correlation with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol and had similar stability with all other formulations as compared to SFD. This study demonstrated that the RFSFD technique can significantly reduce the duration of primary drying cycle from 48 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.</p> <p>Lastly, Chapter 6 consists of a summary of the conclusions formed from the work presented in this thesis. Furthermore, suggestions for future work are provided based on observations of results, less-explored areas of formulation and processing conditions as well as characterization tools to understand effects on protein physical stability.</p> <p><br></p>

<p>Glavni cilj ove doktorske disertacije je određivanje hemijskog sastava, biolo&scaron;kih i funkcionalnih karakteristika novih proizvoda od zove. Dobijanje proizvoda na bazi zove zasniva se na primeni tradicionalnih i savremenih tehnolo&scaron;kih procesa proizvodnje. Iskori&scaron;ćenje prirodnog potencijala zove započeto je primenom tradicionalne i savremene (liofilizacija) tehnike su&scaron;enja. U cilju dobijanja visoko-vrednih ekstrakata ploda i cveta zove primenjene su tradicionalna (maceracija) i savremene (ultrazvučna i mikrotalasna) ekstrakcione tehnike sa dva ekstragensa (50% etanol i voda). Dobijanje matičnog soka od plodova zove podrazumevalo je primenu tradicionalnog načina ceđenja, dok je vino od plodova zove dobijeno po standardnom postupku proizvodnje vina. Vino je izloženo različitim temperaturnim tretmanima u različitom vremenskom periodu (60 &deg;C u toku 5 minuta, 60 &deg;C u toku 10 minuta, 70 &deg;C u toku 5 minuta i bez toplotnog tretmana) u cilju evaluacije biolo&scaron;ke aktivnosti dobijenog proizvoda. Etarsko ulje ploda i cveta zove je dobijeno hidrodestilacijom. Ispitivanje efikasnosti primenjenih tehnolo&scaron;kih postupaka su&scaron;enja i ekstrakcije je zasnovano na određivanju biolo&scaron;kih i funkcionalnih karakteristika dobijenih ekstrakata ploda i cveta zove. U ispitivanim ekstraktima ploda i cveta zove dominantne fenolne kiseline su hlorogenska i protokatehinska kiselina, a rutin i kvercetin-3-O-heksozid su dominantna flavonoidna jedinjenja. Biolo&scaron;ke i funkcionalne karakteristike su ispitane primenom različitih in vitro antioksidativnih, neuroprotektivnih, antitirozinaznih i antidijabetogenih testova. Primenom liofilizacije kao savremene tehnike su&scaron;enja i mikrotalasne ekstrakcije kao savremene ekstrakcione tehnike povećava se biopotencijal ispitivanih ekstrakata. Matični sok od plodova zove kao potencijalno novi funkcionalni proizvod je analiziran u cilju definisanja hemijskog, fitohemijskog i nutritivnog sastava, biolo&scaron;kog potencijala i senzorskih karakteristika. Ispitivanja dobijenog vina su bila usmerena na utvrđivanje optimalnih uslova za proizvodnju voćnog vina. Definisanjem hemijskog i fitohemijskog sastava i evaluacijom biopotencijala vina određen je optimalan temperaturni profil za dobijanje jednog od novih funkcionalnih proizvoda. Na osnovu utvrđenog hemijskog sastava etarsko ulje ploda i cveta zove se pokazalo kao potencijalno novi prirodni agens za održavanje svežine i produženja roka trajanja prehrambenih proizvoda. Zova je nesumnjivo samonikla biljna vrsta koja u budućnosti osnovano može biti polazna sirovina za kreiranje i dobijanje novih prehrmabenih proizvoda na domaćem i inostranom trži&scaron;tu.</p> / <p>The main goal of this doctoral dissertation is to determine the chemical composition, biological and functional characteristics of new elderberry products. Elderberry products were obtained via traditional and modern technological processes. The exploitation of the natural potential of the elderberry started with the application of traditional and modern (lyophilization) drying techniques. To obtain high-value extracts of fruits and flowers, traditional (maceration) and modern (ultrasonic and microwave) extraction techniques with two solvents (50% ethanol and water) were applied. Obtaining the juice from the elderberry fruits implied the application of the traditional cold pressing method, whereas wine from the elderberry fruits was obtained in accordance with the standard procedure of wine production. The wine was exposed to different temperature treatments in different periods (60&deg;C for 5 minutes, 60&deg;C for 10 minutes, 70&deg;C for 5 minutes and without heat treatment) to evaluate the biological activity of the product. The essential oil of the fruit and flower was obtained by hydrodistillation. The examination of the efficiency of the applied technological procedures of drying and extraction is based on observing the biological and functional characteristics of the obtained extracts of the said fruits and flowers. In the examined fruit and flower extracts, the dominant phenolic acids are chlorogenic and protocatechuic acid, while rutin and quercetin-3-O-hexoside are the dominant flavonoid compounds. Biological and functional characteristics were examined using various in vitro antioxidant, neuroprotective, antityrosinase, and antidiabetic tests. The application of lyophilization and microwave extraction (as modern drying and extraction techniques) increased the biopotential of the analyzed extracts. Elderberry juice, a potentially new functional product, was analyzed to define the chemical, phytochemical and nutritional composition, biological potential, and sensory characteristics. The wine was tested in order to determine the optimal conditions for the production of fruit wine. By defining the chemical and phytochemical composition and evaluating the biopotential of wine, the optimal temperature profile for obtaining one of the new functional products was determined. Based on the obtained chemical composition, it is determined that the essential oil of fruits and flowers is a potentially new natural agent for maintaining freshness and extending the shelf life of food products. Without a doubt, elderberry is a wild plant species that could be used in the future as the starting material for creating and obtaining new food products on the domestic and foreign markets.</p>

[ES] Existen muchos estudios que relacionan el consumo regular de frutas con importantes beneficios para la salud. Esto es debido a la gran cantidad de fibra, vitaminas y otros compuestos bioactivos presentes en ellas, los cuales pueden ser de gran ayuda en la prevención de algunas enfermedades. En los últimos años, el consumidor se ha decantado por consumir productos con alto valor nutricional y, como consecuencia del estilo de vida actual, existe una alta demanda de productos con fácil manejo para su consumo. Algunos de los principales inconvenientes de las frutas son su estacionalidad y corta vida útil, por lo que resulta interesante ofrecer productos de fruta de alto valor nutritivo y sensorial, estables y fácil manejo. La liofilización es una técnica que permiten obtener productos altamente estables microbiológicamente, debido a su bajo contenido de humedad, y con una máxima calidad sensorial, nutritiva y funcional. Sin embargo, se trata de un proceso largo y energéticamente costoso. Con base a lo anterior, el objetivo de esta tesis ha sido seleccionar las mejores condiciones de liofilización para la obtención de dos productos de naranja, uno la propia torta liofilizada y otro el polvo obtenido por trituración de la misma. Así, la sección uno se centra en evaluar la influencia tanto del contenido de agua como de la composición en solutos de un producto naranja en algunas propiedades de la torta liofilizada y del polvo correspondiente. Las propiedades analizadas se seleccionaron teniendo en cuenta tanto la retención de los compuestos bioactivos, como la porosidad, el color y las propiedades mecánicas de la torta obtenida y del polvo conseguido tras su trituración, además de las propiedades de flujo del polvo y su comportamiento frente a la rehidratación. En base a los resultados obtenidos se ha podido concluir que el puré de naranja es más recomendable a los efectos de obtener un producto tipo snack crujiente y el zumo cuando se desee un polvo para rehidratar, en este caso evitando cualquier tratamiento térmico antes de la liofilización. En la sección dos, se ha estudiado el impacto de la temperatura del secado, 30 y 50 °C, sobre los compuestos bioactivos del producto obtenido a partir del zumo de naranja formulado con goma Arábiga y fibra de bambú, así como sobre las mismas propiedades antes descritas de la torta liofilizada y de su correspondiente producto en polvo. En este caso, además, se llevó a cabo una evaluación sensorial de los productos rehidratados. Los resultados obtenidos permiten recomendar 50°C para el secado de este producto por liofilización. Esta temperatura acorta el tiempo del proceso en un 64 %, favorece la extracción de la vitamina C sin afectar al contenido total de fenoles y carotenoides, mejora las propiedades mecánicas de las tortas y no afecta a la fluidez ni a la rehidratación del producto en polvo. Finalmente, en la sección tres, se ha trabajado en la aplicación de una solución analítica basada en la ley de Darcy para modelar la cinética de pérdida de agua durante la liofilización. La solución analítica debe complementarse con una solución numérica de las ecuaciones de Fourier para la transferencia de calor. El modelo se ha ajustado a la dinámica experimental de liofilización de un producto a base de naranja formulado a partir del zumo. La pseudo-permeabilidad de Darcy obtenida fue de 2,0X10-12 con un índice de desviación de la conducta ideal de 0,5. Las ecuaciones propuestas, resueltas con la pseudo-permeabilidad de Darcy ajustada y el índice de desviación ideal, permiten reproducir la dinámica de liofilización experimental del mismo producto a diferentes temperaturas. / [CA] Existeixen molts estudis que relacionen el consum regular de fruites amb importants beneficis per a la salut. Això és degut a la gran quantitat de fibra, vitamines i altres compostos *bioactivos que es troben elles, els quals poden ser de gran ajuda en la prevenció d'algunes malalties. En els últims anys el consumidor s'ha decantat per consumir productes amb alt valor nutricional i, a conseqüència de l'estil de vida actual, existeix una alta demanda de productes amb fàcil maneig per al seu consum. Alguns dels principals inconvenients de les fruites són la seua estacionalitat i curta vida útil, per la qual cosa resulta interessant oferir productes de fruita d'alt valor nutritiu i sensorial, estables i fàcil maneig. La liofilització és una tècnica que permeten obtindre productes altament estables microbiologicament, a causa del seu baix contingut d'humitat, i amb una màxima qualitat sensorial, nutritiva i funcional. No obstant això, es tracta d'un procés llarg i energèticament costós. Amb base a l'anterior, l'objectiu d'aquesta Tesi ha sigut seleccionar les millors condicions de liofilització per a l'obtenció de dos productes de taronja, un la pròpia coca liofilitzada i un altre la pols obtinguda per trituració d'aquesta. Així, la primera part de la Tesi se centra en avaluar la influència tant del contingut d'aigua com de la composició en soluts d'un producte taronja en algunes propietats de la coca liofilitzada i de la pols corresponent. Les propietats a analitzar es van seleccionar tenint en compte tant la retenció dels compostos bioactius, com la porositat, el color i les propietats mecàniques de la coca obtinguda i de la pols aconseguida després de la seua trituració, a més de les propietats de flux de la pols i el seu comportament enfront de la rehidratació. Sobre la base dels resultats obtinguts s'ha pogut concloure que el puré de taronja és més recomanable a l'efecte d'obtindre un producte tipus snack cruixent i el suc quan es desitge una pols per a rehidratar, en aquest cas evitant qualsevol tractament tèrmic abans de la liofilització. En la segona part de la Tesi, s'ha estudiat l'impacte de la temperatura de l'assecat, 30 i 50 °C, sobre els compostos bioactius del producte obtingut a partir del suc de taronja formulat amb goma Aràbiga i fibra de bambú, així com sobre les mateixes propietats abans descrites de la coca liofilitzada i del seu corresponent producte en pols. En aquest cas, a més, es va dur a terme una avaluació sensorial dels productes rehidratats. Els resultats obtinguts permeten recomanar 50 °C per a l'assecat per liofilització d'aquest producte. Aquesta temperatura acurta el temps del procés en un 64%, afavoreix l'extracció de la vitamina C sense afectar el contingut total de fenols i carotenoids, millora les propietats mecàniques de les coques i no afecta a la fluïdesa ni a la rehidratació del producte en pols. Finalment, en la tercera part de la Tesi, s'ha treballat en l'aplicació d'una solució analítica basada en la llei de Darcy per a modelar la cinètica de pèrdua d'aigua durant la liofilització. La solució analítica ha de complementar-se amb una solució numèrica de les equacions de Fourier per a la transferència de calor. El model s'ha ajustat a la dinàmica experimental de liofilització d'un producte a base de taronja formulat a partir del suc. La pseudo-permeabilitat de Darcy obtinguda va ser de 2,0X10-12 amb un índex de desviació de la conducta ideal de 0,5. Les equacions proposades, resoltes amb la pseudo-permeabilitat de Darcy ajustada i l'índex de desviació ideal, permeten reproduir la dinàmica de liofilització experimental del mateix producte a diferents temperatures. / [EN] There are many studies linking regular fruit consumption with important health benefits. This is due to the large amount of fibre, vitamins and other bioactive compounds found in them, which can be of great help in the prevention of some diseases. In recent years, consumers have become more inclined to consume products with high nutritional value and, as a consequence of today's lifestyle, there is a high demand for products that are easy to handle for consumption. Some of the main disadvantages of fruits are their seasonality and short shelf life, so it is interesting to offer fruit products of high nutritional and sensory value that are stable and easy to handle. Freeze-drying is a technique that makes it possible to obtain highly microbiologically stable products, due to their low moisture content, and with maximum sensory, nutritional and functional quality. However, it is a long and energy-intensive process. Based on the above, the aim of this Doctoral Thesis has been to select the best freeze-drying conditions for obtaining two orange products, one the freeze-dried cake itself and the other the powder obtained by crushing it. Thus, the first part of the Thesis focuses on evaluating the influence of both the water content and the solute composition of an orange product on some properties of the freeze-dried cake and the corresponding powder. The properties to be analysed were selected taking into account the retention of the bioactive compounds, the porosity, the colour and the mechanical properties of the cake obtained and of the powder obtained after grinding, as well as the flow properties of the powder and its behaviour against rehydration. Based on the results obtained, it can be concluded that orange puree is more recommendable for obtaining a crunchy snack-type product and juice when a powder for rehydration is desired, in this case avoiding any heat treatment before freeze-drying. In the second part of the Thesis, the impact of the drying temperature, 30 and 50 °C, on the bioactive compounds of the product obtained from orange juice formulated with gum Arabic and bamboo fibre, as well as on the same properties described above of the freeze-dried cake and its corresponding powdered product, has been studied. In this case, a sensory evaluation of the rehydrated products was also carried out. The results obtained allow to recommend 50°C for the freeze-drying of this product. This temperature shortens the processing time by 64 %, favours the extraction of vitamin C without affecting the total phenol and carotenoid content, improves the mechanical properties of the cakes and does not affect the flowability and rehydration of the powdered product. Finally, in the third part of the Thesis, we have studied the application of an analytical solution based on Darcy's law to model the kinetics of water loss during freeze-drying. The analytical solution must be complemented with a numerical solution of the Fourier equations for heat transfer. The model has been fitted to the experimental dynamics of freeze-drying an orange-based product formulated from the juice. The Darcy pseudo-permeability obtained was 2,0X10-12 with an index of deviation from ideal behaviour of 0,5. The proposed equations, solved with the adjusted Darcy pseudo-permeability and the ideal deviation index, allow to reproduce the experimental freeze-drying dynamics of the same product at different temperatures. / Uscanga Ramos, MA. (2022). Influencia de la composición y de la temperatura de secado en la calidad de un producto de naranja liofilizado. Propuesta de un modelo mecanístico para la modelización de la cinética del proceso [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181238 / TESIS

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