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The use of acid alumina and sephadex LH-20 for the separation and characterization of ethanol-soluble peptides produced by Bacillus brevis.

ABSTRACT: Cell extracts from Bacillus brevis (A.T.C.C. 10068), grown with various media, incorporated certain (14)C-labelled amino acids that are normally components of tyrothricin into material that was extracted by ethanol from the precipitate formed by adding acid. When this material was separated by paper and silica-gel thin-layer chromatography and paper electrophoresis (14)C was located in those regions that also contained gramicidin and tyrocidine. From a study of the properties of the system responsible for the incorporation it was deduced that non-tyrothricin materials were present. It was shown that the methods normally used to characterize tyrothricin do not adequately distinguish between tyrothricin and non-tyrothricin materials. However, a method for separating these materials was devised. This involved elution with ethanol from columns of acid alumina followed by gel filtration on Sephadex LH-20 with dimethylformamide-water solvent. The behaviour of gramicidin and tyrocidine on the Sephadex LH-20 column was examined, and it was concluded that the separation was not caused simply by gel filtration of unassociated molecules. Also, tyrocidine molecules with different amino acid compositions seemed to have different affinities for the Sephadex LH-20 column.

SUBMITTER: Bartley IM

PROVIDER: S-EPMC1178689 | biostudies-other | 1972 Apr

REPOSITORIES: biostudies-other

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Project description:In this study, Karenia brevis 165 (K. brevis 165), a Chinese strain, was used to research brevetoxin (BTX) metabolites. The sample pretreatment method for the enrichment of BTX metabolites in an algal culture medium was improved here. The method for screening and identifying intracellular and extracellular BTX metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The results show that the recovery rates for BTX toxins enriched by a hydrophilic-lipophilic balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTX metabolites at different growth stages of K. brevis 165. This is the first time a Chinese strain of K. brevis has been reported that can produce toxic BTX metabolites. Five and eight kinds of BTX toxin metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites. The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages show that the total toxic BTX metabolite contents in single cells ranged between 6.78 and 21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27 and 449.11 μg/L. There were significant differences in the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

Project description:UnlabelledDental caries is still a major oral health problem in most industrialized countries. The development of dental caries primarily involves Lactobacilli spp. and Streptococcus mutans. Although antibacterial ingredients are used against oral bacteria to reduce dental caries, some reports that show partial antibacterial ingredients could result in side effects.ObjectivesThe main objective is to test the antibacterial effect of water-soluble chitosan while the evaluation of the mouthwash appears as a secondary aim.Material and methodsThe chitosan was obtained from the Application Chemistry Company (Taiwan). The authors investigated the antibacterial effects of water-soluble chitosan against oral bacteria at different temperatures (25-37 ºC) and pH values (pH 5-8), and evaluated the antibacterial activities of a self-made water-soluble chitosan-containing mouthwash by in vitro and in vivo experiments, and analyzed the acute toxicity of the mouthwashes. The acute toxicity was analyzed with the pollen tube growth (PTG) test. The growth inhibition values against the logarithmic scale of the test concentrations produced a concentration-response curve. The IC50 value was calculated by interpolation from the data.ResultsThe effect of the pH variation (5-8) on the antibacterial activity of water-soluble chitosan against tested oral bacteria was not significant. The maximal antibacterial activity of water-soluble chitosan occurred at 37 ºC. The minimum bactericidal concentration (MBC) of water-soluble chitosan on Streptococcus mutans and Lactobacilli brevis were 400 µg/mL and 500 µg/mL, respectively. Only 5 s of contact between water-soluble chitosan and oral bacteria attained at least 99.60% antibacterial activity at a concentration of 500 µg/mL. The water-soluble chitosan-containing mouthwash significantly demonstrated antibacterial activity that was similar to that of commercial mouthwashes (>99.91%) in both in vitro and in vivo experiments. In addition, the alcohol-free mouthwash exhibited no cytotoxicity and no oral stinging. To the best of our knowledge, this was the first study to combine in vitro and in vivo investigations to analyze the antibacterial properties of water-soluble chitosan-containing mouthwash.ConclusionsThis study illustrated that water-soluble chitosan may be a viable alternative to commercial mouthwashes in the future.

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The use of acid alumina and sephadex LH-20 for the separation and characterization of ethanol-soluble peptides produced by Bacillus brevis.

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