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Encapsulation comprises a promising potential for the targeted delivery of entrapped sensitive agents into the food system. A unique combination of cellulose/chitosan (Cl-Ch)-based hybrid wall material was employed to encapsulate L. plantarum by emulsion technique. The developed beads were further subjected to morphological and in vitro studies. The viability of free and encapsulated probiotics was also evaluated in kefir during storage. The developed beads presented porous spherical structures with a rough surface. A 1.58 ± 0.02 log CFU/mL, 1.26 ± 0.01 log CFU/mL, and 1.82 ± 0.01 log CFU/mL reduction were noticed for Cl-Ch hybrid cells under simulated gastro-intestinal and thermal conditions, respectively. The encapsulated cells were found to be acidic and thermally resistant compared to the free cells. Similarly, encapsulated probiotics showed better viability in kefir at the end of the storage period compared to free cells. In short, the newly developed Cl-Ch hybrid-based encapsulation has a promising potential for the targeted delivery of probiotics, as career agents, in gastric transit, and in foods.

Orally delivered probiotics must survive transit through harsh environments during gastrointestinal (GI) digestion and be delivered and released into the target site. The aim of this work was to evaluate the survivability and delivery of gel-encapsulated Lactobacillus rhamnosus GG (LGG) to the colon. New hybrid symbiotic beads alginate/prebiotic pullulan/probiotic LGG were obtained by the extrusion method. The average size of the developed beads was 3401 µm (wet), 921 µm (dry) and the bacterial titer was 109 CFU/g. The morphology of the beads was studied by a scanning electron microscope, demonstrating the structure of the bacterial cellulose shell and loading with probiotics. For the first time, we propose adding an enzymatic extract of feces to an artificial colon fluid, which mimics the total hydrolytic activity of the intestinal microbiota. The beads can be digested by fecalase with cellulase activity, indicating intestinal release. The encapsulation of LGG significantly enhanced their viability under simulated GI conditions. However, the beads, in combination with the prebiotic, provided greater protection of bacteria, enhancing their survival and even increasing cell numbers in the capsules. These data suggest the promising prospects of coencapsulation as an innovative delivery method based on the inclusion of probiotic bacteria in a symbiotic matrix.

Thanks to the beneficial properties of probiotic bacteria, there exists an immense demand for their consumption in probiotic foods worldwide. Nevertheless, it is difficult to retain a high number of viable cells in probiotic food products during their storage and gastrointestinal transit. Microencapsulation of probiotic bacteria is an effective way of enhancing probiotic viability by limiting cell exposure to extreme conditions via the gastrointestinal tract before releasing them into the colon. This research aims to develop a new coating material system of microencapsulation to protect probiotic cells from adverse environmental conditions and improve their recovery rates. Hence, Lactobacillus rhamnosus was encapsulated with emulsion/internal gelation techniques in a calcium chloride solution. Alginate–probiotic microbeads were coated with xanthan gum, gum acacia, sodium caseinate, chitosan, starch, and carrageenan to produce various types of microcapsules. The alginate+xanthan microcapsules exhibited the highest encapsulation efficiency (95.13 ± 0.44%), they were simulated in gastric and intestinal juices at pH 3 during 1, 2, and 3 h incubations at 37 °C. The research findings showed a remarkable improvement in the survival rate of microencapsulated probiotics under simulated gastric conditions of up to 83.6 ± 0.89%. The morphology, size, and shape of the microcapsules were analyzed using a scanning electron microscope. For the protection of probiotic bacteria under simulated intestinal conditions, alginate microbeads coated with xanthan gum played an important role, and exhibited a survival rate of 87.3 ± 0.79%, which was around 38% higher than that of the free cells (49.4 ± 06%). Our research findings indicated that alginate+xanthan gum microcapsules have a significant potential to deliver large numbers of probiotic cells to the intestines, where cells can be released and colonized for the consumer’s benefit.

Orientador: Mirna Lúcia Gigante Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: A adição do probiótico na fabricação de queijos altera a sua microbiota e, consequentemente, pode afetar o desenvolvimento da maturação, o perfil sensorial e funcional do produto. O objetivo desse trabalho foi avaliar o desenvolvimento da maturação, a viabilidade dos micro-organismos e as características físico-químicas e sensoriais de queijo Prato probiótico tipo lanche adicionado de Lactobacillus acidophilus La-5 e Bifidobacterium Bb-12, separadamente ou juntos. Avaliou-se também, o efeito da matriz queijo sobre a viabilidade dos micro-organismos probióticos durante a simulação da passagem pelo trato gastrointestinal. Para a fabricação dos queijos foram realizados os seguintes tratamentos: 1) adicionado de cultura láctica tipo O (queijo controle); 2) adicionado de cultura láctica tipo O e L. acidophilus; 3) adicionado de cultura láctica tipo O e Bifidobacterium; 4) adicionado de cultura láctica tipo O, L. acidophilus e Bifidobacterium. O experimento foi realizado em esquema fatorial 4 x 6, em blocos inteiramente casualizados com três repetições. Os queijos foram avaliados quanto à composição físico-química, proteólise, textura e viabilidade dos micro-organismos após 1, 7, 14, 28, 40 e 60 dias de armazenamento refrigerado. A viabilidade dos micro-organismos probióticos durante simulação das condições gastrointestinais foi avaliada após 28 e 60 dias de armazenamento. Os resultados foram avaliados por Análise de Variância (ANOVA) e teste de Tukey para comparação de médias a um nível de significância de 5%. A análise sensorial descritiva através do método de Perfil Livre dos queijos foi realizada após 28 e 60 dias de armazenamento e os resultados foram avaliados por Análise de Procrustes Generalizada (APG). Os resultados indicaram que os queijos controle e probióticos apresentaram composição típica de queijo Prato e que os tratamentos não afetaram sua composição. Durante 60 dias de armazenamento, os queijos apresentaram aumento da proteólise, redução da firmeza e os queijos probióticos mantiveram altas contagens de L. acidophilus e Bifidobacterium. Os queijos probióticos apresentaram perfis sensoriais similares, com características de aroma e sabor de queijo Prato mais pronunciados que o queijo controle. Os micro-organismos probióticos adicionados nos queijos foram resistentes à simulação do trato gastrointestinal e à bile. O conjunto dos resultados indicou que a adição de probióticos em queijo Prato mostrou-se uma alternativa viável, uma vez que promoveu a manutenção da viabilidade destes micro-organismos durante o processamento e armazenamento refrigerado sem ocasionar alterações indesejáveis nas características sensoriais do produto e conferiu proteção aos probióticos durante a simulação gastrointestinal Abstract: The addition of probiotics during cheese manufacture changes its microbiota and, consequently, may affect ripening, sensory and functional profile of the product. The aim of this study was to evaluate ripening, viability of microorganism, physicochemical and sensory characteristics of probiotic Prato cheese with L. acidophilus La-5 and Bifidobacterium Bb-12 added separately or together. The effect of the cheese matrix on the viability of the probiotics during the simulation of the passage through the gastrointestinal tract was also evaluated. For the manufacture of the Prato cheeses, the following treatments were studied: 1) addition of type O lactic culture (control cheese); 2) addition of type O lactic culture and L. acidophilus; 3) addition of type O lactic culture and Bifidobacterium; 4) addition of type O lactic culture, L. acidophilus, and Bifidobacterium. The experiment was repeated three times, and assembled in a 4 x 6 factorial design, using a completely randomized block design. Cheeses were evaluated regarding their physicochemical composition, proteolysis, texture profile and viability of microorganisms after 1, 7, 14, 28, 40 and 60 days of refrigerated storage. Probiotics viability during simulated gastrointestinal conditions was evaluated after 28 and 60 days of storage. The results were evaluated using Analysis of Variance (ANOVA), and comparison of means by Tukey¿s test at 5% significance level. Descriptive sensory analysis was performed using free-choice profiling after 28 and 60 days of cheese storage, and the results were submitted to Generalized Procrustes Analysis (GPA). Both control and probiotic cheeses presented typical composition of Prato cheese and the treatments did not affect cheese composition. During 60 days of storage, an increased proteolysis, and decreased firmness was observed for all cheeses, and high populations of L. acidophilus and Bifidobacterium were found for the probiotic cheeses. Probiotic cheese showed similar sensory profiles with characteristics aroma and taste of Prato cheese more pronounced than control cheeses. The probiotics added to the cheeses were resistant during the simulation of the passage through the gastrointestinal tract and bile. In conclusion, the results evidenced that the addition of probiotics in Prato cheese proved to be a viable alternative, since the viability of microorganisms remained during cheese processing and storage, without causing undesirable changes in the sensory characteristics of the product, and provided protection to the probiotics during simulated gastrointestinal conditions Doutorado Tecnologia de Alimentos Doutora em Tecnologia de Alimentos

Encapsulation technologies in functional beverage market have been increasing according to the demand for enhanced flavor and aroma, increased bioavailability, and targeted delivery of bioactive compounds to enhance their health-promoting effects, longer shelf life, cost effectiveness, and among others. Flavor and taste of functional beverages have been improved using encapsulation technology creating innovative products. Specialized characteristics have been added to functional beverages such as enhanced flavor, masked unpleasant odor, long lasting, sustained and burst, or quick release of flavor. Functional ingredients are incorporated into beverages using encapsulation technology to improve their health-promoting properties. They also allow the protection of probiotics, readily oxidable components such as phenolic compounds, vitamins, and unsaturated lipids. Moreover, encapsulation technology has increased the beverage alternatives for consumers with diverse characteristics such as color and flavor differences, foam-producing, cooling and warming effects long-lasting and slow-releasing flavor, multicolor hydrocolloids, intelligent packaging, and effervescence. There is a growing interest of functional beverages with health-promoting and value-added encapsulation technologies that take a role in targeted delivery of bioactive compounds. Besides, encapsulation technologies are taking great attention for nutrigenomics and metabolomics for production of functional beverages specific for individual genetic prototypes and specific metabolic requirements. Many new encapsulation methods have been developed such as bioactive tagging such as PEGylation, nanoemulsions, and electrospinning for producing nanofibers and nanotubes, in addition to traditional encapsulation methods such as spray drying, extrusion, and coacervation. This chapter highlights the encapsulation methods and materials, their functions, and applications in the functional beverage industry.

Abstract The objectives of this study were to develop a probiotic milk powder containing Lactobacillus plantarum NCIMB 8826 immobilized with prebiotic Hi-maize starch and to analyze cell viability after spray drying and exposure to simulated gastric and intestinal conditions. Milk powders containing free L. plantarum and cells immobilized with Hi-maize starch were assessed. Powders were evaluated during storage at 4 °C for 15 days. After spray drying, at 0 and 15 days of storage both treatments had over 8 log CFU/g of viable cells and there were higher viable counts found for immobilized cells compared to free cells after 120 min in simulated gastric fluid. At 15 days of storage, immobilized cells had higher viable counts than free cells after exposure to simulated intestinal fluid for 120 min. The combined probiotic and prebiotic milk powder had stable viable cell counts at refrigerated storage conditions and under simulated gastric and intestinal transit. Graphical abstract

Kafirin microparticles have potential as colon-targeted delivery systems because of their ability to protect encapsulated material from digestive processes of the upper gastrointestinal tract (GIT). The aim was to optimize prednisolone loading into kafirin microparticles, and investigate their potential as an oral delivery system. Response surface methodology (RSM) was used to predict the optimal formulation of prednisolone loaded microparticles. Prednisolone release from the microparticles was measured in simulated conditions of the GIT. The RSM models were inadequate for predicting the relationship between starting quantities of kafirin and prednisolone, and prednisolone loading into microparticles. Compared to prednisolone released in the simulated gastric and small intestinal conditions, no additional drug release was observed in simulated colonic conditions. Hence, more insight into factors affecting drug loading into kafirin microparticles is required to improve the robustness of the RSM model. This present method of formulating prednisolone-loaded kafirin microparticles is unlikely to offer clinical benefits over commercially available dosage forms. Nevertheless, the overall amount of prednisolone released from the kafirin microparticles in conditions simulating the human GIT demonstrates their ability to prevent the release of entrapped core material. Further work developing the formulation methods may result in a delivery system that targets the lower GIT.

Het RIVM onderzoekt hoe mensen ziek worden als zij voedsel eten dat besmet is met de bacterie Clostridium perfringens. Dit wordt gedaan met experimenten die de omstandigheden in maag en darmen nabootsen. Doel van het onderzoek is om een schatting te maken van het aantal ziektegevallen in Nederland per jaar. Momenteel is dat moeilijk omdat de klachten (voornamelijk waterige diarree) kortdurend en mild zijn. Daardoor gaan patienten niet snel naar een arts en worden gevallen van de ziekte niet snel geregistreerd. Voor een gedegen schatting van het aantal ziektegevallen is kennis nodig over de manier waarop de ziekte ontstaat en hoe vaak en in welke mate de bacterie in voedsel voorkomt. Voor veel bacterien vormt de maag een moeilijk te nemen obstakel, maar C. perfringens lijkt de maag gemakkelijk te passeren. Dit rapport is een tussenrapportage. Het onderzoek wordt bemoeilijkt doordat de productie van de gifstof, die de klachten veroorzaakt, een proces is dat moeilijk is na te bootsen en door veel factoren wordt beinvloed. Ook bestaan er veel stammen van deze bacterie die op verschillende manieren reageren. Daarnaast moet nog worden onderzocht hoe vaak en in welke mate de bacterie in voedsel voorkomt. Nader onderzoek is daarom nodig.

Few in vitro screening assays for biological activities of plant extracts consider the potential effect of the gastrointestinal system on orally consumed plant extracts. Crude water and methanol extracts of Tarchonanthus camphoratus (wild camphor) and Agathosma betulina (‘buchu’) were prepared and exposed to simulated gastric fluid and simulated intestinal fluid during dissolution studies to address this aspect. The crude extracts and resulting simulated gastric fluid and simulated intestinal fluid products were screened for antimicrobial activity against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Proteus vulgaris (ATCC 33420). The T. camphoratus crude extract exhibited antimicrobial activity which was reduced after exposure to simulated gastric fluid. After exposure to simulated intestinal fluid no antimicrobial activity was detected, which suggests chemical alteration or degradation of the active compounds. For A. betulina , the crude water extract and simulated gastric fluid product exhibited no antimicrobial activity, while the simulated intestinal fluid product exhibited antimicrobial activity. This suggests activation of antimicrobial constituents during exposure to simulated intestinal fluid. The chemical composition profiles of the crude extracts and products were determined by means of liquid chromatography coupled to an ultraviolet detector (LC-UV) and a mass spectrometer (LC-MS) to qualitatively assess the effect of exposure to simulated gastrointestinal conditions on the chemical composition of the extracts. In many cases, the peak area of compounds decreased after exposure to simulated gastric fluid and simulated intestinal fluid, while the peak area of other compounds increased. Thus, it can be deduced that the antimicrobial activity and chemical composition was altered after exposure to intestinal conditions during dissolution studies.

This study considers the influence of exposure to simulated gastrointestinal conditions (saliva, gut, intestine and acidic conditions of the gut) on IgG binding of unmodified allergens and three types of LMW allergoids of Artemisia vulgaris pollen extract obtained by means of potassium cyanate, succinic and maleic anhydride. It also concerns the optimization of a self-developed ELISA assay for comparison of the specific IgG binding of mugwort pollen extract and modified mugwort pollen derivatives. The ELISA was conducted with a mugwort pollen extract coupled to the plate, using the sera from 12 mugwort-pollen allergic patients. The exposure to saliva fluid for 2 min did not influence the IgG binding properties of allergens and allergoids. Exposure of mugwort pollen allergens and LMW allergoids to the acidic conditions of the gut did not dramatically change their IgG binding properties. By exposing mugwort pollen extract and LMW derivatives to the SGF conditions for 1 h, the percent of IgG binding epitopes was reduced to a half of its starting value in the extract and to about 30 % in all the allergoid samples. After prolonged exposure only the carbamyl derivative showed reduced IgG binding. Changes of the IgG binding potential of all four samples after exposure in SIF followed a similar pattern.

This study considers the influence of exposure to simulated gastrointestinal conditions (saliva, gut, intestine and acidic conditions of the gut) on IgG binding of unmodified allergens and three types of LMW allergoids of Artemisia vulgaris pollen extract obtained by means of potassium cyanate succinic and maleic anhydride. It also concerns the optimization of a self-developed ELISA assay for comparison of the specific IgG binding of mugwort pollen extract and modified mugwort pollen derivatives. The ELISA was conducted with a mugwort pollen extract coupled to the plate, using the sera from 12 mugwort- pollen allergic patients. The exposure to saliva fluid for 2 min did not influence the IgG binding properties of allergens and allergoids. Exposure of mugwort pollen allergens and LMW allergoids to the acidic conditions of the gut did not dramatically change their IgG binding properties. By exposing mugwort pollen extract and LMW derivatives to the SGF conditions for 1 h, the percent of IgG binding epitopes was reduced to a half of its starting value in the extract and to about 30%in all the allergoid samples. After prolonged exposure only the carbamyl derivative showed reduced IgG binding. Changes of the IgG binding potential of all four samples after exposure in SIF followed a similar pattern. Predmet ovog rada je ispitivanje promena IgG vezivanja nemodifikovanih alergena polena A. vulgaris i tri tipa alergoida malih molekulskih masa dobijenih tretmanom sa kalijum-cijanatom, i anhidridima ćilibarne i meleinske posle izlaganja uslovima gastrointestinalnog trakta (saliva, želudac, tanko crevo i kisela sredina želudačnog soka) i optimizacija domaćeg ELISA testa za određivanje IgG vezivanja alergena i alergoida polena pelina.UELISA testu je za pločicu kuplovan ekstrakt polena pelina i korišćeni su serumi 12 pacijenata alergičnih na pelin. Izlaganje salivi u trajanju od 2 minuta ne utiče na IgG vezujuće osobine alergena i alergoida. Izlaganje kiseloj sredini želudačnog soka značajno ne utiče na IgG vezujuće osobine alergena i alergoida polena pelina. Posle izlaganja simuliranom želudačnom soku alergena i derivata u trajanju od 1 sata, procenat IgG vezujućih epitopa u nemodifikovanom uzorku se smanjuje na polovinu početne vrednosti i na oko 30 % kod sva tri derivata. Jedino se kod karbamil-derivata % IgG vezivanja dodatno smanjuje sa produžavanjem izlaganja SGF-u.Promene u IgG vezujućem potencijalu sva 4 uzorka posle izlaganja simuliranim uslovima tankog creva prate sličan obrazac.

Various microbial polymers, namely xanthan gum, gellan gum, pullulan gum and jamilan, were tested as a suitable encapsulating material for Lactobacillus plantarum CRL 1815 and Lactobacillus rhamnosus ATCC 53103. Resulting capsules were also studied for their pH and simulated gastrointestinal conditions tolerance. The morphology of the microcapsules was studied using scanning electron microscopy. pH tolerance was tested at pH 2.0, 3.5, 5.0 and 6.5 over a 6. h incubation period. Simulated gastrointestinal conditions were assayed with simulated gastric and pancreatic juices and simulated bile over a 24. h incubation period. Suspensions of probiotic organisms were used as a control. The results from encapsulation with microbial polymers indicate that mixtures of 1% xanthan gum with 0.75% gellan gum and 1% jamilan with 1% gellan gum were the most suitable for microencapsulation. Results for the pH tolerance tests showed no improvement in the viability of cells in relation to the control, except for pH 2.0 where lactobacilli encapsulated in xanthan:gellan gum (1%:0.75%) prolonged their viability by 6. h exposure. Xanthan:gellan gum (1%:0.75%) was the most effective of the encapsulating materials tested in protecting L. plantarum and L. rhamnosus against simulated bile, improving its viability in 1-2. log. CFU when compared with control. The results of this study suggest that microbial polymers are an interesting source of encapsulating material that should be taken into account for prospective studies of probiotic encapsulation for oral delivery applications. Fil: Jiménez Pranteda, María Luján. Universidad de Granada; España. École Nationale Nantes Atlantique Vétérinaire, Agroalimentaire et de l'Alimentation; Francia. Centro de Investigación Biomédica. Instituto de Nutrición y Tecnología de los Alimentos; España Fil: Poncelet, Denis. École Nationale Nantes Atlantique Vétérinaire, Agroalimentaire et de l'Alimentation; Francia Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Arcos, Antonio. Universidad de Granada; España Fil: Aguilera Gómez, Margarita. Universidad de Granada; España. Centro de Investigación Biomédica. Instituto de Nutrición y Tecnología de los Alimentos; España Fil: Monteoliva Sánchez, Mercedes. Universidad de Granada; España. Centro de Investigación Biomédica. Instituto de Nutrición y Tecnología de los Alimentos; España Fil: Ramos Cormenzana, Alberto. Universidad de Granada; España. Centro de Investigación Biomédica. Instituto de Nutrición y Tecnología de los Alimentos; España

Orientador: Mirna Lúcia Gigante Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: A microencapsulação de micro-organismos probióticos tem se mostrado como uma técnica promissora para manter sua viabilidade tanto nos produtos aos quais são adicionados, bem como durante sua passagem pelo trato gastrointestinal. O objetivo geral deste trabalho foi avaliar a fabricação e as características durante o armazenamento refrigerado de iogurte batido probiótico adicionado de Lactobacillus acidophilus nas formas livre e encapsulada. O micro-organismo probiótico foi microencapsulado por gelificação iônica e coacervação complexa, utilizando-se pectina e concentrado proteico de soro como materiais de parede. Antes de serem adicionadas ao iogurte, as microcápsulas foram avaliadas quanto à característica físico-química e viabilidade do micro-organismo probiótico. Os iogurtes foram fabricados com leite homogeneizado esterilizado e padronizado adicionado de 2,5% (v/v) da cultura láctica tradicional para fabricação de iogurte e submetidos aos seguintes tratamentos: 1) adição de 1% (v/v) de L. acidophilus livre; 2) adição de 10% (p/v) de microcápsulas úmidas contendo L. acidophilus e 3) adição de 13% (p/v) de microcápsulas liofilizadas reidratadas contendo L. acidophilus. O tempo de fermentação foi considerado o necessário para que o produto atingisse pH 4,8 ± 0,05. Os produtos foram caracterizados quanto à sua composição centesimal e população dos microorganismos. Após 1, 7, 14, 21, 28 e 35 dias de armazenamento refrigerado, os iogurtes foram avaliados quanto à pós-acidificação e viabilidade dos micro-organismos da cultura tradicional de iogurte e probiótica. A viabilidade do L. acidophilus durante a simulação da passagem do iogurte através das condições gastrointestinais foi avaliada após 35 dias. O experimento completo foi repetido três vezes e o delineamento experimental utilizado foi o Split¿plot, em um planejamento do tipo fatorial 3 x 6, em blocos completamente aleatorizados. O efeito dos tratamentos, do tempo de armazenamento bem como da interação desses fatores sobre as variáveis estudadas foi avaliado por ANOVA aplicando-se teste de Tukey para comparação entre as médias ao nível de 5% de significância. O tempo de fermentação dos produtos variou de 180 a 200 minutos. Um dia após a fabricação, o iogurte obtido com o micro-organismo livre apresentou menor pH que os iogurtes adicionados do micro-organismo encapsulado. Os iogurtes produzidos com o micro-organismo encapsulado apresentaram maior teor de proteína quando comparados ao produzido com o L. acidophilus livre. A interação entre os tratamentos e o tempo de armazenamento refrigerado afetou significativamente a pós-acidificação e o número de células viáveis de S. thermophilus, L. bulgaricus e L. acidophilus no iogurte. Os iogurtes probióticos adicionados de L. acidophilus na forma microencapsulada apresentaram menor pós-acidificação e maior sobrevivência do micro-organismo probiótico após 35 dias de armazenamento quando comparado ao iogurte com probiótico adicionado na forma livre. Na forma livre, a população de células viáveis de L. acidophilus reduziu 0,98 ciclos logarítmicos, enquanto na forma encapsulada (úmida e liofilizada) essa redução foi de 0,20 ciclos logarítmicos. Esses resultados indicam a sobrevivência de aproximadamente 10% e 62% do L. acidophilus nas formas livre e encapsulada, respectivamente. Após 35 dias de armazenamento refrigerado, a simulação da passagem dos produtos pelo trato gastrointestinal indicou maior sobrevivência do L. acidophilus encapsulado do que do micro-organismo livre. A microencapsulação do L. acidophilus por gelificação iônica e coacervação complexa utilizando-se pectina e proteína de soro como materiais de parede, conferiu proteção ao micro-organismo, tanto durante o armazenamento refrigerado do produto por 35 dias, como durante a simulação gastrointestinal Abstract: Microencapsulation of probiotic microorganisms has been shown to be a promising technique to maintain their viability either in products to which they are added and during their passage through the gastrointestinal tract. The aim of this study was to evaluate the manufacture and characteristics during refrigerated storage of probiotic stirred yoghurt added of Lactobacillus acidophilus in both free and encapsulated forms. The probiotic microorganism was microencapsulated by ionotropic gelation and complex coacervation, using pectin and whey protein concentrate as wall materials. Before being added to yoghurt, the microcapsules were evaluated for physical chemical characteristics and viability of the probiotic microorganism. The yoghurts were manufactured with sterilized, homogenized and standardized milk added of 2.5% (v/v) yoghurt starter culture and submitted to the following treatments: 1) addition of 1% (v/v) of free L. acidophilus, 2) addition of 10% (w/v) of moist microcapsules containing L. acidophilus and 3) addition of 13% (w/v) of rehydrated freeze-dried microcapsules containing L. acidophilus. The fermentation period was considered the enough time for the product to reach pH 4.8 ± 0.05. The products were characterized to their centesimal composition and population of microorganisms. After 1, 7, 14, 21, 28 and 35 days of refrigerated storage, the yoghurts were evaluated for post-acidification and viability of yoghurt starter culture and probiotic microorganism. The viability of L. acidophilus during simulation of the passage of yoghurt through gastrointestinal conditions was also evaluated after 35 days. The complete experiment was repeated three times and the experimental design was a Split-Plot in a factorial design of the type 3 x 6, in completely randomized blocks. The effect of treatments, storage time and the interaction of these factors on these studied variables was evaluated by ANOVA according to the Tukey test for comparison between the averages, in 5% of significance level. The fermentation period of the products ranged from 180 to 200 minutes. One day after manufacturing, the yoghurt obtained with free microorganism showed lower pH than the yoghurt produced with added encapsulated microorganism. The yoghurts produced with the encapsulated microorganism showed higher protein content than that produced with free L. acidophilus. The interaction between treatments and time of refrigerated storage affected significantly the post-acidification and the number of viable cells of S. thermophilus, L. bulgaricus and L. acidophilus in yoghurt. The probiotics yoghurts added by L. acidophilus in microencapsulated form showed less post-acidification and increased survival of probiotic microorganism after 35 days of storage than yoghurt added of probiotic in free form. The counting of viable cells of L. acidophilus reduced 0.98 log cycles in free form, while in encapsulated form (moist and freeze-dried) the reduction was 0.20 log cycles. These results indicate the survival of approximately 10% and 62% of L. acidophilus in both free and encapsulated forms, respectively. After 35 days of refrigerated storage, the simulation of the passage of yoghurts through the gastrointestinal tract showed a higher survival of L. acidophilus encapsulated than the free microorganism. Microencapsulation of L. acidophilus by ionotropic gelation and complex coacervation, using pectin and whey protein concentrate as wall materials provide protection to microorganism, both during refrigerated storage for 35 days and during simulated gastrointestinal conditions Mestrado Engenharia de Alimentos Mestre em Tecnologia de Alimentos

This study considers the influence of exposure to simulated gastrointestinal conditions (saliva. gut. intestine and acidic conditions of the gut) on hiCy binding of unmodified allergens and three types of LMW allergoids of Artemisia vulgaris pollen extract obtained by means of potassium cyanate. succinic and maleic anhydride. It also concerns the optimization of a self-developed ELISA assay for comparison of the specific IgG binding of mugwort pollen extract and modified mugwort pollen derivatives. The ELISA was conducted with a mugwort pollen extract coupled to the plate. using the sera from 12 mugwort-pollen allergic patients. The exposure to saliva fluid for 2 min did not influence the IgG binding properties of allergens and allergoids. Exposure of mugwort pollen allergens and LMW allergoids to the acidic conditions of the gut did not dramatically change their IgG binding properties. By exposing mugwort pollen extract and LMW derivatives to the SGF conditions for 1 h. the percent of IgG binding epitopes was reduced to a half of its starting value in the extract and to about 30 % in all the allergoid samples. After prolonged exposure only the carbamyl derivative showed reduced IgG binding. Changes of the IgG binding, potential of all four samples after exposure in SIF followed a similar Pattern. Predmet ovog rada je ispitivanje promena IgG vezivanja nemodifikovanih alergena polena A. vulgaris i tri tipa alergoida malih molekulskih masa dobijenih tretmanom sa kalijum-cijanatom, i anhidridima ćilibarne i meleinske posle izlaganja uslovima gastrointestinalnog trakta (saliva, želudac, tanko crevo i kisela sredina želudačnog soka) i optimizacija domaćeg ELISA testa za određivanje IgG vezivanja alergena i alergoida polena pelina.UELISA testu je za pločicu kuplovan ekstrakt polena pelina i korišćeni su serumi 12 pacijenata alergičnih na pelin. Izlaganje salivi u trajanju od 2 minuta ne utiče na IgG vezujuće osobine alergena i alergoida. Izlaganje kiseloj sredini želudačnog soka značajno ne utiče na IgG vezujuće osobine alergena i alergoida polena pelina. Posle izlaganja simuliranom želudačnom soku alergena i derivata u trajanju od 1 sata, procenat IgG vezujućih epitopa u nemodifikovanom uzorku se smanjuje na polovinu početne vrednosti i na oko 30 % kod sva tri derivata. Jedino se kod karbamil-derivata % IgG vezivanja dodatno smanjuje sa produžavanjem izlaganja SGF-u.Promene u IgG vezujućem potencijalu sva 4 uzorka posle izlaganja simuliranim uslovima tankog creva prate sličan obrazac.

L. acidophilus has been considered to be among the predominant lactobacilli in the intestinal tract of healthy humans. The ability of L. acidophilus to survive and retain its viability through the gastrointestinal (GI) tract is one of the main characteristics desirable for probiotic activity. The influence of mucin and dietary constituents, such as casein, whey proteins concentrate (WPC) and skim milk, on survival of L. acidophilus M92 in the simulated GI conditions, was determined. A washed cell suspension was exposed to pepsin (3g/L) and sodium chloride (5g/L) at pH = 1.5, 2.0, 2.5 and 3.0, and to pancreatin (1g/L), sodium chloride (5g/L) and oxgall (1.5, 2.0, 3.0, 5.0 mg/mL) at pH = 8.0, mimicking the GI environment. Much greater tolerance to gastric and small intestinal juice was observed in the presence of mucin and milk proteins, especially with WPC. The influence of WPC, as the best protector, on the cell viability of L. acidophilus M92 in simulated gastric and small intestinal juice was confirmed by a mathematical model. Critical points, indicating GI conditions that L. acidophilus M92 can tolerate, were established. These observations showed that L. acidophilus M92 survives at pH = 1.99 in simulated gastric juice for 2 h and at 3.1 mg/mL oxgall in simulated small intestinal juice for 4 h, while in the presence of WPC it tolerates even lower pH (1.77) and higher concentration of oxgall (4.25 g/L). Furthermore, only 15 % of the L. acidophilus M92 cells survived the direct transit from simulated gastric to simulated small intestinal juice, but with added WPC, 45 % of the L. acidophilus M92 cells survived. These results suggested the addition of WPC as a protector in preparation of L. acidophilus M92 for probiotic use.