Synthesis of silver nanoparticles using Lactobacillus bulgaricus and assessment of their antibacterial potential / Síntese de nanopartículas de prata usando Lactobacillus bulgaricus e avaliação de seu potencial antibacteriano

Many pathogenic strains have acquired multidrug-resistant patterns in recent a year, which poses a major public health concern. The growing need for effective antimicrobial agents as novel therapies against multidrug-resistant pathogens has drawn scientist attention toward nanotechnology. Silver nanoparticles are considered capable of killing multidrug-resistant isolates due to their oligo-dynamic effect on microorganisms. In this research study NPs were synthesized using the gram-positive bacteria Lactobacillus bulgaricus and its activity against selected pathogenic strains. Lactobacillus bulgaricus pure cultures were isolated from raw milk and grown in "De Man, Rogasa, and Sharp" broth for synthesis of nanoparticles. Lactobacillus bulgaricus culture was centrifuged and Cellfree supernatant of it was employed with aqueous silvery ions and evaluated their antibacterial activities against bacterial strains i.e. Staphylococcus aureus, Staphylococcus epidermidis and Salmonella typhi using agar well diffusion assay. Antibiotic profiling against selected pathogenic strains were also conducted using disc diffusion method. The synthesis and characterization of silver nanoparticles were monitored primarily by the conversion of the pale-yellow color of the mixture into a dark-brown color and via ultraviolet-visible absorption spectroscopy and Scanning electron microscopy respectively. The result showed that that AgNPs with size (30.65-100 nm) obtained from Lactobacillus bulgaricus were found to exhibit antibacterial activities against selected bacterial strains. Taken together, these findings suggest that Lactobacillus bulgaricus has great potential for the production of AgNPs with antibacterial activities and highly effective in comparison to tested antibiotics.

Muitas cepas patogênicas adquiriram padrões multirresistentes nos últimos anos, o que representa um grande problema de saúde pública. A crescente necessidade de agentes antimicrobianos eficazes como novas terapias contra patógenos multirresistentes atraiu a atenção dos cientistas para a nanotecnologia. As nanopartículas de prata são consideradas capazes de matar isolados multirresistentes por causa de seu efeito oligodinâmico em microrganismos. Neste estudo de pesquisa, as NPs foram sintetizadas usando a bactéria Gram-positiva Lactobacillus bulgaricus e sua atividade contra cepas patogênicas selecionadas. Culturas puras de Lactobacillus bulgaricus foram isoladas do leite cru e cultivadas em caldo "De Man, Rogasa e Sharp" para síntese de nanopartículas. A cultura de Lactobacillus bulgaricus foi centrifugada, e o sobrenadante livre de células foi empregado com íons prateados aquosos, avaliando-se suas atividades antibacterianas contra cepas bacterianas, isto é, Staphylococcus aureus, Staphylococcus epidermidis e Salmonella typhi usando ensaio de difusão em poço de ágar. O perfil de antibióticos contra cepas patogênicas selecionadas também foi conduzido usando o método de difusão em disco. A síntese e a caracterização das nanopartículas de prata foram monitoradas principalmente pela conversão da cor amarelo-pálida da mistura em uma cor marrom-escura e por espectroscopia de absorção visível e ultravioleta e por microscopia eletrônica de varredura, respectivamente. O resultado mostrou que AgNPs com tamanho de 30,65-100 nm, obtidas de Lactobacillus bulgaricus, exibiram atividades antibacterianas contra cepas bacterianas selecionadas. Tomados em conjunto, esses achados sugerem que o Lactobacillus bulgaricus tem um grande potencial para a produção de AgNPs com atividades antibacterianas e altamente eficazes em comparação aos antibióticos testados.

Synthesis of silver nanoparticles using Lactobacillus bulgaricus and assessment of their antibacterial potential / Síntese de nanopartículas de prata usando Lactobacillus bulgaricus e avaliação de seu potencial antibacteriano

Many pathogenic strains have acquired multidrug-resistant patterns in recent a year, which poses a major public health concern. The growing need for effective antimicrobial agents as novel therapies against multidrug-resistant pathogens has drawn scientist attention toward nanotechnology. Silver nanoparticles are considered capable of killing multidrug-resistant isolates due to their oligo-dynamic effect on microorganisms. In this research study NPs were synthesized using the gram-positive bacteria Lactobacillus bulgaricus and its activity against selected pathogenic strains. Lactobacillus bulgaricus pure cultures were isolated from raw milk and grown in "De Man, Rogasa, and Sharp" broth for synthesis of nanoparticles. Lactobacillus bulgaricus culture was centrifuged and Cell free supernatant of it was employed with aqueous silvery ions and evaluated their antibacterial activities against bacterial strains i.e. Staphylococcus aureus, Staphylococcus epidermidis and Salmonella typhi using agar well diffusion assay. Antibiotic profiling against selected pathogenic strains were also conducted using disc diffusion method. The synthesis and characterization of silver nanoparticles were monitored primarily by the conversion of the pale-yellow color of the mixture into a dark-brown color and via ultraviolet-visible absorption spectroscopy and Scanning electron microscopy respectively. The result showed that that AgNPs with size (30.65-100 nm) obtained from Lactobacillus bulgaricus were found to exhibit antibacterial activities against selected bacterial strains. Taken together, these findings suggest that Lactobacillus bulgaricus has great potential for the production of AgNPs with antibacterial activities and highly effective in comparison to tested antibiotics.

Muitas cepas patogênicas adquiriram padrões multirresistentes nos últimos anos, o que representa um grande problema de saúde pública. A crescente necessidade de agentes antimicrobianos eficazes como novas terapias contra patógenos multirresistentes atraiu a atenção dos cientistas para a nanotecnologia. As nanopartículas de prata são consideradas capazes de matar isolados multirresistentes por causa de seu efeito oligodinâmico em microrganismos. Neste estudo de pesquisa, as NPs foram sintetizadas usando a bactéria Gram-positiva Lactobacillus bulgaricus e sua atividade contra cepas patogênicas selecionadas. Culturas puras de Lactobacillus bulgaricus foram isoladas do leite cru e cultivadas em caldo "De Man, Rogasa e Sharp" para síntese de nanopartículas. A cultura de Lactobacillus bulgaricus foi centrifugada, e o sobrenadante livre de células foi empregado com íons prateados aquosos, avaliando-se suas atividades antibacterianas contra cepas bacterianas, isto é, Staphylococcus aureus, Staphylococcus epidermidis e Salmonella typhi usando ensaio de difusão em poço de ágar. O perfil de antibióticos contra cepas patogênicas selecionadas também foi conduzido usando o método de difusão em disco. A síntese e a caracterização das nanopartículas de prata foram monitoradas principalmente pela conversão da cor amarelo-pálida da mistura em uma cor marrom-escura e por espectroscopia de absorção visível e ultravioleta e por microscopia eletrônica de varredura, respectivamente. O resultado mostrou que AgNPs com tamanho de 30,65-100 nm, obtidas de Lactobacillus bulgaricus, exibiram atividades antibacterianas contra cepas bacterianas selecionadas. Tomados em conjunto, esses achados sugerem que o Lactobacillus bulgaricus tem um grande potencial para a produção de AgNPs com atividades antibacterianas e altamente eficazes em comparação aos antibióticos testados.

Synthesis of silver nanoparticles using Lactobacillus bulgaricus and assessment of their antibacterial potential

Abstract Many pathogenic strains have acquired multidrug-resistant patterns in recent a year, which poses a major public health concern. The growing need for effective antimicrobial agents as novel therapies against multidrug-resistant pathogens has drawn scientist attention toward nanotechnology. Silver nanoparticles are considered capable of killing multidrug-resistant isolates due to their oligo-dynamic effect on microorganisms. In this research study NPs were synthesized using the gram-positive bacteria Lactobacillus bulgaricus and its activity against selected pathogenic strains. Lactobacillus bulgaricus pure cultures were isolated from raw milk and grown in De Man, Rogasa, and Sharp broth for synthesis of nanoparticles. Lactobacillus bulgaricus culture was centrifuged and Cell- free supernatant of it was employed with aqueous silvery ions and evaluated their antibacterial activities against bacterial strains i.e. Staphylococcus aureus, Staphylococcus epidermidis and Salmonella typhi using agar well diffusion assay. Antibiotic profiling against selected pathogenic strains were also conducted using disc diffusion method. The synthesis and characterization of silver nanoparticles were monitored primarily by the conversion of the pale-yellow color of the mixture into a dark-brown color and via ultraviolet-visible absorption spectroscopy and Scanning electron microscopy respectively. The result showed that that AgNPs with size (30.65-100 nm) obtained from Lactobacillus bulgaricus were found to exhibit antibacterial activities against selected bacterial strains. Taken together, these findings suggest that Lactobacillus bulgaricus has great potential for the production of AgNPs with antibacterial activities and highly effective in comparison to tested antibiotics.

Resumo Muitas cepas patogênicas adquiriram padrões multirresistentes nos últimos anos, o que representa um grande problema de saúde pública. A crescente necessidade de agentes antimicrobianos eficazes como novas terapias contra patógenos multirresistentes atraiu a atenção dos cientistas para a nanotecnologia. As nanopartículas de prata são consideradas capazes de matar isolados multirresistentes por causa de seu efeito oligodinâmico em microrganismos. Neste estudo de pesquisa, as NPs foram sintetizadas usando a bactéria Gram-positiva Lactobacillus bulgaricus e sua atividade contra cepas patogênicas selecionadas. Culturas puras de Lactobacillus bulgaricus foram isoladas do leite cru e cultivadas em caldo De Man, Rogasa e Sharp para síntese de nanopartículas. A cultura de Lactobacillus bulgaricus foi centrifugada, e o sobrenadante livre de células foi empregado com íons prateados aquosos, avaliando-se suas atividades antibacterianas contra cepas bacterianas, isto é, Staphylococcus aureus, Staphylococcus epidermidis e Salmonella typhi usando ensaio de difusão em poço de ágar. O perfil de antibióticos contra cepas patogênicas selecionadas também foi conduzido usando o método de difusão em disco. A síntese e a caracterização das nanopartículas de prata foram monitoradas principalmente pela conversão da cor amarelo-pálida da mistura em uma cor marrom-escura e por espectroscopia de absorção visível e ultravioleta e por microscopia eletrônica de varredura, respectivamente. O resultado mostrou que AgNPs com tamanho de 30,65-100 nm, obtidas de Lactobacillus bulgaricus, exibiram atividades antibacterianas contra cepas bacterianas selecionadas. Tomados em conjunto, esses achados sugerem que o Lactobacillus bulgaricus tem um grande potencial para a produção de AgNPs com atividades antibacterianas e altamente eficazes em comparação aos antibióticos testados.

Comparative study of Bacillus clausii and multistrain probiotics in the management of acute diarrhoea in children

Background: Diarrhoea is the second most common cause of under-five mortality especially in developing world. Many studies have been conducted so far using different probiotic strains with variable outcome. So, the aim of the present study was to compare the clinical efficacy of Bacillus clausii and multi strain probiotic formulation as adjunct treatment of acute diarrhoea.Methods: This prospective single blind randomized controlled clinical trial included 300 infants and children between 6 months to 6 years of age admitted in a tertiary care hospital Sylhet, Bangladesh with acute watery diarrhoea having varied dehydration status ranging from no to severe dehydration excluding shocked state. Cases were randomly assigned to three groups which were group I (n=100) comprised of children who were treated with standard treatment (according to WHO guideline) only as control group, group II (n=100) who received standard treatment plus Bacillus clausii and group III (n=100) who received standard treatment plus multi strain probiotic formulation (Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium breve, Bifidobacterium infantis, Streptococcus thermophilus). Primary outcome variables were duration, frequency of diarrhoea and consistency of stool. Secondary outcome variable was duration of hospital stay.Results: Mean duration of diarrhoea was significantly shorter (p=0.001) in group III (2.62 days) compared to group I (3.26 days) and group II (3.22 days). Frequency of diarrhoea was significantly lower on day 3 of probiotic administration in group III (p <0.05) and on day 5 of treatment in group II (p <0.05). Stool consistency significantly improved on day 3 in group III (p <0.05) while it was on day 4 in group II. The duration of diarrhoea, hospital stay, stool consistency and frequency of stool on day 3 were not statistically significant (p >0.05) in group II in comparison to group I and group III.Conclusions: Multistrain probiotic formulation is effective in reducing the duration, frequency of diarrhoea and duration of hospital stay.

Atividade antifúngica dos lacatobacillus presentes no iogurte sobre a candida albicans in vitro / Antifungal activity of lactobacillus present in the yogurt on candida albicans in vitro

Objetivo: Este artigo tem como objetivo avaliar a atividade antifúngica in vitro dos Lactobacillus bulgaricus e Streptococcus thermophilus que estão presentes no iogurte sobre o crescimento da Candida albicans. Método: Foi realizado o repique da Candida albicans, fornecido em um tubo, na placa de petri contendo o ágar sabouraud dextrose para formação de colônias e aumento da população de Candida albicans. As amostras foram diluídas em soro fisiológico e semeada na placa de petri e incubou-se por duas horas. Após as duas horas, realizou-se a semeadura do iogurte, contendo os Lactobacillus bulgaricus e Streptococcus thermophilus, sobre a placa de petri contendo a Candida albicans e incubou-se na estufa a 37º C por 48 horas. Resultado: Após o período de incubação, verificamos que houve a inibição do crescimento da Candida albicans, reduzindo a quantidade de colônias das placas onde foi semeado o iogurte. Conclusão: Os Lactobacillus presentes no iogurte foram eficazes na atividade antifúngica contra a Candida albicans in vitro. Atualmente, poucos estudos foram realizados in vitro, sendo necessário realizar novos experimentos isolando os Lactobacillus bulgaricus e Streptococcus thermophilus presentes no iogurte, para maior certeza da eficácia da atividade antifúngica de cada Lactobacillus.

Objective: this paper aims to assess the antifungal activity in vitro of Lactobacillus bulgaricus and Streptococcus thermophilus that exist in yogurts on the Candida albicans growing. Method: we ringed the Candida albicans, that was provided in a tube and in a Petri´s plate, containing agar Sabouraud dextrose to compose the colonies and increase the Candida albicans population. Samples were diluted in physiological solution and seeded in the Petri´s plate, being incubated for two hours. After this time, we put the yogurt- containing the Lactobacillus bulgaricus and the Streptococcus thermophilus on the Petri´s plate. It was incubated in the heating chamber under 37oC for 48 hours. Results: After the incubation period, we verified that Candida albicans growing was inhibited, reducing the number of colonies in plates where the yogurt was seeded in. Conclusion: the Lactobacillus existent in the yogurt has an effective antifungal activity in vitro. Nowadays, few investigations were conducted in vitro, being necessary another studies to ensure the antifungal activity effectiveness of Lactobacillus present in yogurts

NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying

Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.

Stability of free and immobilized Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of immobilized B. lactis in yoghurt

This study evaluated the stability of Bifidobacterium lactis (Bb-12) and of Lactobacillus acidophilus (La-05) both free and immobilized in calcium alginate, in milk and in acidified milk (pH 5.0, 4.4 and 3.8). The stability of immobilized B. lactis in yoghurt (fermented to pH 4.2), during 28 days of refrigerated storage was also evaluated. The efficiency of two culture media (modified MRS agar and Reinforced Clostridial Agar plus Prussian Blue) for counting of B. lactis in yoghurt was determined. Lee's agar was used to count Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus when B. lactis were counted in the MRS medium. B. lactis and L. acidophilus in both free and immobilized forms presented satisfactory rates of survival in milk and acidified milk because the average reduction of the population was only one log cycle after 21 days of storage. The number of viable cells of immobilized B. lactis in yoghurt presented a gradual decline throughout the storage period, passing from 10(8) cfu/ml to no count after 28 days of storage. When the cultures were not in equilibrium just the selective medium was efficient in counting B. lactis in yoghurt. The results showed that both microorganisms can be added to milk and acidified milk, because their population was only slightly affected during storage. The presence of traditional culture of yoghurt seems to be harmful for survival of immobilized B. lactis and the immobilization in calcium alginate failed as an effective barrier to protect the cells in all analysed treatments.

Este trabalho avaliou a estabilidade de Bifidobacterium lactis (Bb-12) e de Lactobacillus acidophilus (La-05) nas formas livre e imobilizada em alginato de cálcio, em leite e leite acidificado (pHs 5.0, 4.4 e 3.8), e a estabilidade de B. lactis imobilizado em iogurte (fermentado até pH 4.2), durante 28 dias de estocagem refrigerada. Também foi estudada a eficiência de dois meios de cultura (ágar MRS modificado e Reinforced Clostridial Agar, acrescido de Prussian Blue) para enumerar B. lactis em iogurte. Ágar Lee foi usado para enumeração de Streptococcus thermophilus e Lactobacillus delbrueckii ssp. bulgaricus quando B. lactis era enumerado no meio MRS. Ambos os microrganismos, nas formas livre e imobilizada, apresentaram uma taxa de sobrevivência adequada nos leites acidificados, uma vez que houve redução de apenas um ciclo log, após 21 dias de estocagem refrigerada. O número de células viáveis de B. lactis imobilizado mostrou um declínio gradual durante o período de armazenamento do iogurte, passando de 10(8) ufc/ml até não ter mais contagem na diluição 10-1. Quando as culturas não estavam em equilíbrio, o meio MRS modificado foi mais eficiente para a contagem de B. lactis em iogurte. Em vista destes resultados pode-se concluir que ambos os microrganismos podem ser incorporados em leite e leite acidificados, haja visto que a redução na população foi pequena durante o período de armazenagem estudado. A presença da cultura tradicional de iogurte parece ter afetado negativamente a sobrevivência de B. lactis e a imobilização não proveu proteção às células, em nenhum dos tratamentos estudados.

Immobilization of b-D-galactosidase from Lactobacillus bulgaricus on polyacrylamide in the presence of protective agents and properties of the immobilized enzyme.

b-D-Galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized in a Polyacrylamide gel lattice in the presence of dithiothreitol, glutathione, cysteine, bovine serum albumin, casein, lactose and glucono-δ-lactone. Cysteine, bovine serum albumin, and lactose were found very effective in preserving the activity. With cysteine, bovine serum albumin and lactose, the activity yields were 61, 60 and 66% respectively, as compared to 31% without protective agents. The yield improved upto 85% when all the three protective agents, cysteine, bovine serum albumin and lactose were added during immobilization. The addition of protective agents did not have any effect on optimum pH, optimum temperature, kinetic constants and pH stability when compared with β-galactosidase immobilized without the use of protective agents; however the heat and storage stabilities were found to increase.

Immobilization and properties of β-D-galactosidase from Lactobacillus bulgaricus.

β-D-galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized by entrapment in a Polyacrylamide gel lattice. The enzymatic properties of the immobilized β-galactosidase were compared with those of the native enzyme. The temperature and pH optima were not affected by the immobilization. After entrapment of the enzyme no significant change was observed in its thermostability. The pH stability of the immobilized enzyme was higher than that of the native enzyme on the acidic side. The Km values for the immobilized and native β-galactosidase with both lactose and o-nitrophenyl-β-D-galactoside as substrates were comparable. The immobilized enzyme could be repeatedly used 12 times without any loss of activity. No loss in the activity of the immobilized β-galactosidase was found after its storage for 30 days at 4°C and for 20 days at 25°C.