This study is concerned with certain factors affecting the content of lactobacillic acid in Lactobacillus plantarum. Three main areas of investigation are reported herein. The effects of both the oxygen tension and the pH of the culture medium on the accumulation of lactobacillic acid were determined. In addition, monolayer studies were conducted to determine the influence of cyclopropane fatty acid content on the molecular packing of membrane lipids.

Foram realizados dois experimentos para avaliação de aditivos microbianos na ensilagem de forragens tropicais. No primeiro experimento avaliou-se o efeito de doses crescentes de Lactobacillus buchneri, em diferentes tempos de armazenamento, sobre o valor nutritivo, perfil fermentativo e estabilidade aeróbia de silagens de milho. No segundo experimento avaliou-se o efeito de L. buchneri, de forma exclusiva, ou em combinação com Lactobacillus plantarum, em silagens de milho, cana-de-açúcar e capim elefante cv. Napier. O tratamento das silagens de milho com L. buchneri não afetou a contagem de fungos filamentosos, com decréscimo nas contagens com maiores tempos de armazenamento. Os teores de ácido lático decresceram com o aumento da dose de L. buchneri. Os maiores períodos de armazenamento resultaram em decréscimo na contagem de BAL, e maiores valores de pH. O tempo de armazenamento diminuiu as leveduras na silagem de milho. Maiores tempos de armazenamento propiciaram maiores tempos para atingir a temperatura máxima e a perda da estabilidade aeróbia. As silagens com doses superiores de L. buchneri apresentaram perda de estabilidade em momento posterior às silagens dos tratamentos controle e LB1x105. O maior tempo de armazenamento da silagem de milho resultou em maior valor nutritivo e perfil microbiológico favorável quanto à fermentação e estabilidade em aerobiose. Inoculação com L. buchneri (NCIMB 40788) reduz a sobrevivência de leveduras durante a fase de armazenamento e o crescimento durante a exposição ao oxigênio, melhorando a estabilidade aeróbia da silagem em ambiente tropical. Doses mais elevadas poderiam compensar parcialmente o menor tempo de armazenamento das silagens. No segundo experimento, o tratamento das silagens de milho com L. buchneri (LB), exclusivamente, ou associado com L. plantarum (LB+LP) não afetou as variáveis relacionadas com o valor nutritivo das silagens. As concentrações de ácido acético nas silagens de milho foram superiores no tratamento controle. Nas silagens de cana-de-açúcar, o tratamento LB apresentou maiores teores de MS e DVIVMO. O tratamento controle apresentou maior população de bactérias ácido láticas, e menor valor de pH. A silagem de cana-de-açúcar sem aditivo atingiu o pico de temperatura após o tratamento LB, sendo esse pico superior na silagem aditivada. Foram observadas menores perdas de matéria seca e produção de gases nas silagens aditivadas, resultando assim em maior recuperação de MS no tratamento LB. Foi observado maior tempo para atingir a temperatura máxima e menor acúmulo de temperatura até o quinto dia de exposição aeróbia no tratamento controle. A temperatura máxima atingida durante a exposição aeróbia foi superior no tratamento LB. Nas silagens de capim, as silagens do tratamento LB+LP apresentaram maiores teores de MS, e menor teor de PB. Foi observada maior população de bactérias ácido láticas nas silagens do tratamento LB+LP. A inoculação com L. buchneri, sozinho, ou em associação com L. plantarum não foi eficiente em melhorar a qualidade e estabilidade aeróbia das forragens avaliadas. / Two trials were conducted for the evaluation of microbial additives on tropical forages ensiling. At the first trial the effect of increasing L. buchneri doses, and different storage periods on the fermentation profile, aerobic stability and nutritive value of corn silage were evaluated. At the second trial the effect of L. buchneri with or without L. plantarum was evaluated on the fermentation profile, aerobic stability and nutritive value of corn, sugarcane and tropical grass silages. Silages inoculation with L. buchneri did not affect the filamentous funghi counts, with decreased counts with higher storage periods. Lactic acid content was influenced only by treatment, with decrease in concentrations as the L. buchneri dose increased. Lactic acid bacteria, yeasts, pH, butyric and acetic acid were influenced by both treatment and storage period. Higher storage periods resulted in decreased LAB counts and higher pH values. Storage time decreased yeast count in corn silage. For the dry matter losses, gas losses and maximum temperature there was no treatment effect, only storage period effect, decreasing along time. It was observed an increase in effluent production with higher storage time. Time to reach maximum temperature, AC5 and AC10 were influenced by both treatment and storage period. Higher storage periods resulted in higher time to reach the maximum temperature and to break the aerobic stability. Silages with the higher L. buchneri doses showed stability breakdown after control and LB1x105 silages. The higher storage period of corn silages resulted in higher nutritive value and microbial profile favorable to the fermentation and stability during aerobic exposure. Inoculation with Lactobacillus buchneri (NCIMB 40788) reduces the survival of yeasts during the storage phase, and its growth during oxygen exposure, improving silage aerobic stability in tropical regions. Higher doses could partly compensate the lower storage time in silages. In the second trial, inoculation of corn silages with L. buchneri (LB), alone or in association with L. plantarum (LB+LP) did not affect the variables related to the silage nutritive value. Acetic acid content of corn silages was higher for the control treatment. In sugarcane silages, LB treatment showed higher DM content and IVTOMD. Control treatment showed higher lactic acid bacteria counts, and lower pH. Sugarcane silage with no additive reached temperature peak after LB treatment, with this peak being higher at the additivated silage. There were observed lower dry matter losses and gas production in LB silages, resulting in higher dry matter recovery. It was observed higher time to reach maximum temperature and lower temperature accumulation until fifth day of aerobic exposure for the control treatment. Maximum temperature reached during aerobic exposure was higher for LB treatment. In tropical grass silages, LB+LP silages showed higher DM and lower CP contents. It was observed higher lactic acid bacteria in LB+LP treatment. Inoculation with L. buchneri, alone or in association with L. plantarum was not efficient in improving quality and aerobic stability of evaluated forages.

The purpose of the project was to exam steams ability to sterilize carrots infected withLactobacillus plantarum. The project was a cowork with Brämhults Juice AB. They hadobserved an increase of Lactobacillus in the production of carrot juice during the summer. Tokeep the flavour of fresh fruits, the juice is treated carefully with heat. This means that thejuice only stays fresh for a few days, around two weeks for an unopened package and threedays for an opened package.In an attempt to remove the remaining Lactobacillus of the carrots, steam is tested as an extrastep in the production of juice. During the project it was found that it is difficult to reach thewanted temperature of above 100°C, when the steam is generated under pressure. When thesteam was released in normal pressure, a decrease in temperature occurred that were so largethat the steam had a temperature of only 40°C. At the second treatment the steam wasgenerated at 1,8 bar pressure and 130°C, the steam then decreased to 80°C.At the third and last treatment the steam was generated under normal pressure and atemperature of 90°C was reached. At this temperature an 96,5% sterilization occurred after 15seconds of steam treatment. This is not enough for Brämhults Juice, since they need only afew percent surviving Lactobacills after 6 seconds. / Uppsatsnivå: C

Foram realizados dois experimentos para avaliação de aditivos microbianos na ensilagem de forragens tropicais. No primeiro experimento avaliou-se o efeito de doses crescentes de Lactobacillus buchneri, em diferentes tempos de armazenamento, sobre o valor nutritivo, perfil fermentativo e estabilidade aeróbia de silagens de milho. No segundo experimento avaliou-se o efeito de L. buchneri, de forma exclusiva, ou em combinação com Lactobacillus plantarum, em silagens de milho, cana-de-açúcar e capim elefante cv. Napier. O tratamento das silagens de milho com L. buchneri não afetou a contagem de fungos filamentosos, com decréscimo nas contagens com maiores tempos de armazenamento. Os teores de ácido lático decresceram com o aumento da dose de L. buchneri. Os maiores períodos de armazenamento resultaram em decréscimo na contagem de BAL, e maiores valores de pH. O tempo de armazenamento diminuiu as leveduras na silagem de milho. Maiores tempos de armazenamento propiciaram maiores tempos para atingir a temperatura máxima e a perda da estabilidade aeróbia. As silagens com doses superiores de L. buchneri apresentaram perda de estabilidade em momento posterior às silagens dos tratamentos controle e LB1x105. O maior tempo de armazenamento da silagem de milho resultou em maior valor nutritivo e perfil microbiológico favorável quanto à fermentação e estabilidade em aerobiose. Inoculação com L. buchneri (NCIMB 40788) reduz a sobrevivência de leveduras durante a fase de armazenamento e o crescimento durante a exposição ao oxigênio, melhorando a estabilidade aeróbia da silagem em ambiente tropical. Doses mais elevadas poderiam compensar parcialmente o menor tempo de armazenamento das silagens. No segundo experimento, o tratamento das silagens de milho com L. buchneri (LB), exclusivamente, ou associado com L. plantarum (LB+LP) não afetou as variáveis relacionadas com o valor nutritivo das silagens. As concentrações de ácido acético nas silagens de milho foram superiores no tratamento controle. Nas silagens de cana-de-açúcar, o tratamento LB apresentou maiores teores de MS e DVIVMO. O tratamento controle apresentou maior população de bactérias ácido láticas, e menor valor de pH. A silagem de cana-de-açúcar sem aditivo atingiu o pico de temperatura após o tratamento LB, sendo esse pico superior na silagem aditivada. Foram observadas menores perdas de matéria seca e produção de gases nas silagens aditivadas, resultando assim em maior recuperação de MS no tratamento LB. Foi observado maior tempo para atingir a temperatura máxima e menor acúmulo de temperatura até o quinto dia de exposição aeróbia no tratamento controle. A temperatura máxima atingida durante a exposição aeróbia foi superior no tratamento LB. Nas silagens de capim, as silagens do tratamento LB+LP apresentaram maiores teores de MS, e menor teor de PB. Foi observada maior população de bactérias ácido láticas nas silagens do tratamento LB+LP. A inoculação com L. buchneri, sozinho, ou em associação com L. plantarum não foi eficiente em melhorar a qualidade e estabilidade aeróbia das forragens avaliadas. / Two trials were conducted for the evaluation of microbial additives on tropical forages ensiling. At the first trial the effect of increasing L. buchneri doses, and different storage periods on the fermentation profile, aerobic stability and nutritive value of corn silage were evaluated. At the second trial the effect of L. buchneri with or without L. plantarum was evaluated on the fermentation profile, aerobic stability and nutritive value of corn, sugarcane and tropical grass silages. Silages inoculation with L. buchneri did not affect the filamentous funghi counts, with decreased counts with higher storage periods. Lactic acid content was influenced only by treatment, with decrease in concentrations as the L. buchneri dose increased. Lactic acid bacteria, yeasts, pH, butyric and acetic acid were influenced by both treatment and storage period. Higher storage periods resulted in decreased LAB counts and higher pH values. Storage time decreased yeast count in corn silage. For the dry matter losses, gas losses and maximum temperature there was no treatment effect, only storage period effect, decreasing along time. It was observed an increase in effluent production with higher storage time. Time to reach maximum temperature, AC5 and AC10 were influenced by both treatment and storage period. Higher storage periods resulted in higher time to reach the maximum temperature and to break the aerobic stability. Silages with the higher L. buchneri doses showed stability breakdown after control and LB1x105 silages. The higher storage period of corn silages resulted in higher nutritive value and microbial profile favorable to the fermentation and stability during aerobic exposure. Inoculation with Lactobacillus buchneri (NCIMB 40788) reduces the survival of yeasts during the storage phase, and its growth during oxygen exposure, improving silage aerobic stability in tropical regions. Higher doses could partly compensate the lower storage time in silages. In the second trial, inoculation of corn silages with L. buchneri (LB), alone or in association with L. plantarum (LB+LP) did not affect the variables related to the silage nutritive value. Acetic acid content of corn silages was higher for the control treatment. In sugarcane silages, LB treatment showed higher DM content and IVTOMD. Control treatment showed higher lactic acid bacteria counts, and lower pH. Sugarcane silage with no additive reached temperature peak after LB treatment, with this peak being higher at the additivated silage. There were observed lower dry matter losses and gas production in LB silages, resulting in higher dry matter recovery. It was observed higher time to reach maximum temperature and lower temperature accumulation until fifth day of aerobic exposure for the control treatment. Maximum temperature reached during aerobic exposure was higher for LB treatment. In tropical grass silages, LB+LP silages showed higher DM and lower CP contents. It was observed higher lactic acid bacteria in LB+LP treatment. Inoculation with L. buchneri, alone or in association with L. plantarum was not efficient in improving quality and aerobic stability of evaluated forages.

Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2001. / Résumé en français et en anglais. Textes en français et en anglais. Bibliogr. f. 232-270. Notes bibliogr.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006. / Kefir is a traditional fermented milk that is carbonated, has a sharp acidic taste, yeasty flavour and contains a low percentage alcohol (less than 2% (v/v)). The beverage is manufactured by fermenting milk with Kefir grains, comprised of microorganisms, polysaccharides and milk proteins. The microbial population of Kefir grains primarily include lactic acid bacteria (LAB), namely lactococci and lactobacilli, yeasts, Acetobacter and filamentous fungi. Kefir exhibits antimicrobial activity in vitro against some fungi, and Grampositive and Gram-negative bacteria. Although the exact cause of this inhibition in Kefir is not known, the ability of LAB to inhibit the growth of closely related bacteria is well known. This inhibition of pathogenic and spoilage microbes may be due to the production of organic acids, hydrogen peroxide, acetaldehyde, diacetyl, carbon dioxide or bacteriocins. Acid is not the only contributor to the antimicrobial activity of Kefir and Kefir grains, and bacteriocins may play a role in the inhibitory activity. The bacteriocin producer Lactobacillus plantarum ST8KF, isolated from Kefir and Kefir grains, produces a bacteriocin 3.5 kDa in size. The mode of activity of bacteriocin ST8KF (bacST8KF) is thought to be bacteriostatic in exponential cultures of Enterococcus faecalis E88, Lactobacillus casei LHS, Lactobacillus curvatus DF38, Lactobacillus sakei DSM 20017, Lactobacillus salivarius 241 and Listeria innocua F and LMG 13568. The peptide is sensitive to proteolytic enzymes and does not adsorb to the surface of the producer cell. The bacteriocin is stable between pH 2.0 and 10.0, and for 20 min at 121°C. Maximum bacteriocin activity was observed in modified MRS medium supplemented with glucose or saccharose, meat extract, KH2PO4, glycerol, thiamine or cyanocobalamin, or in modified MRS medium without tri-ammonium citrate. Maximum levels of adsorption of bacST8KF (80%) to Lb. casei LHS and Lb. sakei DSM 20017 were recorded. Adsorption (80%) of the bacteriocin to Lactobacillus paraplantarum ATCC 700211T and Streptococcus caprinus ATCC 700066, which are not sensitive to the bacteriocin was also recorded. Optimal adsorption to E. faecalis E88 was recorded at 25°C at pH 2.0, and to L. innocua LMG 13568 at 4°C, 10°C and 25°C at pH 6.0. Potassium ions, MgCl2, Tris, NH4- citrate, Na-acetate, Na2CO3, EDTA and SDS led to decreased adsorption to both sensitive strains, while NaCl and mercaptoethanol resulted decreased adsorption to E. faecalis E88, but not to L. innocua LMG 13568. Methanol resulted in lower levels of adsorption to L. innocua LMG 13568 but not to E. faecalis E88. Triton X-100 and Triton X-114 increased the adsorption of bacST8KF by 40%, and ethanol and chloroform had no effect on bacteriocin adsorption. The growth of Lb. plantarum ST8KF and L. innocua LMG 13568 in a mixed culture resulted in an increase of bacST8KF production. Cells treated with bacST8KF secreted DNA and galactosidase. As bacST8KF remains stable under a variety of conditions, the bacteriocin may have application, if awarded GRAS (generally regarded as safe) status, in various food products as a natural additive or preservative. The genes encoding bacteriocin production are located on a 3.9 kilo base (kb) plasmid. Curing of the plasmid resulted in a mutant strain of Lb. plantarum ST8KF, and the Lb. plantarum strains ST8KF(+) and ST8KF(-) differed with regards to antibiotic resistance and carbohydrate fermentation reactions. The wild type and the cured strain were incorporated into Kefir grains during mass cultivation. The survival of the bacST8KF sensitive Enterococcus mundtii ST4SA added to the milk during Kefir production using the enriched mass cultured grains was monitored using fluorescent in situ hybridization. Enterococcus mundtii ST4SA was present in higher numbers in the ST8KF(-) Kefir system when compared to the ST8KF(+) system. It can, therefore, be concluded that Lb. plantarum ST8KF(+) contributes to the antimicrobial activity of Kefir through the production of bacteriocin ST8KF.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T00:02:13Z (GMT). No. of bitstreams: 1 277525.pdf: 903897 bytes, checksum: 7d856e166a29a1927236c8c628ced144 (MD5) / As lipases e esterases pertencem ao grupo das hidrolases e são enzimas capazes de catalisar a clivagem e formação de ligações ésteres. Essas enzimas são empregadas em diferentes ramos industriais, principalmente na indústria química, farmacêutica e de alimentos. A lipase (AF208229) de Staphylococcus xylosus foi previamente isolada, clonada, seqüenciada e caracterizada. O objetivo deste trabalho foi avaliar a importância do resíduo de aminoácido valina 309 para atividade enzimática desta lipase, modificando a valina 309 por aspartato ou lisina através da PCR mutagênese sitio dirigida. A mutação foi realizada com sucesso e os mutantes (Mut-Asp e Mut-Lys) apresentaram características diferentes da lipase recombinante selvagem (WT-Val) em relação a hidrólise do butirato de p-nitrofenila. Foi observado que a substituição do aminoácido 309 aumentou a atividade das lipases mutantes, principalmente com a presença do aminoácido básico (Mut-Lys). No entanto, a afinidade da enzima ao substrato diminui após a substituição do aminoácido valina, ocasionando aumento em duas vezes do valor de Km para as enzimas mutantes. A atividade ótima para as três lipases recombinantes foi observada em pH 9,0 e a 42°C, sendo que, a Mut-Lys apresentou a maior atividade no pH alcalino. A lipase recombinante selvagem (WT-Val) e as lipases mutantes Mut-Asp e Mut-Lys mostraram ser termoestáveis após incubação a 80°C, visto que não foi observada perda na atividade nos períodos de incubação analisados (10, 20 e 30 min), fato observado pela primeira vez para lipases do gênero Staphylococcus. A imobilização de enzimas é uma estratégia bastante utilizada para modificar a atividade enzimática e propiciar o uso de biocatalisadores nos processos industriais. A esterase de Lactobacillus plantarum foi previamente isolada, clonada, seqüenciada e caracterizada. Neste trabalho, esta esterase foi expressa em E. coli e o lisado celular foi imobilizado no suporte hidrofóbico polipropileno (Accurel MP1000) pelo método de adsorção e apresentou eficiência de 68,7%. A enzima imobilizada mostrou ser mais estável nas diferentes temperaturas testadas, como também, apontou ser mais termoestável que o lisado celular de enzima livre, mostrando que o processo de imobilização ocasiona restrição na mobilidade conformacional e maior rigidez na estrutura da enzima, impedindo a desnaturação e a perda da capacidade catalítica. A enzima imobilizada apresentou menor atividade específica e menor afinidade pelo substrato em relação a enzima livre, frente a hidrólise do butirato de p-nitrofenila. Não foi observado desprendimento de proteína do suporte após 3 ciclos contínuos e a atividade de hidrolise da enzima imobilizada foi estável até a terceira semana após ser imobilizada. / The lipases and esterases belong to the group of hydrolases and these enzymes are capable of catalyzing the cleavage and formation of ester bonds. These enzymes are used in different industrial fields, mainly in the chemical, pharmaceutical and food industries. Lipase (AF208229) of Staphylococcus xylosus was previously isolated, cloned, sequenced and characterized. The objective of this study was to evaluate the importance of the amino acid valine 309 for activity of lipase, changing valine 309 to aspartate or lysine by PCR site directed mutagenesis. The mutation was successfully performed and the mutant (Mut and Mut-Asp-Lys) had different characteristics from recombinant lipase wild (WT-Val) for the hydrolysis of butyrate p-nitrophenyl. It was observed that the substitution of amino acid 309 increased the activity of lipase mutants, especially with the presence of basic amino acid (Mut-Lys). However, the affinity of the enzyme to the substrate decreases after substitution of the amino acid valine, leading to increased at twice the value of Km for mutant enzymes. The optimum activities for the three recombinant lipases were observed at pH 9.0 and 42° C, and the Mut-Lys showed the highest activity at alkaline pH. Recombinant lipase wild-type (WT-Val) and the mutants Mut-Asp and Mut-Lys proved to be heat-stable after incubation at 80°C, whereas there was no loss of activity in the incubation periods examined (10, 20 and 30 min ). This result was first observed for lipases of Staphylococcus. The immobilization of enzymes is a strategy often used to modify the enzymatic activity and promote the use of biocatalysts in industrial processes. The esterase of Lactobacillus plantarum was previously isolated, cloned, sequenced and characterized. In this work, this esterase was expressed in E. coli and the cell lysate was immobilized on the support hydrophobic polypropylene (Accurel MP1000) by the method of adsorption and presented efficiency of 68.7%. The immobilized enzyme was more stable at different temperatures, and also was more thermostable than the cell lysate of free enzyme, showing that the immobilization process can causes restricted mobility and greater rigidity in the structure of the enzyme, preventing the denaturation and loss of catalytic ability. The immobilized enzyme showed a lower specific activity and a lower affinity by the substrate than the free enzyme for hydrolysis of butyrate p-nitrophenyl. It was not observed detachment of protein support after 3 continuous cycles and the hydrolysis activity of immobilized enzyme was stable until the third week after being immobilized.