Isolation, identification and prokaryotic expression of a bacteriocin-like substance from Bacillus licheniformis / 生物工程学报

The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.

Bacteriocin-like inhibitory substance in aquaculture: a classic method of protein precipitation for a new aplicability / Substância inibidora tipo bacteriocina na aquicultura: método clássico de precipitação de proteínas para novas aplicações

Techniques to decrease losses from bacterial diseases are always important to improve the fish production. The use of antagonistic substances (bacteriocins) has been proven to be a viable option. The aim of this study was to evaluate different methods of purification for bacteriocin like inhibitory substances (BLIS). For the purification process, we isolated and used two Gram-positive bacilli that produce antagonistic substances for pathogens in aquaculture. Tests for detection of interfering factors were also performed. After the confirmation that the antagonistic action was due the BLIS activity, we carried out the purification methods. The methods tested were: cell free supernatant, acid extraction and ammonium sulfate precipitation at two concentrations (20 and 50%). Salmonella Tiphy CFP/IAL1472 and Aeromonas hydrophila (isolated in a tilapia production environment) were used as indicators of the efficiency of extracts in controlling pathogenic potentials. Ammonium sulfate precipitation at 50% was the most appropriate for purifying the antagonistic substance for both indicators. The extracts of the two isolates remained active for 22 days at 25ºC. These are promising results regarding the water and fish health without the use of antibiotics, in this manner being a safer environmental practice.

Técnicas para diminuir as perdas causadas por doenças bacterianas são importantes para melhorar continuamente a produção de pescado. O uso de substâncias antagônicas (bacteriocinas) tem-se mostrado uma opção viável. O objetivo do trabalho foi avaliar diferentes métodos de purificação de bacteriocinas como substâncias inibidoras (BLIS). Dois bacilos Gram-positivos, produtores de substâncias antagonistas para agentes patogênicos da aquicultura, foram utilizados em processos de purificação. Depois de confirmada a ação antagônica pela atividade de BLIS, os métodos de purificação foram realizados. Os métodos testados foram: células livres de sobrenadante, extração ácida e precipitação por sulfato de amônia em duas concentrações (20 e 50%). Salmonella Typhi PCP/IAL1472 e Aeromonas hydrophila (isolada de um ambiente de tilapicultura) foram utilizadas como indicadores de eficiência dos extratos. O precipitado por sulfato de amônio a 50% foi o mais adequado para purificar a substância antagonista para ambos os isolados indicadores. Os extratos dos dois isolados permaneceram ativos por 22 dias em 25ºC. Estes resultados são promissores do ponto de vista da manutenção da sanidade da água e dos peixes, sem uso de antibióticos, constituindo uma prática ambientalmente mais segura.

Optimization of the yield of bacteriocin-like substance (BLIS) produced by Pediococcus pentosaceus and its application as food bioconservative / Otimização do rendimento de substância semelhante a bacteriocina (BLIS) produzido por Pediococcus pentosaceus e sua aplicação como bioconservante de alimentos

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative

Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos

Bacteriocin-like inhibitory substance (BLIS) activity of Streptococcus macedonicus MBF10-2 and its synergistic action in combination with antibiotics

Objective To characterize the bacteriocin-like inhibitory-substances (BLIS) activity of Streptococcus macedonicus MBF10-2 [named BLIS(MBF10-2)], a bacteriocinogenic strain isolated from an Indonesian tofu byproduct. Methods BLIS(MBF10-2) was obtained by culturing the bacterium, and standard deferred antagonism assays were used to demonstrate its activity. The antibacterial testing of fractions collected by filtration using 3–30 kDa cut-off membrane sizes were carried out by performing well diffusion method. Results The growth of Micrococcus luteus, Streptococcus pyogenes, Lactococcus lactis, Leuconostoc mesenteroides and Weissella confusa were inhibited by BLIS(MBF10-2). Interestingly, BLIS-containing fractions obtained from sequential application on ultrafiltration membranes indicated that this bacterium Streptococcus macedonicus MBF10-2 could produces at least two antimicrobial peptides activities, one of which is likely to be a lantibiotic peptide. Potential synergistic activity against certain Gram-positive (but not Gram-negative) species when partnered with antibiotics (ampicillin, tetracycline or kanamycin) were observed. Conclusion Combination of some BLIS(MBF10-2) active fractions with antibiotics (ampicillin, tetracycline or kanamycin) could demonstrate synergistic activities against certain Gram-positive species.

Bacteriocin-like inhibitory substance (BLIS) activity of Streptococcus macedonicus MBF10-2 and its synergistic action in combination with antibiotics

OBJECTIVE@#To characterize the bacteriocin-like inhibitory-substances (BLIS) activity of Streptococcus macedonicus MBF10-2 [named BLIS(MBF10-2)], a bacteriocinogenic strain isolated from an Indonesian tofu byproduct.@*METHODS@#BLIS(MBF10-2) was obtained by culturing the bacterium, and standard deferred antagonism assays were used to demonstrate its activity. The antibacterial testing of fractions collected by filtration using 3-30 kDa cut-off membrane sizes were carried out by performing well diffusion method.@*RESULTS@#The growth of Micrococcus luteus, Streptococcus pyogenes, Lactococcus lactis, Leuconostoc mesenteroides and Weissella confusa were inhibited by BLIS(MBF10-2). Interestingly, BLIS-containing fractions obtained from sequential application on ultrafiltration membranes indicated that this bacterium Streptococcus macedonicus MBF10-2 could produces at least two antimicrobial peptides activities, one of which is likely to be a lantibiotic peptide. Potential synergistic activity against certain Gram-positive (but not Gram-negative) species when partnered with antibiotics (ampicillin, tetracycline or kanamycin) were observed.@*CONCLUSION@#Combination of some BLIS(MBF10-2) active fractions with antibiotics (ampicillin, tetracycline or kanamycin) could demonstrate synergistic activities against certain Gram-positive species.

Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese

Lactic acid bacteria (LAB) were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21 percent showed antimicrobial, proteolytic and lipolytic activities. Based on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS). The BLS produced by both isolates showed antimicrobial activity against Listeria monocytogenes, whereas that produced by L. plantarum LCN 17 presented higher stability to different temperature, pH and enzyme treatments. These strains present potential for production of BLS, and for use as starter cultures.

Purification and Biological Characteristics of Bacteriocin-like Substance Produced by Lactobacillus buchneri KLDS1.0364 / 微生物学通报

The purpose of this study is to obtain effective and stable bacteriocin-like substance from lactic acid bacteria. Lactobacillus buchneri KLDS1.0364,which was isolated from fermented cream,a traditional dairy product in Inner Mongolia,could produce bacteriocin-like substance. The bacteriocin-like substance produced by KLDS1.0364 was partially purified and the characteristics were studied. The bacteriocin-like substance was purified by SP-Sepharose fast flow cation exchange chromatography. The molecular weight of the bacteriocin-like substance was 21.6kD,as determined by tricine-SDS-PAGE. The bacteriocin-like substance remained stable after 30 min at 121℃ and after 2 h of incubation during pH 2～10. Complete inactivation of antimicrobial activity was observed after treatment of the bacteriocin-like substance with several proteinases. Treatment with catalase and ?-amylase did not result in any changes of antimicrobial activity. The mode of action of the bacteriocin-like substance is bactericidal. It exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria,Gram-negative bacteria and fungi.