Avaliação dos efeitos de 5-hidroxitriptofano em-hidroxibenzilhidrazine associados a Lactobacillus spp. na morfometria intestinal e imunomarcação de serotonina em frangos de corte desafiados com Salmonella Enteridis

Resumo As células enterocromafins são um dos componentes da mucosa intestinal que liberam serotonina para o lúmen, promovendo atividades secretórias e crescimento celular de vários tecidos, incluindo vilosidades intestinais. O presente estudo avaliou as influências do 5-hidroxitriptofano (5HTP) e do m-hidroxibenzilhidrazine (NSD1015), associados a Lactobacillus spp., sobre o peso corporal e o desenvolvimento das vilosidades intestinais na porção proximal do duodeno de frangos de corte desafiados com Salmonella Enteritidis. Verificou-se também se a presença de Lactobacillus spp. e Salmonella Enteritidis influenciaram a imunomarcação de serotonina no duodeno e, para isso, o estudo foi dividido em dois experimentos, com e sem desafio por S. Enteritidis. No Experimento 1, em aves sem desafio, os pesos corporais não diferiram significantemente (p>0,05) e, no Experimento 2, aves com desafio, os tratamentos com o precursor isolado e associado a Lactobacillus spp. determinaram maior peso corporal das aves. Nos dois experimentos, as aves tratadas com 5HTP apresentaram aumento na densidade e altura das vilosidades no duodeno, sugerindo a atuação de 5HTP como um agente trófico. A administração de Lactobacillus spp. também determinou altura maior de vilosidades duodenais. Quanto a imunomarcação de serotonina, as aves tratadas com Lactobacillus spp. no Experimento 1 e as aves tratadas com Lactobacillus spp. e desafiadas com S. Enteritidis no Experimento 2, apresentaram valores superiores aos demais tratamentos, sugerindo que a presença destas bactérias promove maior liberação de serotonina para o duodeno, porém o mecanismo exato de como este processo ocorre necessita ser mais elucidado.

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 108 cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and on atherogenesis in apolipoprotein E knock-out mice

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E) knock-out (KO) mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

Lactobacillus delbrueckii UFV-H2b20 induces type 1 cytokine production by mouse cells in vitro and in vivo

Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-α by peritoneal cells and IFN-γ by spleen cells from germ-free or monoassociated Swiss/NIH mice and LPS-hyporesponsive mice (around 40 ng/mL for IL-12 p40, 200 pg/mL for TNF-α and 10 ng/mL for IFN-γ). Heat treatment of L. delbrueckii did not affect the production of these cytokines. Lysozyme treatment decreased IL-12 p40 production by peritoneal cells from C3H/HeJ mice, but did not affect TNF-α production by these cells or IFN-γ production by spleen cells from the same mouse strain. TNF-α production by peritoneal cells from Swiss/NIH L. delbrueckii-monoassociated mice was inhibited by lysozyme treatment. When testing IL-12 p40 and IFN-γ levels in sera from germ-free or monoassociated Swiss/NIH mice systemically challenged with Escherichia coli we observed that IL-12 p40 was produced at marginally higher levels by monoassociated mice than by germ-free mice (40 vs 60 ng/mL), but IFN-γ was produced earlier and at higher levels by monoassociated mice (monoassociated 4 and 14 ng/mL 4 and 8 h after infection, germfree 0 and 7.5 ng/mL at the same times). These results show that L. delbrueckii UFV-H2b20 stimulates the production of type 1 cytokines in vitro and in vivo, therefore suggesting that L. delbrueckii might have adjuvant properties in infection in which these cytokines play a major role.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Translocação de Lactobacillus acidophilus em ratos alimentados com dieta rica em colesterol e ácido cólico suplementada com probiótico

Lactobacillus acidophilus é um microrganismo bastante estudado e componente comum de probióticos. No entanto, ainda há pouca informação sobre a translocação deste microrganismo para órgãos do corpo. O presente trabalho visou investigar a taxa de translocação de Lactobacillus acidophilus em ratos que receberam uma dieta rica em colesterol, suplementada com um concentrado de células de L. acidophilus NCFM, visto que, o consumo deste, tem sido indicado como adjunto dietético para indivíduos hipercolesterolêmicos. Foram utilizados 130 ratos machos Wistar, com peso médio inicial de 250±32g, distribuídos em 4 grupos experimentais, sendo: Padrão, com 40 animais e Controle, LDR (Leite Desnatado Reconstituído) e P (Probiótico) com 30 animais cada. O grupo Padrão recebeu a dieta AIN-93G durante todo o período experimental (42dias). Os demais grupos receberam a mesma dieta acrescida de 1% de colesterol e 0,1% de ácido cólico. Durante 14 dias (do 1º ao 14º dia), o grupo LDR recebeu 0,1mL/dia/animal de leite desnatado reconstituído a 10% de sólidos não gordurosos e o grupo P, recebeu 0,1mL/dia/animal de probiótico contendo 10(10) UFC/mL de Lactobacillus acidophilus NCFM. A taxa de translocação foi monitorada no 14º, 28º e 42º dias de experimentação (tempo 0, 14 e 28 dias após o término do consumo do probiótico, respectivamente) no baço, coração, fígado e rins. Nos órgãos dos animais dos grupos Padrão, Controle e LDR não foi observado crescimento de microrganismos nos tempos avaliados. Verificou-se translocação para o baço, coração, fígado e rins dos animais do grupo que recebeu probiótico (grupo P). O número de células de Lactobacillus encontrado nos órgãos foram, em UFC/órgão: baço (8,6 x 10²); fígado (4,8 x 10²); coração (4,7 x 10²) e rins (1,3 x 10²). Constatou-se diferença significativa (p<0,05) na contagem de células encontradas no baço, em comparação aos demais órgãos analisados. Entre o fígado e o coração, não foi observada diferença significativa (p>0,05), mas ambos diferiram estatisticamente dos rins (p<0,05). Observou-se uma eliminação constante das células translocadas após o término do consumo do probiótico, de acordo com os diferentes órgãos. O baço apresentou o maior número de células translocadas (860 UFC/órgão), no entanto, foi o órgão que as eliminou mais rapidamente. Ao final do período analisado, 69,8% das células presentes no baço, haviam sido eliminadas.

Avaliação sensorial, microbiológica e de pós-acidificação durante a vida-de-prateleira de leites fermentados contendo Streptococcus thermophilus, Bifidobacterium longum e Lactobacillus acidophilus

Três leites fermentados, o primeiro por Str. thermophilus, o segundo por Bif. longum e o terceiro por Lb. acidophilus e o leite fermentado elaborado pela mistura de volumes iguais dos leites fermentados, separadamente, por Str. thermophilus, Bif. longum e Lb. acidophilus tiveram seu pH, acidez titulável, atributos sensoriais de sabor e acidez, e contagens de células viáveis determinadas, ao longo de 21 dias de estocagem a 4ºC. Não houve diferença significativa (p<0,05) para os atributos sensoriais entre o leite fermentado elaborado por mistura e aquele contendo apenas Str. thermophilus. O leite fermentado por Str. thermophilus apresentou a maior acidez e menor pH, enquanto o leite fermentado por Bif. longum apresentou a menor acidez e maior pH. As populações de Str. thermophilus e Bif. longum mantiveram-se constantes até 21 dias de estocagem, tanto nos leites fermentados isoladamente como no leite elaborado pela mistura, enquanto as populações de Lb. acidophilus apresentaram redução de um ciclo logarítmico.

Efeito do uso de cultura adjunta (Lactobacillus helveticus) na proteólise, propriedades viscoelásticas e aceitação sensorial de queijo prato light

A proteólise, as propriedades viscoelásticas e a aceitação sensorial de queijo prato light fabricado com e sem adição de cultura adjunta (CAD) foram avaliadas. Os queijos foram fabricados a partir de leite microfiltrado. Dois tratamentos foram testados em duplicata: o queijo controle foi fabricado apenas com cultura mesófila tradicional (acidificante e aromatizante), e o outro foi fabricado com adição de CAD (Lactobacillus helveticus), além da cultura tradicional. A composição dos queijos foi determinada no quinto dia após a fabricação. A proteólise e as propriedades reológicas foram avaliadas nos dias 5, 25 e 45 após a fabricação. Os parâmetros viscoelásticos foram obtidos a partir de testes de relaxação. As amostras foram avaliadas sensorialmente por meio de testes de aceitação. Não houve diferença significativa (p>0,05) na composição dos queijos. Os índices de profundidade de proteólise foram significativamente (p<0,05) maiores ao final do tempo de maturação para o queijo fabricado com adição de CAD. As propriedades viscoelásticas dos queijos não foram influenciadas pelo uso de CAD (p>0,05). Nos testes de aceitação sensorial, o queijo produzido com CAD obteve notas significativamente (p<0,05) mais altas para os atributos aroma, textura e impressão global. Em relação ao atributo intenção de compra, 70% dos consumidores, certamente ou provavelmente, comprariam o queijo com CAD, enquanto apenas 43,4% teriam a mesma atitude em relação ao queijo controle.

Permanent magnet synchronous generator design solution for large directdrive wind turbines: Thermal behavior of the LC DD-PMSG

Wind is one of the most compelling forms of indirect solar energy. Available now, the conversion of wind power into electricity is and will continue to be an important element of energy self-sufficiency planning. This paper is one in a series intended to report on the development of a new type of generator for wind energy; a compact, high-power, direct-drive permanent magnet synchronous generator (DD-PMSG) that uses direct liquid cooling (LC) of the stator windings to manage Joule heating losses. The main param-eters of the subject LC DD-PMSG are 8 MW, 3.3 kV, and 11 Hz. The stator winding is cooled directly by deionized water, which flows through the continuous hollow conductor of each stator tooth-coil winding. The design of the machine is to a large degree subordinate to the use of these solid-copper tooth-coils. Both steady-state and timedependent temperature distributions for LC DD-PMSG were examined with calculations based on a lumpedparameter thermal model, which makes it possible to account for uneven heat loss distribution in the stator conductors and the conductor cooling system. Transient calculations reveal the copper winding temperature distribution for an example duty cycle during variable-speed wind turbine operation. The cooling performance of the liquid cooled tooth-coil design was predicted via finite element analysis. An instrumented cooling loop featuring a pair of LC tooth-coils embedded in a lamination stack was built and laboratory tested to verify the analytical model. Predicted and measured results were in agreement, confirming the predicted satisfactory operation of the LC DD-PMSG cooling technology approach as a whole.

Salame elaborado com Lactobacillus plantarum fermentado em meio de cultura de plasma suíno

Este trabalho teve por objetivo produzir uma cultura starter com uma cepa de Lactobacillus plantarum em um meio de cultura com plasma suíno e verificar a viabilidade de sua aplicação em salame. O meio de cultura foi preparado com plasma suíno e água destilada (1:1, pH 11,0). Após a esterilização, 300 mL foram adicionados de 400 mL de uma solução estéril de glicose e difosfato de potássio. A cepa de Lb. plantarum foi semeada no meio de cultura e submetida à fermentação em pH 7,0, durante 36 horas (100 rpm, 37 ± 0,1 °C). Ao alcançar a fase estacionária, a cultura foi centrifugada e ressuspendida em leite desnatado estéril, liofilizada e aplicada em salame. A influência do inóculo foi avaliada nas características microbiológicas, físico-químicas e sensoriais de salames. Os resultados encontrados foram comparados com tratamentos sem adição de cultura starter e com uma cultura comercial. O microrganismo Lb. plantarum teve um crescimento máximo de 9,82 Log UFC.mL-1, após 30 horas de fermentação. Os salames elaborados com a cultura starter produzida apresentaram uma queda de pH significativamente maior, e menor valor de atividade de água que os demais tratamentos. O microrganismo Lb. plantarum melhorou significativamente o sabor dos salames.

Lactobacillus plantarum AJ2 isolated from naturally fermented sausage and its effects on the technological properties of Milano-type salami

L. plantarum, strain AJ2, was isolated from naturally fermented sausage manufactured in the Southern region of Brazil and inoculated in Milano-type salami. The lactic culture exhibited the ability of growing in the product, decreasing pH in the first seven days of fermentation/maturation and the residual levels of nitrite and nitrate, as well as peroxide and TBARS values. The inoculated sausage had the highest intensity for brightness and red color, but did not present a difference in chemical composition and fatty acid composition, compared to the control.

Desenvolvimento de queijo minas frescal adicionado de Lactobacillus acidophilus produzido a partir de retentados de ultrafiltração

Este trabalho teve por objetivos: desenvolver um queijo probiótico tipo minas frescal a partir de retentados de ultrafiltração, estudar a sobrevivência da bactéria probiótica Lactobacillus acidophilus durante a vida de prateleira do produto e avaliar as alterações físico-químicas e sensoriais do produto. O queijo foi produzido a partir de retentados, obtidos a partir da ultrafiltração de leite integral até o fator de concentração volumétrico de 5:1. Os retentados pasteurizados foram inoculados com L. acidophilus, de forma a obter uma população inicial no queijo de 10(6), 10(7) e 10(8) UFC.g-1. Foram realizadas análises microbiológicas, para contagem das bactérias probióticas em intervalos de 7 dias durante sua estocagem, análises físico-químicas e análise sensorial, para verificar a existência de preferência. Foi utilizado o delineamento de blocos completos com uma variável em três níveis e três repetições. Os resultados obtidos mostraram que a concentração de inóculo não influenciou de forma significativa (p > 0,05) os valores de pH, índices de extensão e de profundidade de proteólise. Ao longo dos 28 dias de estocagem, o decréscimo da população de L. acidophilus não foi significativo (p > 0,05) para os três queijos avaliados. A análise sensorial mostrou a existência de diferença e preferência não significativas (p > 0,05) entre os queijos com diferentes populações de L. acidophilus. Concluiu-se que o queijo minas frescal produzido a partir de retentados de ultrafiltração apresenta grande potencial como alimento probiótico.

Lactobacillus plantarum strains isolated from naturally fermented sausages and their technological properties for application as starter cultures

In the present study, technological properties of L. plantarum strains isolated from naturally fermented sausages manufactured in the South region of Brazil were investigated in order to obtain a starter culture. The technological properties evaluated were the following: ability to growth at different pH values, at different temperatures, in different salt concentrations and in the presence of commercial curing salt, fast production of acid, determination of D - and L - lactic acid; nitrate reductase activity; antagonistic activity and stability of the isolated cultures after fermentation, concentration, and freeze-drying process. The isolated strains showed effectiveness to improve technological properties as starter cultures.

Development of effervescent products, in powder and tablet form, supplemented with probiotics Lactobacillus acidophilus and Saccharomyces boulardii

Probiotics are living microorganisms, which when administered in adequate amounts confer health benefits on the host through a beneficial influence on the intestinal microbiota related to competition and to antagonistic and immunological effects. Thus, the objective of this study was the development of effervescent products (tablets and powder) supplemented with the probiotics Lactobacillus acidophilus and Saccharomyces boulardii, and the identification of the best formulation in terms of viability of these microorganisms. The physical properties of the tablets (compressive force applied, mean weight, hardness, and friability) were assessed, and the viability of the microorganisms in the gastrointestinal tract and their stability during storage were also determined. The results show that the microorganisms remained stable and viable during the 60-day storage period in the effervescent powder. However, the results indicated that the effect of compression force affected the viability of the microorganisms during the production of the effervescent tablets.