Purification, characterization, mode of action, and enhanced production of Salivaricin mmaye1, a novel bacteriocin from Lactobacillus salivarius SPW1 of human gut origin

Background: Emergence of antibiotic resistance among pathogenic and food spoilage bacteria such as Staphylococcus aureus, Micrococcus luteus, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mutans, Bacillus cereus, and Listeria monocytogenes triggered the search for alternative antimicrobials. An investigation aimed at purifying, characterizing, elucidating the mode of action, and enhancing the production of salivaricin from Lactobacillus salivarius of human gut origin was conducted. Results: Salivaricin mmaye1 is a novel bacteriocin purified from L. salivarius isolated from human feces. It is potent at micromolar concentrations and has a molecular weight of 1221.074 Da as determined by MALDI-TOF mass spectrometry. It has a broad spectrum of antibacterial activity. Salivaricin mmaye1 showed high thermal and chemical stability and moderate pH stability. The proteinaceous nature of salivaricin mmaye1 was revealed by the complete loss of activity after treatment with pepsin, trypsin, α-chymotrypsin, protease, and proteinase. Salivaricin mmaye1 is cell wall associated, and adsorption­desorption of the bacteriocin from the cell wall of the producer by pH modification proved successful. It exhibited a bactericidal mode of action mediated by pore formation. Its biosynthesis is regulated by a quorum sensing mechanism. Enhanced production of salivaricin mmaye1 was achieved in a newly developed growth medium. Conclusions: A novel, cell wall adhering, highly potent bacteriocin with a broad spectrum of inhibitory activity, membrane-permeabilizing ability, and enhanced production in a newly constituted medium has been isolated. It has a quorum sensing regulatory system and possesses interesting physicochemical characteristics favoring its future use in food biopreservation. These findings pave the way for future evaluation of its medical and food applications.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

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.

Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from 'Marcha'

In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Comparative antimicrobial activity of two new mutacins / Actividad microbiana comparativa de dos nuevas mutacinas

OBJECTIVES: To compare the in vitro activity of mutacins D-123.1 and F-59.1 against different bacteria including antibiotic-resistant strains, in order to evaluate their application potential. DESIGN AND METHODS: The antibacterial activity spectrum of purified F-59.1 and the MIC and MBC of F-59.1 and D-123.1 against target bacteria were determined. RESULTS: Most bacteria were inhibited by the purified mutacins. Mutacin F-59.1 shows a relatively wide activity spectrum. Mutacin D-123.1 has low Minimum Inhibitory Concentrations [MICs] (0.25-4 µ/ml) against human pathogens while F-59.1 has higher MICs (3.2-12.8 fig/ml) mainly against food-borne pathogens. CONCLUSION: The effectiveness of mutacins D-123.1 and F-59.1 against human and food-borne pathogens is demonstrated. Mutacin D-123.1 shows potential as a new antibiotic while F-59.1 shows promising application in food products. ABBREVIATIONS: MALDI-TOF MS, matrix assisted laser desorption ionisation-time of flight mass spectrometry; MB(I)C, minimum bactericidal (inhibitory) concentrations; OD, optical density; RP-HPLC, reverse-phase high-pressure liquid chromatography; TSBYE, trypticase soy broth yeast extract.

OBJETIVOS: Comparar la actividad in vitro de las mutacinas D-123.1 y F-59.1 frente a diferentes bacterias incluyendo las cepas resistentes a los antibióticos, a fin de evaluar el potencial de su aplicación. DISEÑO Y MÉTODOS: Se determinó el espectro de actividad antibacteriana de F-59.1 purificada y la CIM y la CBM de F-59.1 y D-123.1 frente a determinadas bacterias. RESULTADOS: La mayor parte de las bacterias eran inhibidas por las mutacinas purificadas. La mutacina F-59.1 muestra un espectro de actividad relativamente amplio. La mutacina D-123.1 posee bajas concentraciones de inhibición mínimas [CIM] (0.25-4 fig/ml) contra los patógenos humanos, mientras que el F-59.1 posee concentraciones CIM más altas (3.2-12.8 fig/ml) principalmente frente a los patógenos alimentarios. CONCLUSIÓN: Queda demostrada la efectividad de las mutacinas D-123.1 y F-59.1 frente a los patógenos humanos y alimentarios. La mutacina D-123.1 muestra poseer un potencial como nuevo antibiótico, en tanto que F-59.1 se presenta como una aplicación promisoria en relación con los productos alimentarios. ABREVIATURAS: MALDI-TOF MS, espectrometría de masas con desorción/ionización mediante láser asistida por matriz asociada a un analizador de tiempo de vuelo (del inglés: matrix assisted laser desorption ionisation-time de flight mass spectrometry). CIM: concentración inhibitoria mínima (inglés MIC). CBM: concentración bactericida mínima (inglés MBC). DO: densidad óptica (inglés OD); RP-HPLC: cromatografía líquida de alta resolución en fase revertida; TSBYE:caldo tripticasa soya- extracto de levadura.

Purification and characterization of bacteriocin produced by strain of Lactobacillus brevis MTCC 7539.

Bacteriocin, an antimicrobial agent having potential for food biopreservation was purified from Lactobacillus brevis (a safe food-grade bacteria isolated from Vari Kandal, a traditional fermented food of Himachal Pradesh by adopting a novel repeated washing method. Its purity was confirmed by SDS-PAGE and Native-PAGE. The relative molecular mass of bacteriocin was 93.74 kD, while specific activity and recovery were 35.52 folds and 17.13%, respectively. It showed high thermal stability and was active over wide range of pH and exhibited sensitivity to trypsin.

Bacteriocin production by indigenous marine catfish associated vibrio Spp

Fifty strains of genus Vibrio were isolated [identified] from healthy and diseased marine catflsh[es]. The isolates were screened for bacteriocin [vibriocin] production. About 32% isolates were found bacteriocin producers. The best producer was identified as Vibrio anguillarum AVP10. The maximum production of vibiiocin AVP10 was manifested at 29°C at pH 7, after 18-20h of incubation. Vibriocin activity was enhanced in the presence of citrate-phosphate buffer. The vibriocin AVP10 withstands autoclaving temperature and showed activity even after prolonged chloroform treatment. Proteolytic enzymes inhibited its activity, while lipolytic enzyme had no effect, It was found bioactive only against intrageneric bacterial strains. Mode of action of vibriocin AVP10 varies with the indicator [sensitive] culture used i.e. bactericidal effects was exerted against V. anguillarum AVS9 while bacteriostatic effect was shown against entero-toxigenic E. coil