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1.
Braz. j. biol ; 83: e250550, 2023. tab, graf
Article in English | MEDLINE, LILACS, VETINDEX | ID: biblio-1345536

ABSTRACT

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.


Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.


Subject(s)
Benzaldehydes/metabolism , Flavoring Agents/metabolism , Bacillus subtilis/metabolism , Industrial Microbiology , Pseudomonas fluorescens/metabolism , Enterococcus faecium/metabolism , Culture Media , Alcaligenes faecalis/metabolism , Fermentation
2.
Braz. j. biol ; 83: e244261, 2023. tab, graf
Article in English | MEDLINE, LILACS, VETINDEX | ID: biblio-1285633

ABSTRACT

Abstract Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.


Resumo As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.


Subject(s)
Lycopersicon esculentum , Verticillium , Prunus domestica , Plant Diseases/prevention & control , Ascomycota , Bacillus subtilis , Fusarium
3.
Braz. J. Pharm. Sci. (Online) ; 58: e18373, 2022. tab
Article in English | LILACS | ID: biblio-1364428

ABSTRACT

Abstract The aim of this study was to determine antimicrobial activities of Alchemilla mollis, Alchemilla persica as well as ellagic acid and miquelianin against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans by using microbroth dilution method and anti-inflammatory activity by using human red blood cell (HRBC) membrane stabilization method. Microbroth dilution method was used to determine the antimicrobial activities. Extracts possessed activity having MIC values of 2.5-5-10mg/ mL, compounds possessed activity having MIC values of 1.25-2.5-4-5mg/mL. A.mollis aerial parts displayed the highest anti-inflammatory activity (IC50=1.22±0.07mg/mL). Ellagic acid and miquelianin were also determined as anti-inflammatory agents with 0.57±0.01mg/mL and 1.23±0.02mg/mL IC50 values, respectively. Total phenolic content and tannin content of the A.mollis and A.persica were determined as 357.00±75.80mg, 282.50±28.70mg PGE/g plant material and 18.02%, 18.63% respectively according to the method described by European Pharmacopoeia. Ellagic acid, miquelianin and catechin were analyzed by HPLC. The highest catechin content was detected in A.persica roots (6.69±0.05g/100g plant material). A.mollis aerial parts contain higher miquelianin (0.39±0.02g/plant material) and ellagic acid (1.56±0.01g/ plant material) than A.persica.


Subject(s)
Alchemilla/classification , Staphylococcus aureus , Bacillus subtilis , Candida albicans , Chromatography, High Pressure Liquid/methods , Dilution/methods , Ellagic Acid/pharmacology , Membranes , Anti-Inflammatory Agents
4.
Electron J Biotechnol ; 49: 56-63, Jan. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1291900

ABSTRACT

BACKGROUND: 2R,3R-butanediol dehydrogenase (R-BDH) and other BDHs contribute to metabolism of 3R/3S-Acetoin (3R/3S-AC) and 2,3-butanediol (2,3-BD), which are important bulk chemicals used in different industries. R-BDH is responsible for oxidizing the hydroxyl group at their (R) configuration. Bacillus species is a promising producer of 3R/3S-AC and 2,3-BD. In this study, R-bdh gene encoding R-BDH from Bacillus sp. DL01 was isolated, expressed and identified. RESULTS: R-BDH exerted reducing activities towards Diacetyl (DA) and 3R/3S-AC using NADH, and oxidizing activities towards 2R,3R-BD and Meso-BD using NAD+ , while no activity was detected with 2S,3S-BD. The R-BDH showed its activity at a wide range of temperature (25 C to 65 C) and pH (5.0­8.0). The R-BDH activity was increased significantly by Cd2+ when DA, 3R/3S-AC, and Meso-BD were used as substrates, while Fe2+ enhanced the activity remarkably at 2R,3R-BD oxidation. Kinetic parameters of the R-BDH from Bacillus sp. DL01 showed the lowest Km, the highest Vmax, and the highest Kcat towards the racemic 3R/3S-AC substrate, also displayed low Km towards 2R,3R-BD and Meso-BD when compared with other reported R-BDHs. CONCLUSIONS: The R-BDH from Bacillus sp. DL01 was characterized as a novel R-BDH with high enantioselectivity for R-configuration. It considered NAD+ and Zn2+ dependant enzyme, with a significant affinity towards 3R/3S-AC, 2R,3R-BD, and Meso-BD substrates. Thus, R-BDH is providing an approach to regulate the production of 3R/3S-AC or 2,3-BD from Bacillus sp. DL01.


Subject(s)
Bacillus subtilis/enzymology , Alcohol Oxidoreductases/isolation & purification , Alcohol Oxidoreductases/metabolism , Temperature , Kinetics , Hydrogen-Ion Concentration , Acetoin
5.
Chinese Journal of Biotechnology ; (12): 4303-4313, 2021.
Article in Chinese | WPRIM | ID: wpr-921507

ABSTRACT

D-allulose-3-epimerase (DPEase) is the key enzyme for isomerization of D-fructose to D-allulose. In order to improve its thermal stability, short amphiphilic peptides (SAP) were fused to the N-terminal of DPEase. SDS-PAGE analysis showed that the heterologously expressed DPEase folded correctly in Bacillus subtilis, and the protein size was 33 kDa. After incubation at 40 °C for 48 h, the residual enzyme activity of SAP1-DSDPEase was 58%. To make the recombinant B. subtilis strain reusable, cells were immobilized with a composite carrier of sodium alginate (SA) and titanium dioxide (TiO2). The results showed that 2% SA, 2% CaCl2, 0.03% glutaraldehyde solution and a ratio of TiO2 to SA of 1:4 were optimal for immobilization. Under these conditions, up to 82% of the activity of immobilized cells could be retained. Compared with free cells, the optimal reaction temperature of immobilized cells remained unchanged at 80 °C but the thermal stability improved. After 10 consecutive cycles, the mechanical strength remained unchanged, while 58% of the enzyme activity could be retained, with a conversion rate of 28.8% achieved. This study demonstrated a simple approach for using SAPs to improve the thermal stability of recombinant enzymes. Moreover, addition of TiO2 into SA during immobilization was demonstrated to increase the mechanical strength and reduce cell leakage.


Subject(s)
Bacillus subtilis/metabolism , Carbohydrate Epimerases/genetics , Enzyme Stability , Enzymes, Immobilized/metabolism , Fructose , Hydrogen-Ion Concentration , Racemases and Epimerases , Temperature
6.
Chinese Journal of Biotechnology ; (12): 3242-3252, 2021.
Article in Chinese | WPRIM | ID: wpr-921421

ABSTRACT

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.


Subject(s)
Asparaginase/genetics , Bacillus subtilis/genetics , Industrial Microbiology , Protein Engineering , Rhizomucor/enzymology , Sequence Alignment
7.
Chinese Journal of Biotechnology ; (12): 1619-1636, 2021.
Article in Chinese | WPRIM | ID: wpr-878658

ABSTRACT

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Editing , Metabolic Engineering , Promoter Regions, Genetic
8.
Chinese Journal of Biotechnology ; (12): 923-938, 2021.
Article in Chinese | WPRIM | ID: wpr-878604

ABSTRACT

Bacillus subtilis is a model strain for studying the physiological and biochemical mechanisms of microorganism, and is also a good chassis cell for industrial application to produce biological agents such as small molecule compounds, bulk chemicals, industrial enzymes, precursors of drugs and health product. In recent years, studies on metabolic engineering methods and strategies of B. subtilis have been increasingly reported, providing good tools and theoretical references for using it as chassis cells to produce biological agents. This review provides information on systematically optimizing the Bacillus subtilis chassis cell by regulating global regulatory factors, simplifying and optimizing the genome, multi-site and multi-dimensional regulating, dynamic regulating through biosensors, membrane protein engineering. For producing the protein reagent, the strain is optimized by optimizing the promoters, signal peptides, secretion components and building the expression system without chemical inducers. In addition, this review also prospects the important issues and directions that need to be focused on in the further optimization of B. subtilis in industrial production.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Biotechnology , Metabolic Engineering , Promoter Regions, Genetic , Protein Sorting Signals/genetics
9.
Chinese Journal of Biotechnology ; (12): 625-634, 2021.
Article in Chinese | WPRIM | ID: wpr-878587

ABSTRACT

Microcystis aeruginosa, a type of algal bloom microalgae, is widely distributed in water, causing serious deteriorated effects on humans and the ecological environment. As a biocontrol microorganism, Bacillus subtilis can synthesize various bioactive substances through non-ribosomal peptide synthetase, to inhibit the growth of M. aeruginosa. Thus, it is imperative to investigate the non-ribosomal peptide (NRP) metabolites of B. subtilis fmb60. Three NRP metabolites from B. subtilis fmb60 including bacillibactin, surfactin and fengycin were extracted and identified by genome mining technology. The growth inhibition of M. aeruginosa was studied by adding various concentrations of NRP metabolites. The half-effect concentration value (EC50.4 d) of M. aeruginosa was 26.5 mg/L after incubation for 4 days. With the increasing concentration, the inhibitory effects of NRP metabolites of B. subtilis fmb60 on M. aeruginosa was enhanced significantly. Compared with the control group, with the addition of 50 mg/L NRP metabolites to the M. aeruginosa, the content of Fv/Fm, Fv/Fo and Yield parameter after cultured for 4 days were decreased by 2.8%, 1.7% and 2.0%, respectively. Those findings indicate that the NRP metabolites of B. subtilis fmb60 can significantly inhibit the photosynthesis and metabolism of M. aeruginosa, which provides a theoretical foundation for the development of biological algae inhibitor of B. subtilis.


Subject(s)
Bacillus subtilis , Humans , Microcystis , Peptides , Photosynthesis
10.
Chinese Journal of Biotechnology ; (12): 2825-2835, 2021.
Article in Chinese | WPRIM | ID: wpr-887845

ABSTRACT

Biosynthesis of nanomaterials has attracted much attention for its excellent characteristics such as low energy consumption, high safety, and environmental friendliness. As we all know, the toxic selenite can be transformed into higher-value nanomaterials by using bacteria. In this study, nano-selenium was synthesized by halophilic Bacillus subtilis subspecies stercoris strain XP in LB medium supplemented with selenite (electron acceptor). The physicochemical characteristics of nano-selenium were analyzed by scanning electron microscope (SEM), X-ray energy dispersive spectral analysis (EDAX), X-ray diffraction (XRD), and fourier transform infrared spectroscopy (FTIR). Meanwhile, the antifungal activity of nano-selenium to strawberry pathogens (fusarium wilt, erythema, and purple spot fungi) was determined. The products from reduction of selenite by strain XP was amorphous spherical selenium nanoparticles (SeNPs) with a diameter range of 135-165 nm. The production of SeNPs was positively correlated with time (0-48 h) and no changes were observed on cell morphology. Selenium was dominant in the surface of SeNPs where the organic elements (C, O, N, and S) existed at the same time. SeNPs were coated with biomolecules containing functional groups (such as -OH, C=O, N-H, and C-H) which were associated with the stability and bioactivity of particles. Although the highest concentration of SeNPs had significant (P<0.05) inhibitory effects on three strains of strawberry pathogens, antifungal activity to erythema and fusarium wilt pathogenic fungi was higher than that to purple spot pathogenic fungi from strawberry. In conclusion, strain XP not only has strong tolerance to high salt stress, but can be also used to synthesize biological SeNPs with good stability and biological activity. Thus, the strain XP has bright perspectives and great potential advantage in pathogens control and green selenium-rich strawberry planting as well as other fields.


Subject(s)
Bacillus subtilis , Fragaria , Nanoparticles , Selenious Acid , Selenium
11.
Braz. arch. biol. technol ; 64: e21190749, 2021. tab, graf
Article in English | LILACS | ID: biblio-1278444

ABSTRACT

Abstract Bacteriocin has been identified as an excellent alternative to chemical preservatives due to its astonishing antimicrobial activity against food spoiling and food-borne pathogens. So there is a need to identify the newer and potent sources of bacteriocin producers. This study aims the isolation of potent bacteriocin producing microorganism from fresh fruits and vegetables, its production, purification, and characterization. Firstly, 43 isolates were analysed for its antimicrobial potential, out of which7 were found to inhibit the growth of various pathogens. Considering the results of antimicrobial activity; the microorganism isolated from mango was regarded as the most potent one; which was identified as Bacillus subtilis VS.70% ammonium sulphate precipitated and dialysed bacteriocin was purified using DEAE cellulose and sephadex G75 chromatography. Bacteriocin was purified by 24.64 fold with 8.65% recovery and its molecular weight was found to be 31.2kDa. The Purified bacteriocin was found to be stable at broad pH and temperature. It was found to be degraded by various proteases studied confirming its proteinaceous nature. Considering all these attributes; the purified bacteriocin isolated from Bacillus subtilis VS can be exploited by various food industries.


Subject(s)
Peptide Hydrolases/analysis , Bacteriocins/analysis , Anti-Infective Agents/analysis , Bacillus subtilis , Chromatography
12.
Braz. J. Pharm. Sci. (Online) ; 57: e18899, 2021. tab, graf
Article in English | LILACS | ID: biblio-1339302

ABSTRACT

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied


Subject(s)
Spectrum Analysis/instrumentation , Pharmaceutical Preparations/analysis , Discriminant Analysis , Spectroscopy, Fourier Transform Infrared/methods , Fourier Analysis , Pseudomonas aeruginosa/classification , Bacillus subtilis/classification , Candida albicans/classification , Quality Management , Limit of Detection
13.
Rev. Ciênc. Méd. Biol. (Impr.) ; 19(2): 258-264, set 24, 2020. tab
Article in English | LILACS | ID: biblio-1358136

ABSTRACT

Introduction: bacterial infections are a public health problem. Besides, the emergence of strains resistant to antimicrobials has contributed to the search for new alternatives, such for the terpenes with antimicrobial potential. Objectives: the objective of this study was to determine the possible interaction of isolated monoterpenes (-)-Carveol, Geraniol, Citronellol, α-terpineol, R-(-) Carvone, (-)-Menthol, Linalool, D-Dihydrocarvone, and (-)-Terpine-4-ol with conventional antimicrobials (Chloramphenicol, Minocycline, Amoxicillin and Ciprofloxacin) when they are evaluated on Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa strains. Methodology: the minimum inhibitory concentrations of these test drugs were determined using the microdilution method. The Checkerboard method was used to assess the interactions, by determining the fractional inhibitory concentration index (FIC index). Results: aamong the monoterpenes, only Carveol, Citronellol, and Geraniol presented antimicrobial activity (MIC < 1024 µg/mL). They presented synergistic effects against Pseudomonas aeruginosa ATCC-9027 (FIC index ≤ 0.5) when in combination with Minocycline. Conclusion: this study contributes to the development of new approaches to control bacterial resistance and to the possibility of discovering new drugs.


Introdução: as infecções bacterianas são um problema de saúde pública. Além disso, o surgimento de cepas resistentes aos antimicrobianos tem contribuído para a busca de novas alternativas, como a pesquisa de terpenos com potencial antimicrobiano. Objetivos: o objetivo deste estudo foi determinar a possível interação de monoterpenos isolados (-) - Carveol, Geraniol, Citronelol, α-terpineol, R - (-) Carvona, (-)-Mentol, Linalol, D-Diidrocarvona e (-)-Terpina-4-ol com antimicrobianos convencionais (cloranfenicol, minociclina, amoxicilina e ciprofloxacina) quando avaliados em Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli e Pseudomonas aeruginosa. Metodologia: as concentrações inibitórias mínimas destas drogas foram determinadas usando o método de microdiluição. O método checkerboard foi utilizado para avaliar as interações, determinando o índice de concentração inibitória fracionária (índice FIC). Resultados: entre os monoterpenos, apenas Carveol, Citronelol e Geraniol apresentaram atividade antimicrobiana (CIM < 1024 µg/mL). Eles apresentaram efeitos sinérgicos contra Pseudomonas aeruginosa ATCC-9027 (índice FIC ≤ 0,5) quando em combinação com Minociclina. Conclusão: este estudo contribui para o desenvolvimento de novas abordagens para o controle da resistência bacteriana e para a possibilidade de descoberta de novas drogas.


Subject(s)
Pseudomonas aeruginosa , Staphylococcus aureus , Staphylococcus epidermidis , Bacillus subtilis , Complementary Therapies , Monoterpenes , Escherichia coli , Gram-Negative Bacteria , Gram-Positive Bacteria , Anti-Infective Agents
14.
Biosci. j. (Online) ; 36(2): 606-618, 01-03-2020. tab, ilus
Article in English | LILACS | ID: biblio-1146428

ABSTRACT

The radish (Raphanus sativus L.) is a vegetable of the Brassicaceae family cultivated worldwide and has several medicinal properties. Its biological activities are related to various secondary metabolites present in the species, especially phenolics. Thus, the objectives of this study were the chemical analysis and evaluation of the antioxidant and antimicrobial activities of the dry extract and fractions of the fodder turnip leaves (R. sativus var. oleiferus Metzg.). Samples were analyzed by mass spectrometry and the antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method and the reducing power method. Antimicrobial activity was determined by the agar diffusion and microdilution methods. The total phenols were concentrated in the butanol fraction (121.27 mg GAE/g) and the flavonoids were concentrated in the ethyl acetate fraction (98.02 mg EQ/g). The ethyl acetate fraction showed the best antioxidants results, with 83.45% of free radical scavenging and 11.34% of ferric ions reduction. The analysis of antimicrobial activity showed that the dry extract had the highest average zone of inhibition against Bacillus subtilis (18.67 mm). Smaller values of the minimum inhibitory concentration for Micrococcus luteus were, and the ethyl acetate fraction showed a lower minimum inhibitory concentration (0.1 mg/ml) for that microorganism. There was a strong correlation between the antioxidant activity and the content of phenols and flavonoids. The results showed the potential antioxidant and antimicrobial activities of this extract with the ethyl acetate fraction being most promising for further studies.


O rabanete(Raphanus sativus L.) é um vegetal da família Brassicaceae cultivado em todo o mundo e possui diversas propriedades medicinais. Suas atividades biológicas estão relacionadas aos vários metabólitos secundários presentes na espécie, especialmente os compostos fenólicos. Desta forma, os objetivos deste estudo foram realizar análises químicas e avaliar as atividades antioxidante e antimicrobiana do extrato seco e das frações das folhas de R. sativus var. oleiferus Metzg. As amostras foram analisadas em espectrômetro de massas e o potencial antioxidante foi avaliado pelos métodos do radical DPPH (2,2-difenil-1-picrilhidrazila) e do poder redutor. A atividade antimicrobiana foi determinada pelos métodos de difusão em ágar e da microdiluição. Observou-se que os fenóis totais se concentraram na fração butanólica (121,27 mg EAG/g), enquanto que e os teores de flavonoides concentraram-se na fração acetato de etila (98,02 mg EQ/g). A fração acetato de etila apresentou os melhores resultados antioxidantes, com porcentagem de sequestro dos radicais DPPH de 83,45% e com porcentagem de redução dos íons férrico de 11,34%. A análise da atividade antimicrobiana revelou que o extrato seco teve maior média de halos de inibição frente ao Bacillus subtilis(18,67 mm). Os menores valores da concentração inibitória mínima foram para Micrococcus luteus, sendo que a fração acetato de etila demonstrou menor concentração inibitória mínima (0,1 mg/mL) para esse micro-organismo. Houve uma forte correlação entre a atividade antioxidante e o teor de fenóis e de flavonoides. Os resultados demonstraram potenciais ações antioxidante e antimicrobiana do extrato e das frações avaliados, sendo a fração acetato de etila promissora para estudos posteriores.


Subject(s)
Raphanus , Anti-Infective Agents , Antioxidants , Plants, Medicinal , Bacillus subtilis , Micrococcus luteus , Brassicaceae , Phenolic Compounds , Chemical Phenomena
15.
Braz. arch. biol. technol ; 63(spe): e20190493, 2020. tab, graf
Article in English | LILACS | ID: biblio-1142505

ABSTRACT

Abstract We evaluated the effect of combined Rhizobium tropici, Trichoderma asperellum and plant growth-promoting rhizobacteria (PGPR) in beans crop. The hypothesis that strains of T. asperullum, R. tropici and PGPR combined could improve growth, biomass accumulation and beans yield was tested under greenhouse and field conditions. The treatments consisted of control, mineral nitrogen application and inoculation, isolated and associated with the following microorganisms: Rhizobium tropici, Bacillus subtilis, Trichoderma asperellum and Burkholderia sp. 10N6. Results were evaluated by shoot dry weight (SDW) and root dry weight (RDW), number of nodules and yield components. In greenhouse environment all the microorganisms behaved similarly, and the treatments inoculated with Burkholderia sp. 10N6 (IBu) and R. tropici (IR) stood out regarding the production components. In field conditions the treatments IR and IRTBa presented the highest values of SDW and RDW. Our results suggest that inoculation with R. tropici, T. asperellum and PGPR may promote beans growth and bring benefits to shoot and root accumulation, increase the number of nodules as well as improve yield components, contributing to a sustainable agriculture.


Subject(s)
Phaseolus/growth & development , Phaseolus/microbiology , Bacillus subtilis , Trichoderma , Burkholderia , Rhizobium tropici , Plant Development
16.
Braz. arch. biol. technol ; 63: e20200061, 2020. graf
Article in English | LILACS | ID: biblio-1132157

ABSTRACT

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.


Subject(s)
Serratia marcescens/enzymology , Bacillus subtilis/enzymology , Chitinases/genetics , Hydrogen-Ion Concentration , Temperature , Gene Expression
17.
Chinese Journal of Biotechnology ; (12): 2644-2656, 2020.
Article in Chinese | WPRIM | ID: wpr-878518

ABSTRACT

Continuous planting of muskmelon and excessive application of chemical fertilizers have caused a series of problems, such as imbalance of the soil micro-ecological environment, serious soil-borne diseases and yield loss. Application of Bacillus subtilis agent is an important way to improve soil micro-ecological environment, prevent soil-borne diseases, and promote plant growth. In this study, B. subtilis was used as experimental agent to analyze the effects of different application methods on the soil microbial diversity and growth of muskmelon in greenhouse. The number of culturable microorganisms in soil was measured by dilution-plate method. The diversity of soil uncultivated microorganisms was determined by Illumina Miseq sequencing technology. The yield of muskmelon was measured by weighing method. The number of culturable bacteria in the root irrigation, hole application and dipping root application groups was higher than that of the control in different muskmelon growth stages, but there was no significant difference among the three different application methods. The number of soil fungi from B. subtilis agent treatment groups in flowering stage was significantly lower in comparison to the control group. However, B. subtilis agent treatment did not cause significant difference on soil fungi number at the fruiting and pulling stage. Diversity analysis of uncultured microorganisms showed that the Shannon index values of bacteria were higher and Simpson index values were lower respectively in the three B. subtilis treatment groups than that in the control. Moreover, the dipping root treatment produced the lowest Shannon index value and the highest Simpson index value of fungi. NMDS and cluster analysis showed that B. subtilis agents dipping root treatment significantly affected the bacterial and fungal flora, both of which were clustered into one independent branch. The application of B. subtilis agents, especially dipping root treatment, significantly decreased the abundance of Bacteroidetes, increased the abundance of Actinobacteria and Acidobacteria. The B. subtilis agent treatment didn't produce significant effect on the diversity of fungal flora except Chytridiomycota. The height, stem diameter and leaf area of muskmelon increased by applying B. subtilis agents, and dipping root treatment produced the most significant effect. As a new type of environmental protection fertilizer, B. subtilis agent can increase the number of soil culturable microorganisms, improve soil microbial diversity, and promote growth and yield. This study would provide a scientific basis for the rational application of B. subtilis.


Subject(s)
Bacillus subtilis/genetics , Fertilizers , Fungi , Soil , Soil Microbiology
18.
Chinese Journal of Biotechnology ; (12): 2377-2386, 2020.
Article in Chinese | WPRIM | ID: wpr-878494

ABSTRACT

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.


Subject(s)
Amino Acids , Bacillus subtilis/metabolism , Culture Media , Fermentation , Lipopeptides , Peptides, Cyclic
19.
Braz. J. Pharm. Sci. (Online) ; 56: e18092, 2020. tab, graf
Article in English | LILACS | ID: biblio-1142491

ABSTRACT

We synthesized a series of compounds bearing pharmacologically important 1,3,4-oxadiazole and piperidine moieties. Spectral data analysis by 1H-NMR, 13C-NMR, IR and EI-MS was used to elucidate the structures of the synthesized molecules. Docking studies explained the different types of interaction of the compounds with amino acids, while bovine serum albumin (BSA) binding interactions showed their pharmacological effectiveness. Antibacterial screening of these compounds demonstrated moderate to strong activity against Salmonella typhi and Bacillus subtilis but only weak to moderate activity against the other three bacterial strains tested. Seven compounds were the most active members as acetyl cholinesterase inhibitors. All the compounds presented displayed strong inhibitory activity against urease. Compounds 7l, 7m, 7n, 7o, 7p, 7r, 7u, 7v, 7x and 7v were highly active, with respective IC50 values of 2.14±0.003, 0.63±0.001, 2.17±0.006, 1.13±0.003, 1.21±0.005, 6.28±0.003, 2.39±0.005, 2.15±0.002, 2.26±0.003 and 2.14±0.002 µM, compared to thiourea, used as the reference standard (IC50 = 21.25±0.15 µM). These new urease inhibitors could replace existing drugs after their evaluation in comprehensive in vivo studies.


Subject(s)
Computer Simulation/classification , Salmonella typhi/classification , Sulfonamides/adverse effects , Thiourea , Bacillus subtilis/classification , Urease , Serum Albumin, Bovine , Pharmaceutical Preparations/administration & dosage , Cholinesterase Inhibitors/pharmacology , Inhibitory Concentration 50 , Proton Magnetic Resonance Spectroscopy/methods , Data Analysis , Amino Acids/antagonists & inhibitors
20.
Electron. j. biotechnol ; 42: 49-55, Nov. 2019. tab, ilus, graf
Article in English | LILACS | ID: biblio-1087461

ABSTRACT

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.


Subject(s)
Bacillus licheniformis/enzymology , Glutaminase/metabolism , Arginine , Plasmids , Prostaglandins A/chemistry , Bacillus subtilis , Protein Sorting Signals , Base Sequence , Mutagenesis, Site-Directed , Aspartic Acid , Escherichia coli , Bacillus licheniformis/genetics , Glutaminase/genetics
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