A madeira quando usada em contato direto com o solo, é atacada por agentes biológicos, principalmente fungos apodrecedores e cupins subterrâneos. Uma maneira de ampliar as possibilidades de utilização das espécies de baixa durabilidade natural, como as do gênero Pinus, é através do tratamento químico preservante. No entanto, faltam informações disponíveis que indiquem a durabilidade destas madeiras em serviço. A fim de se determinar a durabilidade da madeira do gênero Pinus tratada com preservantes, a Escola Superior de Agricultura "Luiz de Queiroz" e o Instituto Florestal do Estado de São Paulo instalaram, em 1980, campos de apodrecimento segundo método de ensaio sugerido pelo IUFRO (International Union of Forestry Research Organizations). As espécies utilizadas no ensaio foram o Pinus elliottii Engl. var. elliottii, o Pinus caribaea Mor. var. hondurensis, B & G., o Pinus oocarpa Shied e o Pinus kesiya Royle ex Gordon; tratadas com os preservantes CCA tipos A, B e C, CCB e pentaclorofenol, em cinco níveis de retenção. Em continuidade ao projeto, foi inspecionado o campo instalado na Estação Experimental de Mogi-Guaçu, visando avaliar o estado de sanidade do material. A análise dos resultados obtidos até o momento, 21 anos de exposição demonstrou que não houve diferença no comportamento entre espécies. Os índices de comportamento mais altos (maior expectativa de durabilidade) correspondem aos tratamentos com CCA tipos A, B e C em retenções acima de 7,5 kg/m3. Mesmo para os tratamentos que apresentaram os menores índices de comportamento (CCA tipo C com retenções de 5,0 kg/m 3 e CCB com 5,9 kg/m 3 ), é prevista uma durabilidade de 30 anos em serviço. / When used in ground contact, wood is deteriorated by biological agents, as root fungi and subterranean termites. A way to increase the use of wood species of low natural durability, as the Pinus lumber, is the preservative treatment. However, there is a lack of information indicating the durability of treated wood. In order to determine the durability of preservative treated Pinus lumber, the Escola Superior de Agricultura "Luiz de Queiroz" and the Instituto Florestal do Estado de São Paulo installed, in 1980, fields tests according to the IUFRO (International Union of Forestry Research Organizations). The species under test were Pinus elliottii Engl. var. elliottii, Pinus caribaea Mor. var. hondurensis, B & G., Pinus oocarpa Shied and Pinus kesiya Royle ex Gordon; treated with the preservatives CCA types A, B and C, CCB and pentachlorophenol, in five retention levels. In continuity to the project, the material installed in the test site of Experimental Station of Mogi-Guaçu was inspected, to evaluate the degree of attack. The analysis of results obtained till now, after 21 years of exposure, demonstrated that there are no differences in durability related with species. The highest performance index (longer durability expectation) correspond to treatments with CCA types A, B and C in retentions above 7,5 kg/m3. Even for the treatments that presented lowest performance index (CCA type C with retention of 5,0 kg/m 3 and CCB with 5,9 kg/m 3 ), the durability expectation is 30 years in service.

Avaliou-se a atividade conservante do Borato de Sódio para amostras de tecidos com lesão tuberculosa. Inicialmente, foi feito um teste de inoculação experimental em hamsters. A partir dos resultados observados neste estudo, foram selecionadas as variáveis para a segunda etapa do experimento, com lesões de abatedouro de bovinos. Noventa hamsters foram inoculados experimentalmente com Mycobacterium bovis e foram eutanasiados 40 dias após a inoculação, quando foi feita a colheita dos baços. Esses órgãos foram, individualmente, armazenados em solução saturada de Borato de Sódio, em quatro períodos de armazenamento distintos (30, 60, 90 e 120 dias após a eutanásia), em duas temperaturas (27ºC e 37ºC), exceto o grupo controle processado no dia da eutanasia. Na segunda etapa do experimento, 69 lesões de órgãos de bovinos foram colhidas de um abatedouro e armazenadas em solução de borato de sódio em três períodos de armazenamento distintos (30, 60 e 90 dias após a colheita), na temperatura de 27ºC, exceto o grupo controle, processado no dia de entrada no laboratório. As análises dos dois experimentos foram feitas separadamente e mostraram-se concordantes. Em relação aos períodos de armazenamento dos órgãos em Borato de Sódio foram consideradas duas variáveis, a proporção de isolados de micobactérias a partir dos órgãos e o número de U.F.C. de micobactérias desses mesmos órgãos. Com base nos resultados observados em ambos os testes, concluiu-se que o armazenamento de tecidos com lesões tuberculosas em solução de Borato de Sódio pode ser feito por até 30 dias em altas temperaturas, 27ºC a 37ºC. Além disso, observou-se que conforme a temperatura ambiente aumenta, diminui a sensibilidade do isolamento de M. bovis a partir de amostras armazenadas em solução saturada de Borato de Sódio. / The preservative activity of Sodium Tetraborate was evaluated for tissues with tuberculous lesion. Initially, an experimental infection essay was performed in hamsters. Based on the results observed in this essay, the variables for the second experiment, involving samples from a slaughterhouse, were selected. The 90 animals were euthanized 40 days after inoculation when the spleens were colected. These organs were individually stored in saturated Sodium Tetraborate solution, in four distincts storage periods (30, 60, 90 and 120 days after the euthanasia), in two temperatures (27º and 37ºC), except for the control group processed on the euthanasia day. On the second part of the experiment, sixty nine samples of bovine organs with tuberculous lesions were collected from a slaughterhouse. The samples were stored in saturated Sodium Tetraborate solution, in three distincts storage periods (30, 60 and 90 days after collection), at the temperature of 27ºC, except for the control group processed on the day of entry in the laboratory. The analysis of both experiments were done separately and were concordant. In relation to the storage periods of the organs in Sodium Tetraborate solution, two variables were considered, the proportion of mycobacterial isolates from the organs and the number of mycobacterial C.F.U from the same organs. Based on the results observed on both essays, it was concluded that tissues with tuberculous lesions can be stored in Sodium Tetraborate solution up to 30 days in high temperatures, 27ºC a 37ºC. Furthermore, it was observed that as the environmental temperature increases the M. bovis isolation sensibility from the samples stored in Sodium Tetraborate decreases.

On the basis of the well-known preservative properties of Sphagnum moss, a potential opportunity to use moss polysaccharides (Sphagnan) in art conservation was tested. Polysaccharides were extracted from the moss (S. palustre spp.) in the amount of 4.1% of the Sphagnum plant dry weight. All lignocelluloses were removed from this extract as a result of the treatment of the moss cellulose with sodium chlorite. The extracted polysaccharide possessed a strong acidic reaction (pH 2.8) and was soluble in water and organic solvents. The extract was tested on laboratory bacterial cultures by the disk-diffusion method. The antibacterial effect was demonstrated for E. coli and P. aeruginosa (both gram-negative) while Staphylococcus aurelus (gram-positive) was shown to be insensitive to Sphagnum polysaccharides. The antifungal effect of Sphagnum extract was tested by the disk-diffusion method on the spores of seventeen fungal species. These fungi were isolated from ethnographic museum objects and from archaeological objects excavated in the Arctic. Twelve of these isolates appeared susceptible to the extract. The inhibiting effect of the extract was also tested by the modified broth-dilution method on the most typical isolate (Aspergillus spp.). In this experiment, in one ml of the nutritious broth, 40µl of 3% solution of polysaccharides in water killed 10,000 fungal spores in 6 hours. The inhibiting effect was not connected to the acidity or osmotic effect of Sphagnum polysaccharides. As an example of the application of Sphagnum polysaccharides in art conservation, they were added as preservative agents to conservation waxes. After three weeks of exposure of microcrystalline wax to test fungi (Aspergillus spp.), 44% of wax was consumed. When, however, ~ 0.1% (w/w) of Sphagnum extract was mixed with wax, the weight loss of wax was only 4% in the same time interval. On the basis of this study it was concluded that Sphagnum moss and Sphagnum products can be recommended for use in art conservation as antifungal agents. / Thesis (Master, Art Conservation) -- Queen's University, 2010-01-14 15:55:23.779

L’industrie parapharmaceutique de même que l’industrie cosmétique recherche des alternatives naturelles aux conservateurs synthétiques. Cet intérêt est lié à l’exigence du consommateur et à la volonté de développer des produits innovants éco-conçus. Compte tenu de leurs activités antioxydantes et antimicrobiennes, les huiles essentielles constituent des options intéressantes dont les coûts et impacts organoleptiques doivent cependant être considérés. Dans le cadre de cette thèse, les activités antioxydante et antimicrobienne de combinaisons huile essentielle-hydrolat et huile essentielle-huile essentielle de Lippia alba, Rosmarinus officinalis et Thymus vulgaris ont été évaluées afin d’objectiver leur potentiel de conservateur pour des produits phytothérapeutiques. Initialement, l’impact d’une méthode de distillation utilisant le macérât aqueux de la matière végétale comme source de vapeur a été évalué sur les propriétés physico-chimiques des hydrolats et huiles essentielles de Thymus vulgaris et Rosmarinus officinalis cultivées dans le sud de Bogotá en Colombie. Puis, les distillats et hydrolats ayant présenté une activité biologique significative ainsi que l’huile essentielle de Lippia alba ont été combinés deux à deux. Les activités antiradicalaire et microbicide ont été évaluées respectivement par ABTS et par les méthodes de microdilution et de diffusion en agar. Des résultats biologiques significatifs ont été obtenus pour les combinaisons huile essentielle-huile essentielle tout spécialement dans le cas de la combinaison des huiles essentielles de T. vulgaris et de L. alba avec une diminution de la CI50 de 2,9 (huile essentielle de Thym seule) à 1,4 µL/L ainsi que dans le cas spécifique de la combinaison hydrolat-huile essentielle de Thymus vulgaris avec une CI50 de 2,1 µL/L. De même, cette dernière combinaison a présenté un effet de synergie en diminuant la Concentration Minimale Bactéricide de l’huile essentielle de Thym de 0,4 à 0,1 µL/mL. Les différents résultats sont discutés ainsi que leurs impacts d’un point de vue industriel et thérapeutique. / The parapharmaceutical industry as well as the cosmetics industry is looking for natural alternatives to synthetic preservatives. This interest is linked to the requirement of the consumer and the will to develop eco-designed innovative products. Antioxidant and antimicrobial activities of essential oils are interesting options, however costs and organoleptic impacts should be considered. As part of this thesis, the antioxidant and antimicrobial activities of essential oil-hydrosol and essential oil-essential oil of Lippia alba, Rosmarinus officinalis and Thymus vulgaris were evaluated to objectify their preservative potential for herbal products. Initially, the impact of a distillation method using the aqueous macerate of a part of vegetal material as vapor source was evaluated on the physicochemical properties of hydrosols and essential oils of Thymus vulgaris and Rosmarinus officinalis grown in southern Bogotá Colombia. Then, distillates and hydrosols with significant biological activity as well as the essential oil of Lippia alba were combined by pairs. The antiradical and microbicidal activities were evaluated respectively by ABTS and by the methods of microdilution and agar diffusion. Significant results were obtained with the essential oil-essential oil combinations especially in the case of the combination of essential oils of T. vulgaris and L. alba with a decrease of the IC50 of 2,9 (T. vulgaris essential oil alone) to 1,4 µL/L as well as in the specific case of the T. vulgaris hydrosol and essential oil combination with an IC50 of 2,1 µL/L. Similarly, the latter combination presented a synergy effect by decreasing the Minimal Bactericidal Concentration of the essential oil of Thyme from 0,4 to 0,1µL/mL. The different results are discussed as well as their impacts in both industrial and therapeutic domains.

Avaliou-se a atividade conservante do Borato de Sódio para amostras de tecidos com lesão tuberculosa. Inicialmente, foi feito um teste de inoculação experimental em hamsters. A partir dos resultados observados neste estudo, foram selecionadas as variáveis para a segunda etapa do experimento, com lesões de abatedouro de bovinos. Noventa hamsters foram inoculados experimentalmente com Mycobacterium bovis e foram eutanasiados 40 dias após a inoculação, quando foi feita a colheita dos baços. Esses órgãos foram, individualmente, armazenados em solução saturada de Borato de Sódio, em quatro períodos de armazenamento distintos (30, 60, 90 e 120 dias após a eutanásia), em duas temperaturas (27ºC e 37ºC), exceto o grupo controle processado no dia da eutanasia. Na segunda etapa do experimento, 69 lesões de órgãos de bovinos foram colhidas de um abatedouro e armazenadas em solução de borato de sódio em três períodos de armazenamento distintos (30, 60 e 90 dias após a colheita), na temperatura de 27ºC, exceto o grupo controle, processado no dia de entrada no laboratório. As análises dos dois experimentos foram feitas separadamente e mostraram-se concordantes. Em relação aos períodos de armazenamento dos órgãos em Borato de Sódio foram consideradas duas variáveis, a proporção de isolados de micobactérias a partir dos órgãos e o número de U.F.C. de micobactérias desses mesmos órgãos. Com base nos resultados observados em ambos os testes, concluiu-se que o armazenamento de tecidos com lesões tuberculosas em solução de Borato de Sódio pode ser feito por até 30 dias em altas temperaturas, 27ºC a 37ºC. Além disso, observou-se que conforme a temperatura ambiente aumenta, diminui a sensibilidade do isolamento de M. bovis a partir de amostras armazenadas em solução saturada de Borato de Sódio. / The preservative activity of Sodium Tetraborate was evaluated for tissues with tuberculous lesion. Initially, an experimental infection essay was performed in hamsters. Based on the results observed in this essay, the variables for the second experiment, involving samples from a slaughterhouse, were selected. The 90 animals were euthanized 40 days after inoculation when the spleens were colected. These organs were individually stored in saturated Sodium Tetraborate solution, in four distincts storage periods (30, 60, 90 and 120 days after the euthanasia), in two temperatures (27º and 37ºC), except for the control group processed on the euthanasia day. On the second part of the experiment, sixty nine samples of bovine organs with tuberculous lesions were collected from a slaughterhouse. The samples were stored in saturated Sodium Tetraborate solution, in three distincts storage periods (30, 60 and 90 days after collection), at the temperature of 27ºC, except for the control group processed on the day of entry in the laboratory. The analysis of both experiments were done separately and were concordant. In relation to the storage periods of the organs in Sodium Tetraborate solution, two variables were considered, the proportion of mycobacterial isolates from the organs and the number of mycobacterial C.F.U from the same organs. Based on the results observed on both essays, it was concluded that tissues with tuberculous lesions can be stored in Sodium Tetraborate solution up to 30 days in high temperatures, 27ºC a 37ºC. Furthermore, it was observed that as the environmental temperature increases the M. bovis isolation sensibility from the samples stored in Sodium Tetraborate decreases.

A madeira quando usada em contato direto com o solo, é atacada por agentes biológicos, principalmente fungos apodrecedores e cupins subterrâneos. Uma maneira de ampliar as possibilidades de utilização das espécies de baixa durabilidade natural, como as do gênero Pinus, é através do tratamento químico preservante. No entanto, faltam informações disponíveis que indiquem a durabilidade destas madeiras em serviço. A fim de se determinar a durabilidade da madeira do gênero Pinus tratada com preservantes, a Escola Superior de Agricultura "Luiz de Queiroz" e o Instituto Florestal do Estado de São Paulo instalaram, em 1980, campos de apodrecimento segundo método de ensaio sugerido pelo IUFRO (International Union of Forestry Research Organizations). As espécies utilizadas no ensaio foram o Pinus elliottii Engl. var. elliottii, o Pinus caribaea Mor. var. hondurensis, B & G., o Pinus oocarpa Shied e o Pinus kesiya Royle ex Gordon; tratadas com os preservantes CCA tipos A, B e C, CCB e pentaclorofenol, em cinco níveis de retenção. Em continuidade ao projeto, foi inspecionado o campo instalado na Estação Experimental de Mogi-Guaçu, visando avaliar o estado de sanidade do material. A análise dos resultados obtidos até o momento, 21 anos de exposição demonstrou que não houve diferença no comportamento entre espécies. Os índices de comportamento mais altos (maior expectativa de durabilidade) correspondem aos tratamentos com CCA tipos A, B e C em retenções acima de 7,5 kg/m3. Mesmo para os tratamentos que apresentaram os menores índices de comportamento (CCA tipo C com retenções de 5,0 kg/m 3 e CCB com 5,9 kg/m 3 ), é prevista uma durabilidade de 30 anos em serviço. / When used in ground contact, wood is deteriorated by biological agents, as root fungi and subterranean termites. A way to increase the use of wood species of low natural durability, as the Pinus lumber, is the preservative treatment. However, there is a lack of information indicating the durability of treated wood. In order to determine the durability of preservative treated Pinus lumber, the Escola Superior de Agricultura "Luiz de Queiroz" and the Instituto Florestal do Estado de São Paulo installed, in 1980, fields tests according to the IUFRO (International Union of Forestry Research Organizations). The species under test were Pinus elliottii Engl. var. elliottii, Pinus caribaea Mor. var. hondurensis, B & G., Pinus oocarpa Shied and Pinus kesiya Royle ex Gordon; treated with the preservatives CCA types A, B and C, CCB and pentachlorophenol, in five retention levels. In continuity to the project, the material installed in the test site of Experimental Station of Mogi-Guaçu was inspected, to evaluate the degree of attack. The analysis of results obtained till now, after 21 years of exposure, demonstrated that there are no differences in durability related with species. The highest performance index (longer durability expectation) correspond to treatments with CCA types A, B and C in retentions above 7,5 kg/m3. Even for the treatments that presented lowest performance index (CCA type C with retention of 5,0 kg/m 3 and CCB with 5,9 kg/m 3 ), the durability expectation is 30 years in service.

Les eaux florales sont des matières premières aromatiques issues de la distillation, contenant généralement moins de 1 g/L de composés volatils leur conférant leurs propriétés organoleptiques. Elles sont utilisées principalement en industrie agroalimentaire et cosmétique. Elles sont sujettes à des problèmes d’instabilité microbienne incompatibles avec leurs applications. Ces microorganismes, leurs dynamiques, ainsi que les nutriments disponibles nécessaires à leur croissance restent mal connus. Les eaux florales sont actuellement stabilisées par ajout de conservateurs dont certains sont controversés et visent à être retirés du marché. De plus, leur efficacité dans les eaux florales n’a pas été évaluée.L’objectif de cette thèse est d’apporter une meilleure connaissance de la composition des eaux florales et de ses contaminants afin de proposer une méthode de stabilisation adaptée.La composition en huile essentielle et le microbiote de 22 échantillons d’eau de fleur d’oranger (Citrus aurantium L. ssp. amara L.) et de rose (Rosa damascena Miller et Rosa centifolia L.), provenant de différents producteurs autour du bassin méditerranéen, ont été analysés afin de déterminer les facteurs responsables de leur altération. Bien que les composés volatils soient connus pour leurs propriétés antimicrobiennes, leurs concentrations dans les hydrolats ne sont pas suffisantes pour assurer la stabilité microbiologique. En plus des composés volatils, les hydrolats contiennent des composés non-volatils tels que des sucres, entraînés vers le distillat par effet de primage ou de moussage pendant la distillation, et pouvant être utilisés comme substrat de croissance par les microorganismes. La population microbienne peut atteindre 106 à 107 UFC/mL en quelques jours à température ambiante et jusqu’à 3 mois à 5°C. Des bactéries environnementales, oligotrophes, et acido-tolérantes, appartenant principalement aux genres Pseudomonas sp. et Burkholderia sp. ont été isolées et identifiées. Parmi ces bactéries, B. vietnamiensis et Novosphingobium capsulatum ont été capables de métaboliser des composés volatiles tels que le géraniol ou l’acétate de 2-phényléthyle pour produire la 6-méthyl-5-heptèn-2-one ou le 2-phényléthanol, et modifier ainsi les propriétés organoleptiques des hydrolats. Enfin, la capacité de croissance de bactéries pathogènes et d’altération dans les hydrolats a été évaluée, et différents conservateurs ont été testés sur les souches capables de se multiplier dans les hydrolats.Une distillation aseptique et un conditionnement stérile permettrait d’assurer la stabilité des hydrolats sans ajout de conservateurs. En l’absence de conditions aseptiques, l’ajout de conservateurs est nécessaire pour assurer la stabilité des hydrolats. / Hydrosols are hydrodistillation products mainly used as food flavoring agents or ingredient in cosmetics. They contain less than 1 g/L of dispersed essential oils giving the organoleptic properties. These are subjected to microbial proliferation that can prevent use due to non-compliance to professional microbiological standards. The microorganisms, their growth dynamics, and the available nutrients in hydrosols remain unknown. Hydrosols can contain few preservatives, but there is no data about their efficiency in hydrosols.The aim of this study was to have a better knowledge on hydrosols composition, their microbiota, and spoilage conditions, in order to propose an adapted stabilization method.The composition in volatile compounds and the microbiota of 22 hydrosol samples of Citrus aurantium L. ssp. amara L. (orange blossom), Rosa damascena Miller (rose D.), and Rosa centifolia L. (rose C.) flowers were analyzed to determine factors responsible for decay. Some non-volatile compounds were likely carried over during distillation by a priming and foaming effect, and could be used as nutrients by microorganisms. Concentrations of volatile compounds in hydrosols are not high enough to prevent microbial proliferation, and bacteria concentrations can reach up to 106 CFU/mL in both hydrosols. The isolated microbial population was composed of oligotrophic Gram-negative bacteria, arranged in four major genera: Pseudomonas sp., Burkholderia cepacia complex, and presumably two new genera belonging to Acetobacteraceae and Rhodospirillaceae. Among those bacteria, Burkholderia vietnamiensis and Novosphingobium capsulatum were able to metabolize volatile compounds, such as geraniol to produce 6-methyl-5-hepten-2-one or geranic acid, or 2-phenylethyl acetate to produce 2-phenylethanol. Finally, the growth potential of a range of bacteria isolated from hydrosols and of pathogenic micro-organisms was evaluated, then the anti-microbial activity in nutrient broth and/or in hydrosols of a range of chemical preservatives authorized for food and cosmetic applications was tested.Additional hurdles such as chemical preservatives or aseptic packaging will be necessary to insure microbial stability.

Natamycin is an antimycotic with very low water solubility and extremely high photosensitivity, which is used to extend the shelf life of shredded cheese products. The objectives of this research are: (a) to find a new delivery system for natamycin, which increases its aqueous solubility and (b) to increase the chemical stability of natamycin so that it has a prolonged antifungal effect on the surface of the shredded cheese. Molecular inclusion complexes of natamycin were formed with β-, hydroxypropyl β-, and γ- cyclodextrins (CDs) which allowed large increases in aqueous solubility without the use of organic co-solvents or surfactants. The water solubility of natamycin was increased 16-fold, 73- fold, and 152-fold with β-CD, γ-CD, and hydroxypropyl β-CD, respectively. The natamycin:CD inclusion complexes resulted in nearly equivalent in vitro antifungal activity as natamycin in its free state. Nuclear magnetic resonance (NMR) was utilized to prove the formation of true inclusion complexes. 1H NMR shift titrations of N-(3 -N-dimethylaminosuccimido) natamycin with β- and γ-CDs enabled determination of the stoichiometry of both complexes as 1:1. Aqueous solutions of natamycin (20 mg/L) were found by quantitative HPLC to be completely degraded after 24 hours of exposure to 1000 lux fluorescent lighting at 4 °C. After 14 days of storage in darkness at 4 °C, 92.2% of natamycin remained in active form. Aqueous solutions of natamycin:β-CD complex and natamycin:γ-CD complex were significantly more stable (p < 0.05) than natamycin in its free state when stored in darkness at 4 °C. Clear poly(ethylene terephthalate) packaging with an ultraviolet light absorber allowed 85.0% natamycin to remain after 14 days of storage under 1000 lux fluorescent lighting at 4 °C. Such dramatic increases in water solubility and light stability will enable natamycin to function as a more effective antimycotic in the food industry. / Master of Science

Natamycin is an antimycotic compound that is widely used in the cheese industry to increase the shelf life of cheeses, especially shredded cheeses, by inhibiting the growth of molds. Natamycin is applied to the surface of cheese as an aqueous suspension or as a powder. However, natamycin is not readily water soluble making it harder to distribute evenly over shredded cheese Natamycin is degraded by ultraviolet (UV) light at wavelengths of 350 nm and below. Typical packaging applications do not provide adequate UV protection causing natamycin to degrade. This work was undertaken to determine the efficacy of UV absorber film to prevent UV light degradation of natamycin on the surface of shredded cheese. Current accepted methods to determine concentration of natamycin were evaluated for appropriateness in natamycin degradation studIes. The use of cyclodextrins to increase water solubility was tested to see if a uniform distribution of natamycin over the shredded cheese could be done effectively. Furthermore, a known application of mold was performed to see how well natamycin and each of its applications could prevent visible mold growth from occurring. The International Dairy Federation recognizes two methods to quantify natamycin on shredded cheese: high performance liquid chromatography (HPLC) and spectrophotometry. Concentrations of natamycin in aqueous suspensions were determined using both methods. Results show that spectrophotometry is flawed when quantifying the amount of active natamycin because the method gives erroneously high results. The amount of active natamycin is not accurately quantified using spectrophotometric techniques because it cannot separate the active form from the inactive form of natamycin. Polymer packages containing a UV absorber (11.4% light transmission at 350 nm) allow significantly less UV-associated degradation of natamycin than those packages that lacked a UV protectant (90.0% light transmission at 350 nm) (p<0.05). Incorporating a UV absorber into a package helps protect natamycin and its various complexes from UV light degradation, which can increase the shelf life of shredded cheese. However, even with a UV absorber, natamycin is still able to degrade. Natamycin was complexed with different cyclodextrins to help better solubilize natamycin â β-cyclodextrin, hydroxy-propyl β-cyclodextrin and γ-cyclodextrin. Using cyclodextrins to apply natamycin more uniformly onto shredded cheese did not significantly increase the consistency of distribution (p<0.05). Variability was uniform throughout all treatments with the exception of HPBCD complex. After 27 days, all of the UV packages treated with each of the cyclodextrin treatments containing shredded cheese began to show visible mold growth. Those packages stored in total darkness remained mold free through the duration of the experiment ending on day 62. When untreated with natamycin and an initial concentration of 101-102 spores/gram of Penicillium roqueforti, shredded cheese remained free from visible mold growth for 24 days in total darkness at 4°C. Samples treated with one of the natamycin treatments were able to remain mold free for at least 9 more days, showing visible signs of mold growth at day 33. There was no statistical difference between the treatments of dry natamycin, aqueous suspension natamycin, β-cyclodextrin-natamycin complex, and γ-cyclodextrin-natamycin complex (p<0.05). However, there was a difference with the use of hydroxy-propyl β-cyclodextrin-natamycin complex. Hydroxy-propyl β-cyclodextrin-natamycin complex allowed the shredded cheese to last for 41 days, 17 days longer than the control sample. / Master of Science

Nisina é um peptídeo antimicrobiano natural produzido por Lactococcus lactis subsp. lactis ATCC 11454 durante a fase exponencial de crescimento. A bacteriocina é usada como conservante natural de alimentos, uma vez que mostra atividade antimicrobiana contra bactérias Gram-positivas e esporos. Tem potencial aplicação em inúmeros campos (farmacêutico, veterinário e cosméticos). O objetivo deste trabalho foi estudar a cinética de crescimento bacteriano e a produção de nisina em biorreator, utilizando leite desnatado diluído, como um meio de cultura a baixo custo. Também foram avaliados os consumos de açúcar e proteína, formação de ácido lático e adsorção de nisina nas células produtoras durante os processos de produção de nisina. Pré-cultivos com 107 UFC.mL-1 de Lactococcus lactis foram cultivados em biorreator de 2 L contendo 25% de leite desnatado diluído em água (1,5 L, pH 6,7). Os ensaios foram esenvolvidos a 30°C por 52 horas, variando a agitação e aeração: (i) 200 rpm (0,0, 0,5, 1,0 e 2,0 L.min-1) e (ii) 100 rpm (0,0 e 0,5 L.min-1). A atividade de nisina foi avaliada pelo método de difusão em ágar, utilizando Lactobacillus sakei ATCC 15521 como microrganismo sensível à ação de nisina. A melhor concentração de nisina (62,68 mg.L-1 ou 2511,89 AU.mL-1), foi obtida em 16 horas, 200 rpm e sem aeração (kLa = 5,29 x 10-3 h-1). A adsorção de nisina nas células produtoras foram baixas (6,8 - 15,1%), quando comparadas com a atividade do sobrenadante. Estes resultados mostraram que o meio de cultivo composto por leite desnatado diluído favoreceu o crescimento celular e produção associada ao crescimento da nisina. Foram realizados estudos preliminares de liofilização (bioconservação) e purificação por cromatografia da nisina produzida em biorreator. A liofilização apresentou perda da atividade de nisina (24,8%), enquanto a purificação por cromatografia de interação hidrofóbica com resina Butyl-Sepharose, recuperou 40% da atividade da biomolécula, mostrando que ambos os processos poderão ser aplicados à bacteriocina. / Nisin is a natural antimicrobial peptide produced by Lactococcus lactis subsp. lactis ATCC 11454 during its exponential growth phase. The bacteriocin is used as natural food preservative due to its antimicrobial activity against Gram-positive bacteria and outgrowth of spores. This property allows its application in numerous fields (pharmaceutical, veterinary and cosmetic). The aim of this work was to study the bacterial growth kinetics of L. lactis and respective nisin production in bioreactor, using diluted skimmed milk as an inexpensive medium. During the production, the consumption of sugar and protein, lactic acid formation and nisin adsorption on the producer strain cells were evaluated. Pre-cultivation with 107 UFC.mL-1 of L. lactis were expanded in a 2 L bioreactor containing 25% diluted skimmed milk in water (1.5 L, pH 6.7). The assays were performed at 30°C for 52 hours, varying agitation and airflow rate: (i) 200 rpm (0.0, 0.5, 1.0 and 2.0 L.min-1) and (ii) 100 rpm (0.0, 0.5 L.min-1). Nisin activity was evaluated through diffusion assays using Lactobacillus sakei ATCC 15521 as sensitive strain. The best nisin concentration (62.68 mg.L-1 or 2511.89 AU.mL-1), was achieved at 16 hours, 200 rpm and with no airflow rate (kLa = 5.29 x 10-3 h-1). The quantity of nisin adsorbed by the producer cells were low (6.8 -15.1%) when compared to the quantity released in the supernatant. These results showed that diluted skimmed milk supported cell growth and growth-associated nisin. Preliminary assays of lyophilization (biopreservation) and purification by chromatography of nisin produced in bioreactor were performed. Lyophilization presented a loss of nisin activity (24.8%) while purification by hydrophobic interaction chromatography with Butyl-Sepharose column recovered 40% of the activity, showing that both processes can be applied to the bacteriocin.