Thesis (Ph. D.)--University of North Texas, Aug., 2009. / Title from title page display. Includes bibliographical references.

Thesis (Ph.D) - University of Queensland, 2007. / Includes bibliography.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaves 36-43, 88-90, 139-140, 187, 238-241, 282-285, 323).

Since the 1970's there has been industrial interest in using microorganisms as microcapsules. The encapsulation of actives (e.g. flavours, drugs, perfumes) is a necessary process for pharmaceutical and food companies because the precious and often expensive ingredients must be protected from degradation and also released in a specific site or under a specific stimulus. Saccharomyces cerevisiae, baker's yeast, represents a first choice microorganism for the encapsulation of active ingredients. It is biodegradable and biocompatible with human digestion and skin, and can be produced in an easy and cheap way. A major part of this project has been dedicated to the development of robust methods of extraction and quantification of hydrophobic substances loaded inside yeast cells, which have been subsequently combined with an indirect, fluorescence-based method for the evaluation of the rate of loading of hydrophobic substances in the same cells. In particular, it has been found that this process reaches a limit in the maximal loading capacity of intact yeast cells, most likely reflecting the maximal volume of the lipid droplet organelles in which loaded hydrophobes accumulate. With the new on-line (fluorescence-based) and off-line (chromatography-based) methods developed here it has been established that the loading process fundamentally follows a diffusion model, in which the solubility in water determines the permeation of substances through the cell wall and ultimately their uptake by yeast cells. However, treating yeast cells with organic solvents like DMSO - a new approach introduced in Prof. Tirelli's lab to enhance the encapsulation of hydrophobes - completely changes the chemical-physical parameters of the encapsulation process. In DMSO-treated cells, substances are loaded fundamentally in response to their hydrophobicity. Conversely, once loaded, the same substances are released with a rate that is inversely proportional to their hydrophobicity, as observed by applying a novel approach to measure the release of hydrophobes encapsulated in yeast cells, either in the absence of presence of DMSO-treatment. In conclusion, the new evidence reported here clarifies basic aspects of hydrophobe encapsulation in intact yeast cells and will thus help improving future applications of these microcapsules as a valid, inexpensive and biocompatible drug delivery system.

The overall goal of this research was to develop a plant protein-based microcapsule capable of carrying, protecting and delivering flaxseed oil within the food and gastrointestinal environment. Specifically, the research aimed to: a) screen a variety of plant proteins and pre-treatment conditions based on their emulsifying properties for use as a wall material; b) develop and optimize encapsulation protocols for entrapping flaxseed oil; and c) study the oxidative stability and delivery of entrapped oils from capsules under different environmental and simulated gastrointestinal conditions. In Chapter 3 and 4, the emulsifying and physicochemical properties of legume and oilseed protein isolates, respectively produced from isoelectric precipitation and salt extraction were investigated. Findings in Chapter 3 indicated that both the legume source and method of production showed significant effects on the emulsifying and physicochemical properties of chickpea (ChPI), faba bean (FbPI), lentil (LPI), pea (PPI), and soy (SPI) protein isolates. The emulsion capacity (EC) values ranged between 476-542 g oil/g protein with LPI showing the highest capacity. Isoelectric-precipitated ChPI and LPI displayed higher emulsion activity index (EAI) (~46.2 m2/g), (emulsion stability index) ESI (~84.9 min) and (creaming stability) CS (98.6%), which were comparable to those of SPI. In Chapter 4, findings indicated that both protein source and method of production had significant effects on the physicochemical and emulsifying properties of canola (CaPI) and flaxseed protein isolates (FlPI). CaPI showed significantly higher EC (~515.6 g oil/g protein) than FlPI (~498.9 g oil/g protein). EAI for FlPI was found to be higher (~40.1 m2/g) than CaPI (~25.1 m2/g) however, ESI values of CaPI and FlPI were similar. Creaming stability of emulsions stabilized by CaPI and FlPI ranged between 86.1 and 96.6%. CaPI and FlPI were shown to have emulsion forming properties; however their stability was low. In Chapter 5, ChPI and LPI-stabilized emulsions were optimized based on pH, protein concentration and oil content for their ability to form and stabilize oil-in-water emulsions using response surface methodology. Droplet charge was shown to be only affected by pH, while droplet size and creaming index were affected by protein concentration, oil content and pH. Optimum conditions for minimal creaming (no serum separation after 24 h), small droplet size (<2 μm), and high net droplet charge (absolute zeta potential (ZP) value >40 mV) were identified as: 4.1% protein, 40.0% oil, and pH 3.0 or 8.0, regardless of the plant protein used for emulsion preparation. Flaxseed oil was microencapsulated by freeze (Chapter 6) or spray (Chapter 7) drying employing ChPI or LPI and maltodextrin. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (ChPI vs. LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Optimized capsule designs were found to have high encapsulation efficiencies, low surface oil, and afforded protection against oxidation over a 25 d room temperature storage study relative to free oil. Microcapsules were also able to deliver 84.2% of the encapsulated oil in the simulated gastrointestinal environments.

Hidrolisados proteicos possuem propriedades terapêuticas e são absorvidos mais facilmente pelo organismo quando comparados às proteínas, no entanto sua aplicação em alimentos é dificultada por serem higroscópicos, reativos e apresentarem gosto amargo. A microencapsulação por spray chilling pode ser uma alternativa para solucionar essas limitações. Este método de encapsulação consiste na atomização de uma mistura, formada pela dispersão ou emulsão do material ativo com o carreador fundido, em uma câmara com temperatura inferior ao ponto de fusão do carreador, que nessas condições solidifica, formando micropartículas esféricas. O objetivo deste trabalho foi elaborar micropartículas de hidrolisado de proteína de soja utilizando o método de spray chilling e gordura vegetal (PF 51°C) como carreador. Foram realizados ensaios para obtenção das micropartículas avaliando a alimentação por emulsão e dispersãoe diferentes formulações variando a proporção material ativo:encapsulante (1:5 e 1:10), velocidades de rotação no ultra-turrax (6000 e 8000 rpm) e três diferentes temperaturas (60, 70 e 80°C), totalizando dezoito tratamentos. As misturas foram submetidas à análise reológica para determinação de viscosidade e após serem atomizadas em spray chiller as micropartículas obtidas foram caracterizadas por FTIR, Difração de Raio-X, distribuição e tamanho médio por difração a laser e morfologia por microscopia eletrônica de varredura e confocal. Foram obtidas micropartículas lipídicas sólidas esféricas e aglomeradas, o tamanho médio variou de 53,06 &plusmn; 2,17 &micro;m e 68,03 &plusmn; 14,07 &micro;m, sem diferenças significativas entre os tratamentos. Partículas obtidas pela atomização da emulsão apresentaram poros, todavia exibiram maior capacidade de carregamento do hidrolisado, cerca de 96%, enquanto as obtidas por dispersão apresentaram 54%. Variações durante o preparo da emulsão não proporcionaram alterações na morfologia e tamanho de partícula nas micropartículas, apesar de terem tido influência sobre as propriedades reológicas do sistema. A análise de difração de raios-X indicou que as micropartículas após 90 dias de preparo apresentaram a estrutura na forma polimórfica mais estável. A espectroscopia na região do infravermelho (FTIR) revelou que não ocorreu interação entre os ingredientes independentemente do modo de preparo das micropartículas. Tais resultados demonstram que a técnica de spray chilling é eficiente na microencapsulação de hidrolisado proteico de soja, possibilitando uma futura aplicação em alimentos. / Protein hydrolysates possess therapeutic properties and absorption easier than to proteins; however its application in food is limited due to its bitter taste, hygroscopic and reactivity. Encapsulation byspray chilling could be an alternative to minimize these limitations. This method consists in the atomization of a mixture formed by the dispersion or emulsion of the active material with the molten carrier, into an environment with temperature below the melting point of the carrier, under these conditions it solidifies to form spherical microparticles. The aim of this work was to develop microparticles loaded with hydrolyzed soy protein using the method of spray chilling and vegetable fat (PF 51°C) as carrier. Tests were conducted to obtain microparticles evaluating the feed by emulsion and dispersion and different formulations by varying the proportions active materials:carrier (1:5 and 1:10), homogenization speed by Ultra-Turrax (6000 and 8000 rpm) and temperature (60, 70 and 80°C ), totaling eighteen treatments. The mixtures were subjected to rheological analysis for determination of viscosity and after being atomized at spray chiller obtained microparticles were characterized by infrared spectroscopy and X-ray diffraction, particle size distribution and mean diameter measured using a laser light diffraction instrument and morphology was observed by scanning electron microscopy (SEM) and confocal microscopy. Solid lipid microparticles obtained were spherical and agglomerated the average size between 53.06 &plusmn; 2.17 &micro;m and 68.03 &plusmn; 14.07 &micro;m, there was no significant difference between formulations. Particles obtained by atomization of emulsion had presence of pores, but exhibited a higher loading capacity of the hydrolyzed, about 96%, while that obtained by dispersion had 54%. Changes during the preparation of the emulsion no provided changes at morphology and particle size of the microparticles, despite having influence on the rheological properties of the system. The analysis of X-ray diffraction showed that the microparticles after 90 days of storage had &beta; polymorphic form. The infrared spectroscopy (FTIR) showed that there was no interaction between the ingredients regardless of the mode of preparation of the microparticles. These results demonstrate that the technique spray chilling is efficient in microencapsulation of soy protein hydrolyzate, allowing future use in foods.

Oral administration of live probiotics such as Lactobacillus and Bifidobacterium spp. possess numerous beneficial effects. However, delivering viable probiotics to the host intestine has been a challenge due to poor survival of these bacteria during the gastric transit. An improved oral delivery system (modified alginate microcapsules) was developed in this study for targeted release of viable probiotics to the host intestine. Effect of various encapsulation parameters such as capsule size, alginate concentration, calcium chloride concentration, gelling/hardening time of microcapsules, addition of prebiotics and polymer coating, were individually investigated for improving the stability of microcapsules under simulated gastrointestinal (GI) conditions. Ability of microcapsules in protecting the viability of encapsulated bacteria improved significantly (p<0.05) in improving the stability of microcapsules. Optimisation of encapsulation parameters significantly improved the viability of encapsulated probiotics under simulated GI conditions. Furthermore, co-encapsulation of probiotics with complementary prebiotics (such as Hi-Maize starch) and chitosan coating provided additional protection to the encapsulated bacteria under simulated GI conditions. Release profile of chitosan-coated alginate-starch (CCAS) encapsulated bacteria was investigated in the GI tracts of different animal models. Addition of CCAS encapsulated bacteria to porcine GI contents (ex vivo) resulted in complete release of microencapsulated bacteria in the ileal contents within 8 h, while there was no significant release (p>0.05) of encapsulated bacteria in the gastric contents even after 24 h of incubation. In another experiment, CCAS microcapsules containing Lactobacillus casei Shirota (LCS) was orally administered to mice and the release profile of encapsulated bacteria was monitored throughout the murine GI tract for 24 h. Partial release of microencapsulated LCS was observed in duodenal and jejunal regions, while no significant (p>0.05) release of microencapsulated bacteria was observed in the stomach during the 24 h monitoring period. However, a significant release (nearly complete release) of microencapsulated bacteria was observed in ileal and colon of murine GI tract after 24 h. Elevated counts of LCS in ileum and colon indicated the most favorable site for the release of CCAS encapsulated bacteria. Further studies investigated the immunomodulatory activity of microencapsulated probiotic bacteria in a murine model. Lactobacillus casei Shirota was orally administered to mice either as microencapsulated or as free bacteria (non encapsulated) for two weeks. On day 14, the splenocytes from different experimental groups were harvested and assessed for ConA induced cytokine levels. A significant increase (p>0.05) in IFN-γ levels was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS, compared to the control group (no LCS treatment). However, there was no significant difference (p<0.05) in the IFN-γ concentration between the groups treated with microencapsulated and free (non-encapsulated) LCS. No significant difference (p<0.05) in the IL-10 concentration was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS. Finally, the stability of microencapsulated probiotics in different dairy products was investigated. CCAS microcapsules significantly protected the viability of probiotic bacteria in set and stirred yoghurts over 6-week refrigerated storage conditions compared to free (non-encapsulated) probiotics. Overall, chitosan-coated alginate-starch microcapsules developed in this study effectively protected the viability of probiotics from adverse gastric conditions and released the bacteria in the host intestine without detrimentally affecting its immunomodulatory properties. / Doctor of Philosophy

Commercial flavour-enhancing enzymes were delivered in an encapsulated form to accelerate Cheddar cheese ripening. Polymers such as alginate, chitosan and k- Carrageenan were screened to be used as encapsulant material for microencapsulation of the commercial protease enzyme, Flavourzyme®. Alginate was found to be a suitable polymer for Flavourzyme encapsulation using the Inotech® encapsulator while _-Carrageenan and chitosan were too viscous for extrusion through the encapsulator nozzle. Gelling of alginate-Flavourzyme microcapsules in 0.1M CaCl2 resulted in poor encapsulation efficiency (ranging 17- 18% depending on the alginate concentration). Incorporation of Hi-Maize™ starch or pectin as filler materials into the alginate-Flavourzyme encapsulation matrix to increase encapsulation efficiency by minimising porosity also resulted in poor encapsulation efficiency. An alternative approach to the modification of the cationic gelling solution, by adding chitosan, significantly increased the encapsulation efficiency to 70-88% and produced mostly spherical capsules with an average diameter of 500_m. Encapsulation efficiency increased with an increase in chitosan concentration from 0.1 to 0.3% (w/v) in the cationic gelling solution of 0.1M CaCl2. Though gelling of alginate-Flavourzyme microcapsules in gelling solution of 0.1M CaCl2 containing 0.3% (w/v) chitosan resulted in higher encapsulation efficiency, a chitosan concentration of 0.1% (w/v) was chosen for further work as higher concentrations of chitosan in the gelling solution resulted in aggregation of capsules during formation. Gelling time of 10 min and alginate concentrations in the range 1.6 to 2.0% (w/v) were found to be optimal encapsulation parameters for Flavourzyme encapsulation while 2.0% (w/v) solution of trisodium citrate was found to be optimal for in vitro release of encapsulated enzymes for measurement of enzyme activity. Flavourzyme capsules stored frozen or freeze-dried were shelf stable for at least 10 weeks retaining about 80% of the initial enzyme activity as opposed to retention of 25-34% activity in air-dried capsules. Leakage of encapsulated Flavourzyme prepared from 1.6% (w/v) alginate was slightly higher than those prepared from 1.8 and 2.0% (w/v) alginate in cheese milk. Flavourzyme-alginate capsules prepared from 1.6, 1.8 and 2.0% (w/v) alginate retained over 70% of the initial enzyme activity under simulated cheese-press pressure. Concentration of alginate had no significant effect (p > 0.05) on the retention of encapsulated Flavourzyme when the capsules were pressed for 4h; however when the simulated cheese press duration increased to 8 and 16h the retention of encapsulated Flavourzyme was significantly higher (p [less than] 0.01) in capsules produced from 2.0% (w/v) alginate. Incorporation of encapsulated enzymes into the milk prior to rennetting resulted in an even distribution of capsules in the cheese matrix compared to aggregation of capsules, when added to milled curd prior to salting. All cheeses; control with no added enzymes and experimental cheeses with free and encapsulated Flavourzyme and/or Palatase showed higher levels of moisture and lower levels of fat compared to standard Cheddar cheese due to the variation in the manufacturing protocol. There was no significant difference (p > 0.05) in fat and final pH between control and experimental cheeses and there was no difference in the numbers of coliforms, E.coli, Salmonella, Listeria, coagulase positive staphylococci, Bacillus cereus, yeast and moulds in control or experimental cheeses. Increased and prolonged proteolysis was observed in cheeses with encapsulated Flavourzyme showing increased release of several peptides, also with the formation of new peptides absent in the control cheese with no added enzymes. Accumulation of high molecular weight/hydrophobic peptides was higher in cheeses with free Flavourzyme followed by cheeses with encapsulated Flavourzyme. Concentration of water-soluble peptides increased with the increase in the concentration of encapsulated Flavourzyme in the cheese. Concentration of water-insoluble peptides was higher in control cheese compared to cheeses with encapsulated Flavourzyme even after 180 days ripening. After 30 days of ripening, concentration of most free amino acids was about 3 times greater in cheeses with encapsulated Flavourzyme than in control and about 7 times higher after 90 days ripening. Concentration of total amino acids was consistently higher in cheeses with encapsulated Flavourzyme compared to control. Cheese grading scores for body, texture and appearance of all cheeses with encapsulated enzymes were lower than control and free enzyme treated cheeses during the entire grading period of about 100 days due to crumbly and pasty texture. Control and cheeses with added Flavourzyme received high overall score for flavour. Flavour score of cheese with encapsulated Flavourzyme at a concentration of 0.75 LAPU/g milk protein was higher than all cheeses around 50 days with better overall flavour score until about 94 days ripening with improved flavour and elimination of bitterness. However the flavour of enzyme treated cheeses deteriorated with time and the control cheese scored the highest for flavour. Though increased concentration of free fatty acids was detected in cheeses treated with encapsulated lipase; Palatase, these cheeses developed rancid, unpleasant, strong lipolytic flavours as early as 55 days ripening. / Doctor of Philosophy (PhD)

A microencapsulation-based technology platform for effective delivery of multiple micronutrients for food fortification has been developed. The technology, consisting of extrusion agglomeration followed by encapsulation through surface coating, has been successfully tested on three size scales in typical staple foods: as a surface treatment on salt and sugar, on 20-100µm scale; in salt on a 300-1200 μm scale; and on reconstituted rice on the 5-10 mm scale. The process results in effective delivery systems for one or more active ingredients with organoleptic properties that are unnoticeable to the average consumer. Particularly, salt double fortified with iodine and iron using the microencapsulated ferrous fumarate premix made by the extrusion-based agglomeration process had acceptable sensory properties and stability when stored at 40oC and 60% relative humidity (RH) for up to a year. In these tests >85% of iodine and >90% of ferrous iron were retained. Reconstituted Ultra Rice® grains made by extrusion stabilized by internal gelation has resulted in improved grain integrity and a much simplified process, compared to the original, patented surface crosslinking technique. The most effective internal gelation system is composed of alginate, calcium sulphate (CaSO4), and sodium tripolyphosphate (STPP) at a best ratio of 3%:3%:0.6% (w/w). It is feasible to incorporate folic acid into the existing fortification programs using the technology platform developed in this study. The results indicate that the potential interactions of folic acid with other added micronutrients or with the food vehicles could be prevented by incorporating folic acid as a premix made by the extrusion-based technology. Virtually no folic acid was lost after 9 months storage at 40oC and 60% RH when the folic acid premix was added into salt or sugar samples. The technical feasibility of the microencapsulation-based technology platform has been successfully demonstrated for micronutrient delivery in food vehicles of different size ranges, resulting in fortified staple foods with desired physical, chemical, nutritional, and organoleptic properties. The technology should be adaptable to formulating customized delivery systems of active ingredients for broader applications, and promises to bring immediate benefits in combatting micronutrient deficiencies, that will have far reaching effects in health and social development.

A microencapsulation-based technology platform for effective delivery of multiple micronutrients for food fortification has been developed. The technology, consisting of extrusion agglomeration followed by encapsulation through surface coating, has been successfully tested on three size scales in typical staple foods: as a surface treatment on salt and sugar, on 20-100µm scale; in salt on a 300-1200 μm scale; and on reconstituted rice on the 5-10 mm scale. The process results in effective delivery systems for one or more active ingredients with organoleptic properties that are unnoticeable to the average consumer. Particularly, salt double fortified with iodine and iron using the microencapsulated ferrous fumarate premix made by the extrusion-based agglomeration process had acceptable sensory properties and stability when stored at 40oC and 60% relative humidity (RH) for up to a year. In these tests >85% of iodine and >90% of ferrous iron were retained. Reconstituted Ultra Rice® grains made by extrusion stabilized by internal gelation has resulted in improved grain integrity and a much simplified process, compared to the original, patented surface crosslinking technique. The most effective internal gelation system is composed of alginate, calcium sulphate (CaSO4), and sodium tripolyphosphate (STPP) at a best ratio of 3%:3%:0.6% (w/w). It is feasible to incorporate folic acid into the existing fortification programs using the technology platform developed in this study. The results indicate that the potential interactions of folic acid with other added micronutrients or with the food vehicles could be prevented by incorporating folic acid as a premix made by the extrusion-based technology. Virtually no folic acid was lost after 9 months storage at 40oC and 60% RH when the folic acid premix was added into salt or sugar samples. The technical feasibility of the microencapsulation-based technology platform has been successfully demonstrated for micronutrient delivery in food vehicles of different size ranges, resulting in fortified staple foods with desired physical, chemical, nutritional, and organoleptic properties. The technology should be adaptable to formulating customized delivery systems of active ingredients for broader applications, and promises to bring immediate benefits in combatting micronutrient deficiencies, that will have far reaching effects in health and social development.