RESUMO
Probiotic production in commercial culture media is expensive, so, it is necessary to design culture media based on "low-cost" components like agro-industrial by-products. Therefore, this study aimed to design an agro-industrial by-product-based culture media using whey, sugarcane molasses, and palm kernel cake as components to produce Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10 isolated from Nile tilapia (Oreochromis niloticus) associated gut microbiota. Higher bacterial concentrations were achieved at high whey concentrations and low concentrations of sugarcane molasses and palm kernel cake (PKC) using agitation. The optimal conditions were whey, 3.84% w/v; sugarcane molasses, 7.39% w/v; PKC, 0.77% w/v; and agitation speed, 75 RPM. Bacterial growth under optimal conditions was compared to that in commercial Brain-Heart Infusion (BHI) broth. L. lactis A12 showed similar growth in the optimal media and BHI. The estimated cost of the culture media based on component prices was USD $ 3.01/L, which is 86.93% lower than BHI broth (USD $ 23.04/L). It was possible to design a "low-cost agro-industrial by-product-based culture media to produce L. lactis A12 and the two Priestia species under monoculture conditions.
Assuntos
Meios de Cultura , Probióticos , Probióticos/metabolismo , Animais , Meios de Cultura/química , Lactococcus lactis/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Soro do Leite/microbiologia , Soro do Leite/metabolismo , Ciclídeos/microbiologia , Ciclídeos/metabolismo , Ciclídeos/crescimento & desenvolvimento , Microbioma Gastrointestinal , Melaço , Ração Animal , SaccharumRESUMO
There are different studies that aim to enhance the production of nisin by Lactococcus lactis since its chemical synthesis is not possible. In this study, glutathione (GSH) and pyruvate, which are known to reduce the oxidative stress of cells, have been shown to trigger the production of nisin at both transcriptional and translational levels in L. lactis cells grown under aerobic condition. Presence of GSH and pyruvate caused more nisin yield than the heme-supplemented medium. Moreover, the expression of genes that encode stress-related enzymes were apparently upregulated in the presence of GSH and pyruvate. It can be concluded that GSH and pyruvate contribute to the defense system of L. lactis cells and so that higher biomass was obtained which in turn enhance nisin production. Antioxidant effect of GSH and pyruvate was known; however, their stimulating effect on nisin production was shown for the first time in this study.
Assuntos
Antibacterianos/biossíntese , Glutationa/metabolismo , Heme/metabolismo , Lactococcus lactis/metabolismo , Nisina/biossíntese , Ácido Pirúvico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Meios de Cultura/análise , Meios de Cultura/metabolismo , Glutationa/análise , Heme/análise , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Ácido Pirúvico/análiseRESUMO
The production of a highly concentrated probiotic preparation of Lactococcus lactis CECT 539 was studied in both batch and realkalized fed-batch fermentations in diluted whey (DW) media (DW25, DW50, DW75, DW100, and DW125) supplemented with MRS broth nutrients (except glucose and Tween 80) at 25, 50, 75, 100, and 125% of their standard concentrations in the complex medium. The fed-batch culture using DW100 medium provided the highest concentrations of probiotic biomass (5.98 g/L) and nisin (258.47 BU/mL), which were obtained at lower production costs than those estimated for the fed-batch culture in DW medium. The batch and fed-batch cultures reduced the initial chemical oxygen demand (COD) of the media by 29.1-41.7% and 31.2-54.2%, respectively. Graphical abstract.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Nisina/biossíntese , Probióticos/química , Proteínas do Soro do Leite/metabolismo , Biomassa , Meios de Cultura/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Probióticos/metabolismoRESUMO
The use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2log in BHI and 1log in milk after 24h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods.
Assuntos
Proteínas de Bactérias/genética , Microbiologia de Alimentos/métodos , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/metabolismo , Listeria monocytogenes/genética , Leite/microbiologia , Nisina/metabolismo , Estresse Fisiológico , Animais , Proteínas de Bactérias/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Fatores de TempoRESUMO
Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20-25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence L-lactic acid + H2O2. This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.
Assuntos
Antibacterianos/biossíntese , Citrobacter freundii/efeitos dos fármacos , Lactococcus lactis/crescimento & desenvolvimento , Nisina/análogos & derivados , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/farmacologia , Cromatografia por Troca Iônica , Lactobacillus plantarum/efeitos dos fármacos , Lactococcus lactis/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Nisina/biossíntese , Nisina/farmacologia , Rana catesbeiana/microbiologia , TemperaturaRESUMO
In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.
Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citoplasma/microbiologia , Endocardite Bacteriana/microbiologia , Lactococcus lactis/genética , Miócitos Cardíacos/microbiologia , Animais , Colágeno/metabolismo , Vasos Coronários/citologia , Vasos Coronários/microbiologia , Modelos Animais de Doenças , Células Endoteliais/microbiologia , Humanos , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/patogenicidade , Lactococcus lactis/fisiologia , Laminina/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Nisina/genética , Coelhos , Streptococcus mutans/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The partial substitution of sodium chloride by potassium chloride (0%, 25%, and 50%) and addition of arginine (1% w/w) in probiotic Minas cheese was investigated. Microbiological (Lactococcus lactis and Lactobacillus acidophilus counts, and functionality of the prebiotics L. acidophilus), physicochemical (pH, proteolysis, organic acids, fatty acids, and volatile profiles), rheological (uniaxial compression) and sensory (hedonic test with 100 consumers) characterizations were carried out. The sodium reduction and addition of arginine did not constitute a hurdle to lactic and probiotic bacteria survival, with presented values of about 9 log CFU/g, ranging from 7.11 to 9.21 log CFU/g, respectively. In addition, lower pH values, higher proteolysis, and a decrease in toughness, elasticity and firmness were observed, as well as an increase in lactic, citric, and acetic acid contents. In contrast, no change was observed in the fatty acid profile. With respect to the sensory acceptance, the probiotic low-sodium Minas cheese presented scores above 6.00 (liked slightly) for the attributes flavor and overall acceptance. The addition of arginine can be a potential alternative for the development of probiotic dairy products with reduced sodium content.
Assuntos
Arginina/análise , Queijo/análise , Aditivos Alimentares/análise , Probióticos/análise , Queijo/microbiologia , Contagem de Colônia Microbiana , Aromatizantes/análise , Armazenamento de Alimentos , Humanos , Lactobacillus acidophilus/crescimento & desenvolvimento , Lactococcus lactis/crescimento & desenvolvimento , Sódio/análise , PaladarRESUMO
BACKGROUND: White mold-ripened cheeses were investigated with the objective of proposing a colorimetric method to monitor the surface growth of Penicillium candidum and to evaluate the influence of the mesophilic (homofermentative (QMO) and heterofermentative (QMLD)) and thermophilic (QT) starter cultures on the physicochemical composition and sensory description. RESULTS: The whiteness index was effective in proving the appearance of superficial mycelium and the stability of white mold growth. The lactic cultures showed significant influence on most of the physicochemical analyses. The cheese made with thermophilic lactic culture had a 1 day gain in the growth of mycelium on the surface; nevertheless, the appearance of this product was potentially not acceptable for consumers. The heterofermentative mesophilic cheese had a better appearance and texture profile. However, the homofermentative mesophilic cheese showed aspects of fresh cheese and was acceptable for a wide range of consumers. CONCLUSION: The whiteness index was efficient to monitor the surface growth of P. candidum. The highest proteolytic effect was found in the QMLD and QT cultures. However, the cheese elaborated with the QMLD culture showed the best sensory acceptance. © 2015 Society of Chemical Industry.
Assuntos
Queijo/análise , Inspeção de Alimentos , Qualidade dos Alimentos , Lactobacillus delbrueckii/crescimento & desenvolvimento , Lactococcus lactis/crescimento & desenvolvimento , Penicillium/crescimento & desenvolvimento , Streptococcus thermophilus/crescimento & desenvolvimento , Brasil , Queijo/microbiologia , Fenômenos Químicos , Fermentação , Preferências Alimentares , Humanos , Concentração de Íons de Hidrogênio , Lactobacillus delbrueckii/metabolismo , Lactococcus lactis/metabolismo , Fenômenos Mecânicos , Proteínas do Leite/análise , Proteínas do Leite/metabolismo , Penicillium/metabolismo , Análise de Componente Principal , Proteólise , Sensação , Streptococcus thermophilus/metabolismo , Propriedades de Superfície , Tirosina/análise , Tirosina/metabolismoRESUMO
Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption.
Assuntos
Aminas Biogênicas/análise , Queijo/microbiologia , Lactococcus lactis/crescimento & desenvolvimento , Microbiota , Nisina/biossíntese , Animais , Aminas Biogênicas/biossíntese , Brasil , Bovinos , Queijo/análise , Coagulase/metabolismo , Feminino , Microbiologia de Alimentos , CabrasRESUMO
Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen ß-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.
Assuntos
Células Dendríticas/imunologia , Portadores de Fármacos , Endocitose , Células Epiteliais/imunologia , Lactococcus lactis/fisiologia , Vacinas de DNA/genética , Vacinas de DNA/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/microbiologia , Células Epiteliais/microbiologia , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Camundongos Endogâmicos BALB CRESUMO
Aislar, purificar y conservar cepas de Streptococcus spp. y lactobacillus spp. de la cavidad bucal y enfrentarlas "in vitro" a bacterias lácticas. Materiales y métodos: se seleccionaron individuos con caries y se recolectaron muestras de saliva. Para recuperar Streptococcus spp. se empleó el medio Mitis Salivarius (Difco, Detroit, MI, Estados Unidos) y para Lactobacillus spp. se usó Rogosa (Blokar Diagnostics, Beauvais, Francia). Como cepas productoras de bacteriocinas se utilizaron 7 cepas de Lactococcus lactis subsp. lactis, 2 de Leuconostoc mesenteroides y 1 de Lactococcus lactis subsp. diacetylactis. La actividad antagónica de las bacterias lácticas al crecimiento in vitro de bacterias cariogénicas se determinó con el método de difusión en agar. Resultado: el desarrollo y la multiplicación de las cepas de Streptococcus spp. de origen bucal ensayadas se vieron afectados por la presencia de metabolitos generados por las cepas de Lactococcus lactis subsp. lactis. Conclusión: el crecimiento de las cepas de Streptococcus subsp. fue inhibido por efecto de L. lactis subsp. lactis...
Assuntos
Humanos , Masculino , Adolescente , Adulto , Feminino , Adulto Jovem , Antibiose/fisiologia , Cárie Dentária/microbiologia , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/crescimento & desenvolvimento , Saliva/microbiologia , Streptococcus/classificação , Argentina , Bacteriocinas/isolamento & purificação , Contagem de Colônia Microbiana , Meios de Cultura , Técnicas In VitroRESUMO
Aislar, purificar y conservar cepas de Streptococcus spp. y lactobacillus spp. de la cavidad bucal y enfrentarlas "in vitro" a bacterias lácticas. Materiales y métodos: se seleccionaron individuos con caries y se recolectaron muestras de saliva. Para recuperar Streptococcus spp. se empleó el medio Mitis Salivarius (Difco, Detroit, MI, Estados Unidos) y para Lactobacillus spp. se usó Rogosa (Blokar Diagnostics, Beauvais, Francia). Como cepas productoras de bacteriocinas se utilizaron 7 cepas de Lactococcus lactis subsp. lactis, 2 de Leuconostoc mesenteroides y 1 de Lactococcus lactis subsp. diacetylactis. La actividad antagónica de las bacterias lácticas al crecimiento in vitro de bacterias cariogénicas se determinó con el método de difusión en agar. Resultado: el desarrollo y la multiplicación de las cepas de Streptococcus spp. de origen bucal ensayadas se vieron afectados por la presencia de metabolitos generados por las cepas de Lactococcus lactis subsp. lactis. Conclusión: el crecimiento de las cepas de Streptococcus subsp. fue inhibido por efecto de L. lactis subsp. lactis...(AU)
Assuntos
Humanos , Masculino , Adolescente , Adulto , Feminino , Adulto Jovem , Cárie Dentária/microbiologia , Lactococcus lactis/isolamento & purificação , Antibiose/fisiologia , Streptococcus/classificação , Lactococcus lactis/crescimento & desenvolvimento , Saliva/microbiologia , Meios de Cultura , Contagem de Colônia Microbiana , Bacteriocinas/isolamento & purificação , Técnicas In Vitro , ArgentinaRESUMO
Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.
Assuntos
Animais , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactococcus lactis/metabolismo , Leite/microbiologia , Sequência de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Meios de Cultura/química , Detergentes , DNA Bacteriano/genética , Cabras , Concentração de Íons de Hidrogênio , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Estabilidade Proteica , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/metabolismo , TemperaturaRESUMO
Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.
Assuntos
Animais , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactococcus lactis/metabolismo , Leite/microbiologia , Sequência de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/química , Bacteriocinas/genética , Meios de Cultura/química , Detergentes , DNA Bacteriano/genética , Cabras , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactococcus lactis/metabolismo , Leite/microbiologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Meios de Cultura/química , DNA Bacteriano/genética , Detergentes , Cabras , Concentração de Íons de Hidrogênio , Lactococcus lactis/crescimento & desenvolvimento , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Estabilidade Proteica , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/metabolismo , TemperaturaRESUMO
The presented study aimed to verify the effect of different pH values, enzyme solutions and heat treatments on the antimicrobial activity of the bacteriocinogenic strain Lactococcus lactis subsp. lactis Lc08 and to test their antimicrobial activity against Listeria monocytogenes in reconstituted skim milk at refrigeration temperatures. This strain was previously described as a nisin Z producer and capable of inhibiting L. monocytogenes growth in in vitro tests. The antimicrobial activity of the bacteriocin cell-free supernatant of Lc08 was sensitive to enzyme treatments (except papain). The pH values and heating (65ºC for 30min, 75ºC for 15s) had no apparent effect on the antimicrobial activity of the bacteriocin produced by Lc08. Only treatment at autoclave conditions result in loss of their antimicrobial activity. Lc08 presented antimicrobial activity against L. monocytogenes in the milk system after 12h at 25ºC. No effect was found at 7ºC. The results show the application viability of the Lc08 in food systems as a biopreservative against L. monocytogenes.
O presente estudo teve como objetivo verificar o efeito de diferentes valores de pH, soluções enzimáticas e tratamentos térmicos na atividade antimicrobiana da cepa bacteriocinogênica Lactococcus lactis subsp. lactis Lc08 e testar sua atividade antagonista contra Listeria monocytogenes em leite desnatado reconstituído em diferentes temperaturas de estocagem. Essa cepa já foi descrita como produtora de nisina Z e capaz de inibir o desenvolvimento de L. monocytogenes em testes in vitro. A atividade antimicrobiana do sobrenadante de Lc08 contendo a bacteriocina produzida e livre de células foi sensível ao tratamento pelas enzimas testadas (exceto papaína). A aplicação de diferentes valores de pH e o tratamento térmico (65ºC por 30 min, 75ºC por 15s) não influenciaram na atividade antimicrobiana da bacteriocina produzida por Lc08. Apenas o tratamento em autoclave resultou em perda da sua capacidade em inibir o desenvolvimento de L. monocytogenes. A cepa Lc08 apresentou atividade antagonista contra L. monocytogenes em leite após período de estocagem de 12h a 25ºC. Não foi observado efeito a 7ºC. Os resultados mostram a viabilidade de aplicação da cultura Lc08 ou de sua bacteriocina em produtos lácteos como bioconservador contra L. monocytogenes.
Assuntos
Animais , Lactococcus lactis/crescimento & desenvolvimento , Leite/estatística & dados numéricos , Leite , Listeria monocytogenes/crescimento & desenvolvimento , Nisina , Produtos com Ação AntimicrobianaRESUMO
The presented study aimed to verify the effect of different pH values, enzyme solutions and heat treatments on the antimicrobial activity of the bacteriocinogenic strain Lactococcus lactis subsp. lactis Lc08 and to test their antimicrobial activity against Listeria monocytogenes in reconstituted skim milk at refrigeration temperatures. This strain was previously described as a nisin Z producer and capable of inhibiting L. monocytogenes growth in in vitro tests. The antimicrobial activity of the bacteriocin cell-free supernatant of Lc08 was sensitive to enzyme treatments (except papain). The pH values and heating (65ºC for 30min, 75ºC for 15s) had no apparent effect on the antimicrobial activity of the bacteriocin produced by Lc08. Only treatment at autoclave conditions result in loss of their antimicrobial activity. Lc08 presented antimicrobial activity against L. monocytogenes in the milk system after 12h at 25ºC. No effect was found at 7ºC. The results show the application viability of the Lc08 in food systems as a biopreservative against L. monocytogenes.(AU)
O presente estudo teve como objetivo verificar o efeito de diferentes valores de pH, soluções enzimáticas e tratamentos térmicos na atividade antimicrobiana da cepa bacteriocinogênica Lactococcus lactis subsp. lactis Lc08 e testar sua atividade antagonista contra Listeria monocytogenes em leite desnatado reconstituído em diferentes temperaturas de estocagem. Essa cepa já foi descrita como produtora de nisina Z e capaz de inibir o desenvolvimento de L. monocytogenes em testes in vitro. A atividade antimicrobiana do sobrenadante de Lc08 contendo a bacteriocina produzida e livre de células foi sensível ao tratamento pelas enzimas testadas (exceto papaína). A aplicação de diferentes valores de pH e o tratamento térmico (65ºC por 30 min, 75ºC por 15s) não influenciaram na atividade antimicrobiana da bacteriocina produzida por Lc08. Apenas o tratamento em autoclave resultou em perda da sua capacidade em inibir o desenvolvimento de L. monocytogenes. A cepa Lc08 apresentou atividade antagonista contra L. monocytogenes em leite após período de estocagem de 12h a 25ºC. Não foi observado efeito a 7ºC. Os resultados mostram a viabilidade de aplicação da cultura Lc08 ou de sua bacteriocina em produtos lácteos como bioconservador contra L. monocytogenes.(AU)
Assuntos
Animais , Listeria monocytogenes/crescimento & desenvolvimento , Lactococcus lactis/crescimento & desenvolvimento , Leite/estatística & dados numéricos , Leite , Nisina , Produtos com Ação AntimicrobianaRESUMO
After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15 degrees C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121 degrees C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4 degrees C and up to 6 months at -20 degrees C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.
Assuntos
Antibiose , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Intestinos/microbiologia , Lactococcus lactis/isolamento & purificação , Animais , Bacteriocinas/isolamento & purificação , Meios de Cultura , DNA Bacteriano/genética , Peróxido de Hidrogênio/análise , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/patogenicidade , Filogenia , RNA Ribossômico 16S/genética , TemperaturaRESUMO
Após a constatação da escassez de estudos realizados com vegetais crus na busca por novas estirpes de bactérias láticas (BAL) produtoras de bacteriocinas e diante do potencial tecnológico da aplicação destas cepas tanto como agentes de conservação em alimento, bem como cultura probiótica em alimentos funcionais, este estudo objetivou isolar e identificar cepas de bactérias láticas potencialmente bacteriocinogênicas de amostras de rúcula obtidas no comércio local de São Paulo, SP - Brasil, identificar e caracterizar as bacteriocinas produzidas pelos isolados e avaliar o potencial probiótico dos isolados testando sua sobrevivência no modelo dinâmico do trato gastrointestinal TNO gastro-Intestinal Model - TIM-1 disponível no TNO (The Netherlands Organization for Applied Scientific Research) divisão Quality of Life (Zeist, Holanda). A produção de bacteriocinas neste modelo também foi avaliada, comparando-se com L. sakei 2a, também produtora de bacteriocinas e ainda avaliou-se a interferência na viabilidade de E. faecium LMA1. A cepa Lactococcus lactis subsp. lactis MK02R de rúcula produziu uma bacteriocina sensível à enzimas proteolíticas, termoestável e não influenciada pelo pH, sendo capaz de inibir Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii e Listeria Monocytogenes de diferentes grupos sorológicos. Os ensaios genéticos utilizando primers Nisf e Nisr confirmaram que a bacteriocina MK02R é uma nisina, apresentando uma alteração dos aminoácidos no peptídeo líder em relação às nisinas A, Z, Q, F e U, porém com a estrutura do peptídeo maduro idêntica ao da nisina F. Estes resultados foram confirmados por espectrometria de massas de amostras purificadas por HPLC. L. lactis MK02R resistiu à passagem no modelo dinâmico TIM-1, apresentando uma alta capacidade de sobreviver nas condições simuladas do trato gastrointestinal humano. Entretanto, não foi capaz de causar a redução no número de E. faecium LMA1. Em contrapartida, L. sakei 2a, mesmo apresentando uma sobrevivência menor, foi capaz de causar uma redução de 70% na população de E. faecium LMA1 no ambiente simulado do TGI. Não foi detectada atividade residual da ação antimicrobiana das bacteriocinas produzidas por L. lactis MK02R ou L. sakei 2a após a passagem pelo modelo dinâmico TIM-1. Estes resultados evidenciam a possível aplicação de L. lactis MK02R como um agente de controle biológico na conservação de alimentos e também como uma cultura potencialmente probiótica
Given the scarcity of studies performed with raw vegetables addressing the search for new bacteriocinogenic strains of lactic acid bacteria (LAB) and considering the technological application of these strains as food preservatives and probiotic cultures in functional foods, this study was aimed at isolation and identification of bacteriocinogenic LAB strains from samples of rocket salad obtained in the local market of São Paulo, SP - Brazil, subsequent characterization of the bacteriocins produced by these LABs and evaluation of their probiotic potential by testing their survival in the dynamic gastrointestinal model TNO gastro- Intestinal-Model - TIM-1, available at the TNO (Netherlands Organization for Applied Scientific Research) Quality of Life division (Zeist, Netherlands). The studies in the TIM-1 model were also done with another bacteriocinogenic strain L. sakei 2a for comparison, evaluating their interference on the viability of E. faecium LMA1. The bacteriocin produced by strain Lactococcus lactis subsp. lactis MK02R isolated from rocket salad was sensitive to proteolytic enzymes, heat-stable and not influenced by the pH. The bacteriocin inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii the primers Nisf and Nisr indicated that the bacteriocin produced by the strain MK02R is a nisin, with a change in the amino acid sequence of the leader peptide when compared to nisin A, Z, Q, U and F, but with the structure of the mature peptide homologous to that of nisin F. These results were confirmed by mass spectrometry of purified samples obtained by HPLC. L. lactis MK02R withstood the test in the dynamic model TIM-1, presenting capability to survive in the simulated conditions of the human gastrointestinal tract. However, the strain was not able to cause a reduction in the number of E. faecium LMA1. On the other hand, L. sakei 2a, even presenting lower survival, was able to cause 70% reduction in the population of E. faecium LMA1 in the gut simulated environment. No residual antimicrobial activity of bacteriocin produced by L. lactis MK02R or L. sakei 2a was detected after the transit through the dynamic model TIM-1. These results demonstrate the possible application of L. lactis MK02R both as a biocontrol agent in food preservation and as a potentially probiotic culture
Assuntos
Bacteriocinas/análise , Cochlearia armoracia/farmacologia , Lactococcus lactis/crescimento & desenvolvimento , Plantas/efeitos adversos , Ácido Láctico , Probióticos/farmacologia , Trato Gastrointestinal , NisinaRESUMO
A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l(-1) h(-1) using a cell concentration of 15 g l(-1), 30( degrees )C, pH 5.5 and a dilution rate of 1.24 h(-1). Adding 10 g l(-1) YE and 2 g l(-1) polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.