The enzyme, &amp / #946 / -galactosidase (E.C.3.2.1.23) has been used for dairy industry for removing lactose from milk and milk by-products. In this study, three strains namely L. plantarum NCIMB 1193, L. plantarum DSM 20246 and L. plantarum E081 were used for &amp / #946 / -galactosidase release by sonication method. The peak of the total enzyme activity was found to be corresponding to late logarithmic or early stationary phase of all strains. As a disruption method sonication was used for the release of &amp / #946 / -galactosidase. Meanwhile, the sonication time was optimized for each strain. The peak of the enzyme activity was observed between 210 seconds and 270 seconds of sonication period. It was also found that sonication did not decrease the viability of L.plantarum NCIMB 1193 significantly. Liquid nitrogen cell disruption method was also used to compare the results with those obtained by sonication method. For characterization &amp / #946 / -galactosidase, cell-free crude extract of sonicated cell culture of L.plantanrum NCIMB 1193 was used. Optimum pH found as 7.2, and optimum temperature range was found between between 350 C to 400 C. Km and Vmax values were found as 3.47 mM and 1.721 (&amp / #956 / mol / min per mg protein) respectively from Lineweaver-Burk plot. Km and Vmax values were found as 4.064 mM and 1.863 (&amp / #956 / mol / min per mg cell-free crude extract) respectively from Eadie-Hofstee plot. The number of ligand binding sites (napp) on a molecule of &amp / #946 / -galactosidase was found as 1.03 which indicates that the number of ligand binding sites on the enzyme is one.

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.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-graduação em Ciência dos Alimentos, Florianópolis, 2010 / Made available in DSpace on 2012-10-25T07:00:59Z (GMT). No. of bitstreams: 1 281745.pdf: 3514340 bytes, checksum: f888864f9bbaf44bdf369ae8dec134e3 (MD5) / Esterases e lipases pertencem ao grupo das hidrolases e são enzimas que catalisam a clivagem e a síntese de ligações éster, sendo um grupo de enzimas biotecnologicamente relevantes com inúmeras aplicações em indústrias de alimentos, laticínios, detergentes e farmacêuticas. O objetivo deste trabalho foi clonar, expressar em Escherichia coli, purificar e caracterizar bioquimicamente uma lipase recombinante de Staphylococcus xylosus e uma esterase recombinante de Lactobacillus plantarum. O gene da lipase (1084 pb) foi amplificado de uma cepa de S. xylosus isolada de salame fermentado naturalmente e introduzido no vetor de expressão pET14b para expressar a proteína fusão recombinante (lipase contendo a cauda de His6). A lipase recombinante de S. xylosus foi purificada por cromatografia de afinidade em um sistema HPLC. A lipase recombinante é um monômero em solução, como determinado pela cromatografia de exclusão molecular. Ela apresentou alta atividade em pH 9,0 e 42°C para acetato de p-nitrofenila e butirato de p-nitrofenila entre vários ésteres testados (pNPC2, pNPC4, pNPC10, pNPC12, pNPC14, pNPC16, pNPC18). Além disso, apresentou uma interessante estabilidade térmica após incubação por 10 min a 95°C, mantendo 77 % de sua atividade inicial. O gene da esterase de Lactobacillus plantarum (1014 pb) foi amplificado e clonado no vetor de expressão pET14b para expressar em Escherichia coli a proteína contendo a cauda His6. A esterase recombinante de Lactobacillus plantarum foi purificada em resina Ni-NTA e apresentou uma massa molecular aparente de aproximadamente 38 kDa. A enzima apresentou a atividade enzimática mais elevada em pH 6,0 e 40°C e preferência pelo butirato de p-nitrofenila, embora tenha hidrolisado mais eficientemente o acetato de p-nitrofenila. Além disso, este estudo apresenta, pela primeira vez, dados de dicroísmo circular sobre a estrutura secundária de uma esterase de Lactobacillus plantarum.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-graduação em Aquicultura / Made available in DSpace on 2012-10-24T03:33:42Z (GMT). No. of bitstreams: 1 264394.pdf: 2681897 bytes, checksum: d38fd9b5c0fa6cd6e938349f1b8908e7 (MD5)

A produção de leite e queijo de ovelha pode ser considerada recente no Brasil, por isso a necessidade de estudar a composição e o processamento destes alimentos. As bactérias lácticas presentes no leite são empregadas como culturas iniciadoras em queijos e influenciam nas características de qualidade do produto. A qualidade microbiológica e físico-química do leite de ovelha da raça Lacaune e do queijo Fascal produzidos no RS foi avaliada, durante a produção e maturação do queijo. As bactérias lácticas Lactobacillus plantarum LCN 17 e Lactobacillus rhamnosus LCN 43 foram isoladas a partir das amostras e o potencial antimicrobiano, proteolítico e lipolítico destes isolados foi investigado. Com base nos resultados, foi testada a utilização das bactérias lácticas autóctones como culturas iniciadoras para a produção de queijo de ovelha. A combinação entre Lactobacillus plantarum LCN 28 e Lactobacillus rhamnosus LCN 43 demonstrou ser adequada na fabricação de queijo Fascal, em condições experimentais. A caseína precipitada a partir do leite de ovelha foi usada na preparação de caseinatos de sódio e de cálcio. O processo de agregação e formação de géis de caseinato ovino por acidificação e as modificações que estes géis sofreram na presença de açúcares e/ou pequenos co-solutos foi estudada. A concentração de cálcio afetou a solubilidade do caseinato ovino, o grau de compactação e a dureza dos géis. A adição de sacarose e lactose ao caseinato ovino acarretou a formação de partículas coloidais com estrutura mais compacta e menor hidrofobicidade superficial. Em ambos os casos, o caseinato ovino e bovino apresentaram comportamento um pouco distinto. / The production of sheep milk and cheese may be considered a recent activity in Brazil; hence the need to study the composition and processing of these foods. Lactic acid bacteria present in milk are used as starter cultures in cheese production, and influence product quality characteristics. The microbiological and physicochemical quality of milk of the Lacaune sheep breed and of Fascal cheese produced in the state of Rio Grande do Sul, RS, Brazil, was assessed during cheese production and ripening. The lactic acid bacteria Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were isolated from samples to investigate the antimicrobial, proteolytic and lipolytic potentials of each microorganism. The results were used to test the applicability of these autochthonous bacteria as starter cultures in the production of ewe cheese. The mixed culture of Lactobacillus plantarum LCN 28 and Lactobacillus rhamnosus LCN 43 was shown to be appropriate in the production of Fascal cheese, under experimental conditions. The casein precipitated from sheep milk was used in the preparation of sodium and calcium caseinates. The aggregation process and formation of ovine caseinate gels by acidification and the changes these gels undergo in the presence of sugars and/or small cosolutes were investigated. Calcium concentration affected the solubility of ovine caseinate, compactation degree and hardness of gels. The addition of saccharose and lactose to ovine caseinate led to the formation of colloidal particles presenting a more structured and lesser surface hydrophobicity. In both cases, ovine and bovine caseinate presented fairly distinct behaviors.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Aqüicultura, Florianópolis, 2009. / Made available in DSpace on 2012-10-24T10:06:57Z (GMT). No. of bitstreams: 1 272732.pdf: 416097 bytes, checksum: 89554c700280ba24151a2a22e1505a5c (MD5)