Orientador: Adriana Vitorino Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T21:18:58Z (GMT). No. of bitstreams: 1 Souza_PatriciaTononde_M.pdf: 1500158 bytes, checksum: 99c73fcb4e4c8143227e13cef0571323 (MD5) Previous issue date: 2012 / Resumo: Capsaicinóides (CAPS) são substâncias responsáveis pela pungência em pimentas do gênero Capsicum. Quantificar CAPS é relevante devido às aplicações desses compostos na indústria alimentícia, farmacêutica e cosmética, além do uso em armas químicas. Nesse trabalho, foi desenvolvido método espectrofotométrico baseado na reação colorimétrica entre CAPS, Co e 4-(2-piridilazo)resorcinol, PAR, para quantificação de CAPS totais (CCAPS) em diversas espécies de Capsicum. O produto da reação tem proporção estequiométrica Co:PAR:CAPS 1:2:2 e absorção em 510 nm proporcional a CCAPS. A curva analítica apresentou faixa linear entre 0,001 e 0,03 U, limite de detecção = 0,0004 U e limite de quantificação = 0,0005 U (U = mg CAPS/g pimenta) e coeficiente de correlação = 0,9979. Os resultados foram comparados com o método de cromatografia líquida de alta eficiência (HPLC) e não apresentaram diferença significativa com 95% de confiança, além disso, ambos resultados são coerentes com as escalas organolépticas disponíveis na literatura. No aperfeiçoamento das condições para obtenção de extratos etanólicos de CAPS, estabeleceu-se o uso de cachaça (teor alcoólico 39%), proporção pimenta:cachaça 30:50 g/mL e agitação de 30 min. O estudo da conservação dos extratos indicou que é favorável armazená-los a 6°C, a estocagem das pimentas in natura a -11 °C por até três meses não leva a perda significativa de CAPS e a CCAPS aumenta a medida que o estágio de maturação de pimenta é completado / Abstract: Capsaicinoids (CAPS) are substances responsible for pungency in Capsicum. CAPS is relevant to quantify due to the applications of these comounds in food, pharmaceutical and cosmetic industries, and the use of chemical weapons. Inthis work, we developed a spectrophotometric method based on the colorimetric reaction between CAPS, Co and 4-(2-pyridylazo)resorcinol, PAR, for quantification of total CAPS (CCAPS) in several species of Capsicum. The product of the reaction is stoichiometric Co:PAR:CAPS 1:2:2 and absorption at 510 nm proportional to CCAPS. The analytical curve linear range between 0,001 and 0,03 U, detection limit = 0,0004 and limit of quantification U = 0,0005 U (U = mg CAPS/g pepper) and correlation coefficient = 0,9979. The results were compared with the method of high performance liquid chromatography (HPLC) and not significantly different at 95% confidence, moreover, both results are consistent with the organoleptic scales available in the literature. The improvement of conditions for obtaining ethanol extracts of CAPS, established the use of cachaça (alcohol content 39%), proportion pepper:cachaça 30:50 g/mL and stirred for 30 min. The study of the conservation of the extracts indicated that it supports storing them at 6 °C, the storage of in nature peppers at -11 °C for up to three months does not lead to significant loss of CAPS and CCAPS increases as the stage of maturation of pepper is added / Mestrado / Quimica Analitica / Mestra em Química

Capsaicinoids are a class of compounds responsible for the "heat" of hot peppers. Capsaicin and dihydrocapsaicin have the highest burning effect. The aim of this work is to separate and quantify the two major capsaicinoids in fruits harvested at different stages of development and at different seasons. Simple and rapid HPLC method involves 73:27% methanol water mobile phase with C18 stationary phase and UV-Vis detector set at 210 nm. The method showed good reproducibility with 1.74% - 4.72% relative standard deviations, a linear response within 0.65–45.5 and 0.25-17.5 μg/mL for capsaicin and dihydrocapsaicin, respectively. The method achieved average recovery of 106% for capsaicin and 102% dihydrocapsaicin. Determination of capsaicinoids in four naturally grown chili and commercial source habanero were analyzed. The amount in the sample ranged from 1184-8156 μg/g for capsaicin and 430-3299 μg/g for dihydrocapsaicin.

Objetivos: Desenvolver e caracterizar hidrogéis de quitosana contendo capsaicinóides nanoencapsulados, bem como analisar o efeito das nanocápsulas e do veículo nos perfis de liberação dos capsaicinóides. Metodologia: As nanocápsulas foram obtidas (método de deposição interfacial do polímero préformado) utilizando Eudragit® RS100 e triglicerídeos do ácido cáprico/caprílico como parede polimérica e núcleo oleoso, respectivamente, e foram caracterizadas. Para determinação do teor e da taxa de associação da capsaicina e dihidrocapsaicina às nanocápsulas utilizou-se metodologia validada por CLAE-UV. As nanocápsulas contendo capsaicinóides foram incorporadas em hidrogel de quitosana. Para tanto, determinaram-se previamente as características reológicas e o hidrogel ideal para uso tópico. Através dos estudos de liberação in vitro, utilizando método de saco de diálise, foram obtidos os perfis de liberação dos fármacos a partir das nanocápsulas e destas incorporadas no veículo semissólido. Resultados e Discussão: As nanocápsulas apresentaram diâmetro médio de partícula de 153 ± 7nm (PDI < 0,2), potencial zeta de + 9,62 ± 1,48 mV, pH de 5,7 ± 0,1 e uma leve tendência à sedimentação, típica de suspensões. A nanoencapsulação dos capsaicinóides não provocou alterações nas características da formulação. O teor e a taxa de associação foram próximos a 100% para ambos capsaicinóides, considerando concentração de 0,5mg/mL de capsaicinoides na formulação. Foi possível obter um hidrogel de quitosana contendo nanocápsulas adequado para uso tópico. Previamente, observou-se uma grande influência da concentração de quitosana na consistência dos hidrogéis e um comportamento reológico pseudoplástico que não foi alterado pela adição das nanocápsulas. Observou-se uma liberação controlada devido à nanoencapsulação, de modo que dihidrocapsaicina foi liberada mais lentamente do que a capsaicina. Pode-se observar um retardo na liberação da capsaicina, quando as nanocápsulas foram incorporadas no hidrogel. Conclusões: Nanocápsulas estáveis e capazes de controlar a liberação dos capsaicinóides foram obtidas, sendo que tanto o núcleo oleoso quanto o polímero são responsáveis pelo controle da liberação. A formulação semissólida contendo nanocápsulas, obtida a partir do polímero quitosana, manteve a liberação controlada observada e apresentou adequadas características para aplicação tópica. / Purpose: To develop and characterize chitosan hydrogels containing nanoencapsulated capsaicin and dihydrocapsaicin, as well as to analyze the effect of the nanocapsulation and the vehicle on the in vitro release of the capsaicinoids. Methodology: Nanocapsules were obtained (interfacial deposition of the pre-formed polymer method) using Eudragit® RS100 and capric/caprylic triglycerids as the polymeric shell and oily core, respectively, and they were characterized. An HPLCUV validated method was used to determine the drug content and drug encapsulation efficiency. The nanocapsules containing capsaicinoids were incorporated in a chitosan hydrogel. Previously, the rheological properties and the most suitable hydrogel aiming the topical delivery were determined. By means of in vitro release experiments (dialyses bags), the release profiles of the capsaicinoids from the nanocapsules and the gel containing nanocapsules were obtained. Results and Discussion: The nanocapsule formulation presented an average diameter of 153 ± 7nm (PDI < 0.2), the zeta potential was +9.62 ± 1.48 mV, and the pH 5.7± 0.1. A slight tendency to sedimentation was observed, typical of suspensions. The drug encapsulation did not alter the formulation properties. The drug content and encapsulation efficiency were nearly 100% for both capsaicinoids, considering a drug amout of 0.5mg/mL of capsaicinoids in the formulation. A chitosan hydrogel containing nanocapsules were successfully obtanined. The chitosan concentration significantly influenced the consistence of the formulations, and the pseudoplastic behavior did not change after the nanocapsule incorporation. controlled release was observed due to nanoencapsulation, in a way that capsaicin released faster than dihydrocapsaicin. The release of capsaicin from the hydrogel containing nanocapsules presented a delay in comparison with the release from the nanocapsule suspension. Conclusions: Stable nanocapsules capable of controlling the capsaicinoids release were obtained. Both the oily core and the polymeric shell are responsible for the controlled release. The semi-solid formulation containing nanocapsules obtained with the polymer chitosan maintained the controlled release and presented suitable properties for cutaneous applications.

Objetivos: Desenvolver e caracterizar hidrogéis de quitosana contendo capsaicinóides nanoencapsulados, bem como analisar o efeito das nanocápsulas e do veículo nos perfis de liberação dos capsaicinóides. Metodologia: As nanocápsulas foram obtidas (método de deposição interfacial do polímero préformado) utilizando Eudragit® RS100 e triglicerídeos do ácido cáprico/caprílico como parede polimérica e núcleo oleoso, respectivamente, e foram caracterizadas. Para determinação do teor e da taxa de associação da capsaicina e dihidrocapsaicina às nanocápsulas utilizou-se metodologia validada por CLAE-UV. As nanocápsulas contendo capsaicinóides foram incorporadas em hidrogel de quitosana. Para tanto, determinaram-se previamente as características reológicas e o hidrogel ideal para uso tópico. Através dos estudos de liberação in vitro, utilizando método de saco de diálise, foram obtidos os perfis de liberação dos fármacos a partir das nanocápsulas e destas incorporadas no veículo semissólido. Resultados e Discussão: As nanocápsulas apresentaram diâmetro médio de partícula de 153 ± 7nm (PDI < 0,2), potencial zeta de + 9,62 ± 1,48 mV, pH de 5,7 ± 0,1 e uma leve tendência à sedimentação, típica de suspensões. A nanoencapsulação dos capsaicinóides não provocou alterações nas características da formulação. O teor e a taxa de associação foram próximos a 100% para ambos capsaicinóides, considerando concentração de 0,5mg/mL de capsaicinoides na formulação. Foi possível obter um hidrogel de quitosana contendo nanocápsulas adequado para uso tópico. Previamente, observou-se uma grande influência da concentração de quitosana na consistência dos hidrogéis e um comportamento reológico pseudoplástico que não foi alterado pela adição das nanocápsulas. Observou-se uma liberação controlada devido à nanoencapsulação, de modo que dihidrocapsaicina foi liberada mais lentamente do que a capsaicina. Pode-se observar um retardo na liberação da capsaicina, quando as nanocápsulas foram incorporadas no hidrogel. Conclusões: Nanocápsulas estáveis e capazes de controlar a liberação dos capsaicinóides foram obtidas, sendo que tanto o núcleo oleoso quanto o polímero são responsáveis pelo controle da liberação. A formulação semissólida contendo nanocápsulas, obtida a partir do polímero quitosana, manteve a liberação controlada observada e apresentou adequadas características para aplicação tópica. / Purpose: To develop and characterize chitosan hydrogels containing nanoencapsulated capsaicin and dihydrocapsaicin, as well as to analyze the effect of the nanocapsulation and the vehicle on the in vitro release of the capsaicinoids. Methodology: Nanocapsules were obtained (interfacial deposition of the pre-formed polymer method) using Eudragit® RS100 and capric/caprylic triglycerids as the polymeric shell and oily core, respectively, and they were characterized. An HPLCUV validated method was used to determine the drug content and drug encapsulation efficiency. The nanocapsules containing capsaicinoids were incorporated in a chitosan hydrogel. Previously, the rheological properties and the most suitable hydrogel aiming the topical delivery were determined. By means of in vitro release experiments (dialyses bags), the release profiles of the capsaicinoids from the nanocapsules and the gel containing nanocapsules were obtained. Results and Discussion: The nanocapsule formulation presented an average diameter of 153 ± 7nm (PDI < 0.2), the zeta potential was +9.62 ± 1.48 mV, and the pH 5.7± 0.1. A slight tendency to sedimentation was observed, typical of suspensions. The drug encapsulation did not alter the formulation properties. The drug content and encapsulation efficiency were nearly 100% for both capsaicinoids, considering a drug amout of 0.5mg/mL of capsaicinoids in the formulation. A chitosan hydrogel containing nanocapsules were successfully obtanined. The chitosan concentration significantly influenced the consistence of the formulations, and the pseudoplastic behavior did not change after the nanocapsule incorporation. controlled release was observed due to nanoencapsulation, in a way that capsaicin released faster than dihydrocapsaicin. The release of capsaicin from the hydrogel containing nanocapsules presented a delay in comparison with the release from the nanocapsule suspension. Conclusions: Stable nanocapsules capable of controlling the capsaicinoids release were obtained. Both the oily core and the polymeric shell are responsible for the controlled release. The semi-solid formulation containing nanocapsules obtained with the polymer chitosan maintained the controlled release and presented suitable properties for cutaneous applications.

Objetivos: Desenvolver e caracterizar hidrogéis de quitosana contendo capsaicinóides nanoencapsulados, bem como analisar o efeito das nanocápsulas e do veículo nos perfis de liberação dos capsaicinóides. Metodologia: As nanocápsulas foram obtidas (método de deposição interfacial do polímero préformado) utilizando Eudragit® RS100 e triglicerídeos do ácido cáprico/caprílico como parede polimérica e núcleo oleoso, respectivamente, e foram caracterizadas. Para determinação do teor e da taxa de associação da capsaicina e dihidrocapsaicina às nanocápsulas utilizou-se metodologia validada por CLAE-UV. As nanocápsulas contendo capsaicinóides foram incorporadas em hidrogel de quitosana. Para tanto, determinaram-se previamente as características reológicas e o hidrogel ideal para uso tópico. Através dos estudos de liberação in vitro, utilizando método de saco de diálise, foram obtidos os perfis de liberação dos fármacos a partir das nanocápsulas e destas incorporadas no veículo semissólido. Resultados e Discussão: As nanocápsulas apresentaram diâmetro médio de partícula de 153 ± 7nm (PDI < 0,2), potencial zeta de + 9,62 ± 1,48 mV, pH de 5,7 ± 0,1 e uma leve tendência à sedimentação, típica de suspensões. A nanoencapsulação dos capsaicinóides não provocou alterações nas características da formulação. O teor e a taxa de associação foram próximos a 100% para ambos capsaicinóides, considerando concentração de 0,5mg/mL de capsaicinoides na formulação. Foi possível obter um hidrogel de quitosana contendo nanocápsulas adequado para uso tópico. Previamente, observou-se uma grande influência da concentração de quitosana na consistência dos hidrogéis e um comportamento reológico pseudoplástico que não foi alterado pela adição das nanocápsulas. Observou-se uma liberação controlada devido à nanoencapsulação, de modo que dihidrocapsaicina foi liberada mais lentamente do que a capsaicina. Pode-se observar um retardo na liberação da capsaicina, quando as nanocápsulas foram incorporadas no hidrogel. Conclusões: Nanocápsulas estáveis e capazes de controlar a liberação dos capsaicinóides foram obtidas, sendo que tanto o núcleo oleoso quanto o polímero são responsáveis pelo controle da liberação. A formulação semissólida contendo nanocápsulas, obtida a partir do polímero quitosana, manteve a liberação controlada observada e apresentou adequadas características para aplicação tópica. / Purpose: To develop and characterize chitosan hydrogels containing nanoencapsulated capsaicin and dihydrocapsaicin, as well as to analyze the effect of the nanocapsulation and the vehicle on the in vitro release of the capsaicinoids. Methodology: Nanocapsules were obtained (interfacial deposition of the pre-formed polymer method) using Eudragit® RS100 and capric/caprylic triglycerids as the polymeric shell and oily core, respectively, and they were characterized. An HPLCUV validated method was used to determine the drug content and drug encapsulation efficiency. The nanocapsules containing capsaicinoids were incorporated in a chitosan hydrogel. Previously, the rheological properties and the most suitable hydrogel aiming the topical delivery were determined. By means of in vitro release experiments (dialyses bags), the release profiles of the capsaicinoids from the nanocapsules and the gel containing nanocapsules were obtained. Results and Discussion: The nanocapsule formulation presented an average diameter of 153 ± 7nm (PDI < 0.2), the zeta potential was +9.62 ± 1.48 mV, and the pH 5.7± 0.1. A slight tendency to sedimentation was observed, typical of suspensions. The drug encapsulation did not alter the formulation properties. The drug content and encapsulation efficiency were nearly 100% for both capsaicinoids, considering a drug amout of 0.5mg/mL of capsaicinoids in the formulation. A chitosan hydrogel containing nanocapsules were successfully obtanined. The chitosan concentration significantly influenced the consistence of the formulations, and the pseudoplastic behavior did not change after the nanocapsule incorporation. controlled release was observed due to nanoencapsulation, in a way that capsaicin released faster than dihydrocapsaicin. The release of capsaicin from the hydrogel containing nanocapsules presented a delay in comparison with the release from the nanocapsule suspension. Conclusions: Stable nanocapsules capable of controlling the capsaicinoids release were obtained. Both the oily core and the polymeric shell are responsible for the controlled release. The semi-solid formulation containing nanocapsules obtained with the polymer chitosan maintained the controlled release and presented suitable properties for cutaneous applications.

Orientadores: Julian Martínez, Camila Alves de Rezende / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T08:06:10Z (GMT). No. of bitstreams: 1 Santos_Philipedos_M.pdf: 4512322 bytes, checksum: 66583cf01fbd52620fb21be1cc00b5fc (MD5) Previous issue date: 2013 / Resumo: A tecnologia de extração com fluido supercrítico surgiu como uma alternativa frente aos métodos tradicionais de extração e fracionamento de compostos ativos. Geralmente, uma unidade de extração com fluido supercrítico tem sua capacidade extrativa alterada com a utilização de técnicas em conjunto, como por exemplo, o emprego de diferentes cossolventes e a utilização de ondas ultrassônicas. A técnica de ultrassom baseia-se na formação de ondas ultrassônicas de alta frequência, capazes de provocar cavitação e causar ruptura na parede celular da matriz vegetal. Isto favorece a penetração do solvente e a transferência de massa e, consequentemente, o aumento do rendimento de extrato. O objetivo do presente trabalho foi obter extratos de pimenta malagueta através da utilização do processo de extração com fluido supercrítico (SFE) assistido por ultrassom. A matéria-prima utilizada foi a pimenta malagueta desidratada (5% em b.u.) e triturada. Para estudar a influencia das ondas ultrassônicas na taxa de extração, foram realizadas extrações com e sem ultrassom, nas potências e tempos de radiação variando de 150 a 360 W e 60 a 280 minutos, respectivamente. As condições de temperatura e pressão do dióxido de carbono supercrítico foram 40 ± 3 °C e 15 ± 0,5 MPa, respectivamente. A vazão mássica de CO2 foi fixada em 0,5 ± 0,1 kg/h. As cinéticas de extração foram realizadas na melhor condição de ultrassom em diferentes diâmetros de partícula. Os resultados obtidos indicaram um aumento de 77% no rendimento de extrato quando aplicadas ondas ultrassônicas. Não foi verificada influência significativa da aplicação de ondas ultrassônicas sobre o perfil de capsaicinoides e fenólicos totais. A melhor condição de extração assistida por ultrassom foi a 360 W de potência durante 60 minutos. Verificou-se uma influência do ultrassom sobre a taxa de extração, bem como nos coeficientes de transferência de massa na fase fluída e na fase sólida. O modelo de Sovová (1994) mostrou-se adequado para predizer as cinéticas de extração, porém os valores de rendimento global ( ), obtidos em experimentos assistidos por ultrassom, devem ser utilizados. As imagens obtidas por microscopia eletrônica de varredura com fonte de emissão de campo (FESEM) demonstraram uma perturbação na matriz vegetal quando se aplicou ondas ultrassônicas / Abstract: The supercritical fluid extraction technology came as an alternative to traditional methods of extraction and fractionation of active compounds. The capacity of a supercritical fluid extraction unit is changed with the application of combined techniques, for example, using different co-solvents and ultrasonic waves. The ultrasonic technology is based on high frequency ultrasonic waves formation capable of causing cavitations and disrupting the cell walls of vegetable matrix, favoring the penetration of solvent and enhancing mass transfer, thus increasing the extraction yield. The objective of this study was to obtain extracts of malagueta pepper (Capsicum frutescens L.) using supercritical fluid extraction (SFE) assisted by ultrasound. The raw material used was dried (5% w. b.) and triturated malagueta pepper. To study the influence of ultrasonic waves in the extraction rate, SFE experiments were performed with and without ultrasound, with powers and irradiations times from 150 to 360 W, and from 60 to 280 minutes, respectively. The conditions of temperature and pressure of supercritical carbon dioxide were 40 ± 3°C and 15 ± 0.5 MPa, respectively. The CO2 mass flow rate was fixed at 0.5 ± 0.1 kg/h. The extraction kinetics was performed in the best conditions of ultrasonic power ultrasonic in different particle diameters. The results indicated that the yield of oleoresin increased 77% when ultrasound waves were applied. The influence of the application of ultrasonic waves was not significant in the capsaicinoids and phenolics profiles. The best ultrasound-assisted extraction conditions were at 360 W (power) for 60 minutes (irradiation). The effect of ultrasound was verified on extraction rate and the solid and fluid phase mass transfer coefficient. The Sovová (1994) model was adequate to predict the extraction kinetics, but overall extraction yield values obtained in experiments assisted by ultrasound must be used. The images obtained by field emission scanning electron microscopy (FESEM) showed a disturbance in the vegetable matrix when ultrasound waves were applied / Mestrado / Engenharia de Alimentos / Mestre em Engenharia de Alimentos

This diploma thesis is focused on the influence of the three most represented capsaicinoids – capsaicin, dihydrocapsaicin and nordihydrocapsaicin – on the sensory properties of five varieties of chili peppers. Aji Lemon Drop, Serrano, Jamaican Yellow, Habanero Chocolate and Bhut Jolokia were selected for this thesis. Determination of capsaicinoids content was performed using HPLC-DAD. The content of capsaicin in dried fruits ranged from 359 to 320 gg-1, the content of dihydrocapsaicin in the range of 130–7 767 gg-1 and the content of nordihydrocapsaicin in the range of 7–456 gg-1. The pungency of the samples was 8 928–601 338 SHU. The sensory evaluation of selected varieties of chili peppers was attended by 20 evaluators representing ordinary consumers. The subject of the evaluation was mainly the intensity of burning and sharpness, its pleasantness and identification of the place of manifestation of thermal sensations. Furthermore, the onset of burning and sharpness from the insertion of peppers into the mouth, the duration of thermal sensations, the intensity of sweetness and juiciness, the presence of other flavors and the texture of the fruit. Statistically significant differences were observed between the evaluation results. The low intensity of burning and sharpness of the Jamaican Yellow variety was assessed as neutral to rather pleasant, while the high intensity of thermal sensations of the Habanero Chocolate and Bhut Jolokia varieties was perceived as unpleasant. The Aji Lemon Drop and Serrano varieties were judged very similar from a sensory point of view. By combining the results of capsaicinoids content measurement and sensory analysis, significant correlation relationships were found between the evaluated parameters. With a higher content of capsaicinoids in peppers, their pungency (expressed in SHU) increased, at the same time a higher intensity of thermal sensations and their longer duration were found during consumption. On the contrary, with a high intensity of burning and sharpness, the pleasantness of these perceptions decreased. The onset of these feelings did not affect the observed properties. The higher the intensity of juiciness, the higher the sweetness of the fruit; however, these properties were not significant in terms of the content of combustibles or the intensity of thermal sensations. The effect of individual capsaicinoids on the site of thermal sensation has not been statistically proven.

Kapsaicinoider är ämnen som finns i chilifrukterna och har på senaste tiden fått intresse som läkemedel på grund av sina analgetiska, anti-inflammatoriska och anti-cancer egenskaper. Ett nytt tillvägagångssätt att producera kapsaicinoider kan vara syntesen i rekombinant Saccharomyces cerevisiae med hjälp av metabolisk engineering och rekombinant DNA-tekniker. Gener från Capsicum chinensis, som kodar för enzymerna capsaicinoid-syntas (CS) och acyl-CoA-syntas (ACS), integrerades i S. cerevisiae i tidigare projekt. Den kända laboratoriesträngen CEN.PK modifierades med plasmidtransformation och för vildtyp-stammen ERF 5273 användes den nya CRISPR/Cas9-tekniken. Syftet med detta projekt är att ytterligare karakterisera och jämföra dessa tidigare konstruerade stammar angående deras förmåga att producera nonivamid eller andra jästspecifika kapsaicinoider. Vidare undersöks huruvida kapsaicinoider utsöndras i odlings-medium eller om de ackumuleras intracellulärt. Stammarna odlades i en bioreaktor i lite laboratorieskala. Som odlingsmedium används ett definierat medium med eller utan tillsatser. Odlings-medium kompletterades med vanillyl-amin och nonanoic acid som precursor. För att identifiera de kapsaicinoid-producerande stammarna extraherades supernatanten och cellpelleten och analyserades kromatografisk med HPLC. Resultaten från denna studie visade att jäststammarna, som innehöll båda generna (ACS + CS), sannolikt producerade nonivamid om de odlades i kompletterat medium. Vidare observerades bildning av nonivamid som ackumulerades i själva cellen. Möjligtvis producerades också jästspecifika kapsaicinoider, men topphöjden är nästan inte mätbar. Därför måste dessa resultat bekräftas ytterligare. Framtida arbeten behövs för att säkerställa och förbättra produktionen av kapsaicinoider. Keywords: acyl-CoA syntas, kapsaicinoider, kapsaicinoid syntas, metabolisk engineering, Saccharomyces cerevisiae, nonivamide / Capsaicinoids are compounds found in chili plants and have recently gained interest as pharmaceuticals due to their analgesic, anti-inflammatory and anti-cancer properties. A novel approach producing capsaicinoids could be synthesis in recombinant Saccharomyces cerevisiae with help of metabolic engineering. Genes from Capsicum chinensis, encoding the enzymes capsaicinoid synthase (CS) and acyl-CoA synthase (ACS), were previously inserted into S. cerevisiae. The known laboratory stain CEN.PK was modified with plasmid transformation and the novel CRISPR/Cas9 technology was used for the wild type strain ERF 5273. The aim of this project is to further characterize and compare these previously constructed strains concerning their ability to produce nonivamide or yeast specific capsaicinoids. Furthermore, it is examined whether capsaicinoids are excreted into the broth or accumulated intracellularly. Four different strains were cultivated in bench-scale bioreactors using medium supplemented with or without different precursors (vanillylamine and nonanoic acid). Culture broth supernatants and cell pellets were extracted and analyzed by HPLC in order to identify the capsaicinoid-producing strains. The results from this study revealed that the yeast strains harbouring both genes (ACS+CS) produced most likely nonivamide if they were cultivated in media supplemented with both precursors. Nonivamide formation was equally observed in broth supernatant and cell pellet. Additionally it was shown that yeast specific capsaicinoid production occured, althoug the peak height was close to the limit of detection and these results have to be confirmed further. Future work needs to be done in order to ensure and improve capsaicinoid production. Keywords: acyl-CoA synthase, capsaicinoids, capsaicinoid synthase, metabolic engineering, Saccharomyces cerevisiae, nonivamide.

Au Mexique, les fromages de chèvre ne sont pas très consommés en dehors des zones de production. Dans cette étude a été évalué : si l’ajout de piment (Capsicum annuum var. jalapeño) pouvait rendre les fromages de chèvre plus populaires auprès des consommateurs ; l’effet antibactérien d’un extrait aqueux de piment sur des bactéries d’intérêt alimentaire et la faisabilité économique d’une entreprise intégrée production-fabrication de fromage pimenté. La technologie de fabrication a été mise au point. Onze fromages avec quatre concentrations de capsaicinoïdes (0, 5, 10 et 15 ppm) ont été évalués par deux jurys non experts : Français et Mexicains. Les Français ont bien apprécié les fromages sans piment et peu pimentés avec jalapeño rouge et capsaicinoïdes et les Mexicains les peu et moyennement pimentés, quelle que soit la provenance du caractère brûlant. L’appréciation des fromages diminue avec l’augmentation de la couleur. Les souches de yaourt (S. thermophilus et Lb. delbrueckii) se sont révélées plus sensibles à l’extrait aqueux de piment et les bactéries Gram négatives étudiées n’ont pas montré aucun effet. Une entreprise intégrée est financièrement attractive et peu sensible à une variation de 15 % des coûts de l’alimentation du troupeau, de la main d’œuvre, de la somme des deux et de 8 % sur le taux de rentabilité ; elle est sensible mais faisable à une variation (15 %) du prix de vente du produit (fromage) et très sensible à la diminution en 50 % du rendement ; elle n’est pas économiquement intéressant si produit seulement du lait / In Mexico, the goat’s cheeses are not very much consumed outside production zones. The objective of this study was to obtain a cheese with chilli pepper (Capsicum annuum var. jalapeño). The technology, sensorial parameters for acceptance, hygienic and economical aspects were analyzed. A simple manufacturing process of goat cheeses with chilli pepper was developed. Eleven cheeses with fourth concentrations of capsaicinoids (0, 5, 10 and 15 ppm) were evaluated by two untrained groups: Mexicans and French. Cheeses without chilli pepper and lightly spiced (5 ppm) with red jalapeño and capsaicinoids were well appreciated by French subjects. These cheeses as well as fairly spiced ones (10 ppm), whatever the source of spiciness, were appreciated by Mexicans. The appreciation of cheeses decrease with the increase in red colour intensity. Yoghourt bacteria (S. thermophilus and Lb. delbrueckii) appeared more sensitive to the aqueous red pepper jalapeño extract and no effect could be shown on the studied negative Gram bacteria. The economic survey showed that a goat’s exploitation production-spiced cheese manufacture is profitable. The project is not very sensitive to a variation of 15% in cost of food, labour, sum of both and to 8% of profit rate. It is sensitive but feasible with a variation (15%) of the selling price of the cheese and very sensitive to a decrease of 50% in output and it is not economically interesting if the company produces only milk

Este trabalho visa ao entendimento da interação de capsaicinóides com membranas celulares utilizando sistemas modelo. Dentre os alcalóides derivados de plantas do gênero Capsicum, a capsaicina e a dihidrocapsaicina respondem por 90% dos capsaicinóides, que são usados como analgésicos e antiinflamatórios, devido a sua interação específica com receptores. O mecanismo neurofarmacológico já foi bastante estudado, mas o modo de ação não neural ainda não foi elucidado. Usamos extratos brutos (EBs) de pimenta malagueta e de dedo-de-moça, que têm atividade superficial, e afetaram monocamadas de Langmuir de fosfatidil colina de dipalmitoíla (DPPC) e fosfatidil glicerol de dipalmitoíla (DPPG). Tais efeitos não tiveram dependência expressiva com a carga, pois o EB de dedo-de-moça interagiu mais fortemente com o DPPC do que com o DPPG, ao passo que o contrário se verificou para o EB de malagueta. Também não houve diferença significativa entre os EBs das duas pimentas. Nas monocamadas de Langmuir representativas para a bactéria S. aureus, ambos os EBs tiveram efeito, tanto nas isotermas de pressão quanto nos resultados de espectroscopia de absorção e reflexão no infravermelho com modulação de polarização (PM-IRRAS), sem distinção significativa entre malagueta e dedo-de-moça. No entanto, as medidas de vazamento com lipossomos mostraram maior interação com o EB de dedo-de-moça, o que é consistente com a atividade bactericida para S. aureus. De fato, a concentração inibitória mínima (MIC) foi 0,13 mg mL-1 para o EB da pimenta dedo-de moça e 4,0 mg mL-1 para o EB de malagueta. Para a E. coli, os EBs interagiram com as monocamadas de Langmuir sem diferenças dignas de nota para as duas pimentas, e nas medidas de vazamento o efeito maior foi para a dedo-de-moça. Não houve efeito bactericida para nenhum dos extratos. Isso se explica porque bactérias gram-negativas, como a E. coli, têm uma camada externa protetora de lipossacarídeos (LPS). Das medidas de monocamadas de Langmuir representativas da camada de LPS, observou-se pouca incorporação dos EBs. Conclui-se, assim, que os EBs não conseguem causar rompimento da camada de LPS. Do conjunto dos resultados, infere-se que o mecanismo de ação para a S. aureus envolve solubilização parcial da membrana, e não há relação entre pungência e atividade bactericida, pois a pimenta dedo-de-moça, que é menos pungente, teve maior efeito do que a malagueta. Depreende-se, também, que a ação de extratos de pimenta deve depender da interação com receptores na membrana, o que explica porque o uso de tais extratos tem sido principalmente em aplicações tópicas. / This study is aimed at understanding the interaction of capsaicinoids with cell membranes using model systems. Among the alkaloids derived from plants of the genus Capsicum, capsaicin and dihydrocapsaicin account for 90% of capsaicinoids, which are used as analgesic and anti-inflammatory due to their interaction with specific receptors. The neuropharmacological mechanism has been well studied, but the non-neural mode of action has not been elucidated. Here, we use crude extracts (EBs) of malagueta and dedo-de-moça chilli peppers, which are surface active, and affected Langmuir monolayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG). Such effects did not depend on the charge, since EB from dedo-de-moça interacted more strongly with DPPC than with DPPG, while the opposite applied for malagueta. In addition, there was no significant difference between the two EBs. For Langmuir monolayers representing the bacteria S. aureus, both EBs affected the surface pressure isotherms and the polarizationmodulated infrared reflection-absorption spectroscopy (PM-IRRAS) data, without significant distinction between dedo-de-moça and malagueta. However, in the leakage measurements with liposomes the EB from dedo-de-moça was more efficient in rupturing the liposome, which is consistent with the bactericidal activity for S. aureus. In fact, the minimum inhibitory concentration (MIC) was 0.13 mg mL-1 for dedo-de-moça and 4.0 mg mL-1 for malagueta. For the Langmuir monolayers mimicking the E. coli membrane, the EBs interacted much in the same way, while the EB from dedo-de-moça caused larger leakage in liposomes. There was no bactericidal effect of the EBs. This is explained by the fact that gram-negative bacteria, such as E. coli, have a protective outer layer of liposaccharides (LPS). In monolayers representing LPS, there was little incorporation of EBs, from which one infers that the EBs cannot cause disruption of the LPS layer. Taking all these results together, it appears that the mechanism of action for S. aureus involves partial solubilization of the membrane. Furthermore, there is no relationship between pungency and bactericidal activity because dedo-de-moça, which is less pungent, had greater effect than malagueta. It seems also that the action of pepper extracts must depend on the interaction with membrane receptors, which explains why the use of such extracts has been essentially in topical applications.