Isolation and Characterization of Probiotic Bacillus subtilis MKHJ 1-1 Possessing L-Asparaginase Activity

The purpose of this study was to isolate functional Bacillus strains from Korean fermented soybeans and to evaluate their potential as probiotics. The L-asparaginase activity of MKHJ 1-1 was the highest among 162 Bacillus strains. This strain showed nonhemolysis and did not produce β-glucuronidase. Among the nine target bacteria, MKHJ 1-1 inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Shigella flexneri, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus cereus. 16S rRNA gene sequence analysis resulted in MKHJ 1-1 identified as Bacillus subtilis subsp. stercoris D7XPN1. As a result of measuring the survival rate in 0.1% pepsin solution (pH 2.5) and 0.3% bile salt solution for 3 h, MKHJ 1-1 exhibited high acid resistance and was able to grow in the presence of bile salt. MKHJ 1-1 showed outstanding autoaggregation ability after 24 h. In addition, its coaggregation with pathogens was strong. Therefore, MKHJ 1-1 is a potential probiotic with L-asparaginase activity and without L-glutaminase activity, suggesting that it could be a new resource for use in the food and pharmaceutical industry.

Download Full-text

Degradation of zearalenone in swine feed and feed ingredients by Bacillus subtilis ANSB01G

World Mycotoxin Journal ◽

10.3920/wmj2013.1623 ◽

2014 ◽

Vol 7 (2) ◽

pp. 143-151 ◽

Cited By ~ 18

Author(s):

Y.P. Lei ◽

L.H. Zhao ◽

Q.G. Ma ◽

J.Y. Zhang ◽

T. Zhou ◽

...

Keyword(s):

Bacillus Subtilis ◽

Culture Supernatant ◽

Antimicrobial Activities ◽

Proteinase K ◽

Rrna Gene ◽

Distillers Grains With Solubles ◽

Dried Distillers Grains ◽

Cell Extracts ◽

Gene Sequence Analysis ◽

And Staphylococcus Aureus

Zearalenone (ZEA) and its derivatives are mycotoxins that can cause oestrogenic effects and impair the reproductive physiology of animals, especially in female swine. Strategies to reduce or eliminate ZEA contamination in foods and feeds are very much needed. Among 36 bacterial isolates obtained from a variety of animal intestinal chyme, mouldy foods and feeds, soils, etc., five isolates demonstrated the ability to reduce more than 50% of ZEA in a liquid medium; ANSB01G isolate taken from normal broiler intestinal chyme reduced ZEA the most, by 88.65%. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, the ANSB01G isolate was identified as Bacillus subtilis. Under simulated intestinal tract conditions, the ANSB01G B. subtilis isolate degraded 84.58, 66.34 and 83.04% of ZEA in naturally contaminated maize, dried distillers’ grains with solubles, and swine complete feed, respectively. The highest degradation of ZEA occurred when the mycotoxin was co-incubated with the whole bacterial culture, resulting in a reduction of 88.65%, followed by 75.60% using culture supernatant, 26.11% using cell extracts, and 15.06% using viable cells. Treatments consisting of both heating and addition of proteinase K significantly reduced the rate of ZEA degradation in the culture supernatant, indicating that the ZEA degradation might be enzymatic. B. subtilis ANSB01G displayed resistance to simulated gastrointestinal tract environments and antimicrobial activities against several common bacterial pathogens, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. These properties of B. subtilis ANSB01G suggest the possibility of its potential to effectively degrade ZEA in feed and to develop functional feed products for livestock industries.

Download Full-text

PROBIOTIC CHARACTERIZATION OF BACILLUS SUBTILIS STRAIN ISOLATED FROM INFANT FECAL MATTER REVEALED BY 16S rRNA GENE AND PHYLOGENETIC ANALYSIS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2021.v14i12.43204 ◽

2021 ◽

pp. 77-85

Author(s):

DEVARANJAN DAS ◽

CHANDI CHARAN RATH ◽

NAKULANANDA MOHANTY ◽

SMITA HASINI PANDA

Keyword(s):

Antimicrobial Activity ◽

Bacillus Subtilis ◽

16S Rrna ◽

16S Rrna Gene ◽

Bile Salt ◽

Rrna Gene ◽

Probiotic Properties ◽

Fecal Matter ◽

Antimicrobial Metabolites

Objective: The rationale of our study was to isolate and identify the putative probiotic strain from infant fecal matter exhibiting a broad range of antimicrobial activity and to analyze the effect of different culturing conditions on its probiotic properties and the production of antimicrobial metabolites. Methods: In the present study, bacterial strains were screened for probiotic properties and antimicrobial activity from infant fecal matter (6 months–2 years). The effect of varying culture conditions such as tolerance to acid, bile salt, phenol, NaCl, pH, incubation period, and temperature along with autoaggregation assay, hydrophobicity, and hemolysis was studied. The characterization of the potent strain was studied by morphological, biochemical, and 16S rRNA gene sequencing along the phylogenetic affiliation of the strain was studied. Results: Two putative probiotic bacteria (DAM and IFM) were isolated, identified, characterized, and predicted at pH 2.0, 3.0, and 4.0, the isolate IFM had 50%, 60%, and 70% survivability, while isolate DAM had 55%, 63%, and 75% survivability, respectively. At a bile salt concentration of 0.5%, both isolates had a 75% survival rate. The isolates exhibited a high percentage of hydrophobicity and autoaggregation. The isolates also had non-hemolytic activity and were susceptible to many clinical tested antibiotics (tetracycline, erythromycin, ampicillin, gentamycin, penicillin, etc.). The isolate showed antimicrobial activity against enteric pathogens such as Staphylococcus aureus, Escherichia coli, and Shigella dysenteriae. The accession number of Bacillus subtilis MT279753 and MK453362 was submitted to NCBI. Conclusion: The result revealed that isolates have potent probiotic properties and possess a direct influence on the production of antimicrobial metabolites. These parameters can be modified for the improvement of the potentiality of the isolates.

Download Full-text

Studies of some genes of Bacillus subtilis MN99 local strains against plant diseases

Mongolian Journal of Agricultural Sciences ◽

10.5564/mjas.v33i2.1753 ◽

2021 ◽

Vol 33 (2) ◽

pp. 38-44

Author(s):

Ariunaa Saraadanbazar ◽

Byambasuren Mijidsuren ◽

Battur Banzragch

Keyword(s):

Bacillus Subtilis ◽

Local Strain ◽

Pathogenic Fungi ◽

Plant Diseases ◽

Likelihood Method ◽

Partial Sequence ◽

Rrna Gene ◽

Multiple Sequence ◽

Local Strains ◽

Isolation And Characterization

The objective of this study was the isolation and characterization of Bacillus subtilis local strains from the soil in Mongolia. These local strains of B. subtilis are showed to have high antagonistic activities against some plant pathogenic fungi and bacteria. Six strains of B. subtilis were isolated and characterized morphologically, physiologically and biochemically according the Bergey’s Manual of Systematic Bacteriology. In order to identify species of the isolated strains, we amplified and sequenced 16S rRNA gene, essential funtinal genes bmyB, spoVG and srfAA, which are related to antagonistic activity of these strains. The sequences were aligned using CLASTALW multiple sequence alignment tool. Phylogenetic tree was drawn according to Maximum likelihood”method and “Tamura-Nei” model using “MEGA-X version 10.2.6 program. Among all isolates of B. subtilis MN99 and 7/24 strains had higher antagonist activity against plant diseases. According to partial sequence of srfAA (620bp) gene of MN99, the local strain belongs to B. subtilis and partial sequence of bmyB (370bp), spoVG (22bp) gene of MN7/24 strain showed that the it belongs to B. atrophaeus species. All local strains of B. subtilis had bacillomycin synthesis gene, and B. subtilis MN99 strain had only surfactine synthesis gene, while did not have spore formation and hemolysis gene SpoVG.

Download Full-text

Isolation and Characterization of Diverse Halobenzoate-Degrading Denitrifying Bacteria from Soils and Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3446-3453.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3446-3453 ◽

Cited By ~ 129

Author(s):

Bongkeun Song ◽

Norberto J. Palleroni ◽

Max M. Häggblom

Keyword(s):

Electron Acceptor ◽

Cellular Fatty Acid ◽

Denitrifying Bacteria ◽

Rrna Gene ◽

Terminal Electron Acceptor ◽

Ecological Sites ◽

Isolation And Characterization ◽

Gene Sequence Analysis ◽

Soils And Sediments

ABSTRACT Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2- and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the α-, β-, or γ-branch of theProteobacteria, namely, Acidovorax,Azoarcus, Bradyrhizobium,Ochrobactrum, Paracoccus,Pseudomonas, Mesorhizobium,Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in theProteobacteria and that these bacteria are widely distributed in soils and sediments.

Download Full-text

Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5168-5176.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5168-5176 ◽

Cited By ~ 42

Author(s):

Alessandra Matarante ◽

Federico Baruzzi ◽

Pier Sandro Cocconcelli ◽

Maria Morea

Keyword(s):

Bacillus Subtilis ◽

Dna Sequences ◽

Bacillus Pumilus ◽

Meat Products ◽

Rrna Gene ◽

Emerging Pathogens ◽

Specific Pcr ◽

Gene Sequence Analysis ◽

Rapd Pcr ◽

Cured Meat

ABSTRACT Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.

Download Full-text

Isolation and characterization of Reyranella massiliensis gen. nov., sp. nov. from freshwater samples by using an amoeba co-culture procedure

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.025775-0 ◽

2011 ◽

Vol 61 (9) ◽

pp. 2151-2154 ◽

Cited By ~ 26

Author(s):

Isabelle Pagnier ◽

Didier Raoult ◽

Bernard La Scola

Keyword(s):

Gene Sequence ◽

Novel Species ◽

Phenotypic Characterization ◽

Rrna Gene ◽

Rrna Gene Sequence ◽

Isolation And Characterization ◽

Novel Bacterium ◽

Gene Sequence Analysis ◽

Culture Procedure

The analysis of three water samples from two cooling towers and one river allowed us to isolate three strains of a novel species of the class Alphaproteobacteria which is phylogenetically related to uncultured alphaproteobacteria. Based upon 16S rRNA gene sequence analysis and phenotypic characterization, we propose to name this novel species Reyranella massiliensis gen. nov., sp. nov., type strain 521T ( = CSUR P115T = DSM 23428T). The most closely related cultivable micro-organism to this novel bacterium is a member of the genus Magnetospirillum.

Download Full-text

Phylogenetic Framework and Biosurfactant Gene Expression Analysis of Marine Bacillus spp. of Eastern Coastal Plain of Tamil Nadu

International Journal of Bacteriology ◽

10.1155/2014/860491 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Sreethar Swaathy ◽

Varadharajan Kavitha ◽

Arockiasamy Sahaya Pravin ◽

Ganesan Sekaran ◽

Asit Baran Mandal ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Coastal Plain ◽

Tamil Nadu ◽

Bacterial Species ◽

Individual Species ◽

Rrna Gene ◽

Gene Sequence Analysis ◽

Diversity Assessment ◽

Bacillus Spp

The present study emphasizes the diversity assessment of marine Bacillus species with special reference to biosurfactant production, respective gene expression, and discrimination among Bacillus licheniformis and Bacillus subtilis. Among the 200 individual species of eastern coastal plain of Tamil Nadu screened, five biosurfactant producing potential bacterial species with entirely different morphology were selected. Biochemical and 16S rRNA gene sequence analysis suggested that all the said five species belong to Bacillus genera but differ in species levels. Biosurfactant of all the five species fluctuates in greater levels with respect to activity as well as to constituents but showed partial similarity to the commercially available surfactin. The expression of srf gene was realized in all of the five species. However, the sfp gene expression was observed only in three species. In conclusion, both B. licheniformis and B. subtilis demonstrate srf gene; nevertheless, sfp gene was expressed only by Bacillus subtilis.

Download Full-text

Isolation and Identification of the Potential Novel L-asparaginase producing Bacillus Strains from Soil

Abasyn Journal Life Sciences ◽

10.34091/ajls.4.1.2 ◽

2021 ◽

pp. 11-19

Author(s):

Naushaba Nazli ◽

Rukhsar Masood ◽

Muhmmad Salman ◽

Bilal Nasir ◽

Farah Shireen ◽

...

Keyword(s):

Bacillus Amyloliquefaciens ◽

Agricultural Land ◽

Lymphoblastic Leukemia ◽

Dairy Farm ◽

Sequencing Analysis ◽

Phenol Red ◽

Isolation And Identification ◽

Isolation And Characterization ◽

Asparaginase Activity ◽

Bacillus Strains

L-Asparaginase is a well know enzyme for its antineoplastic potential and is widely used to treat acute lymphoblastic leukemia and lymphosarcoma. The present work describes the isolation and characterization of novel L-asparaginase producing Bacillus strains from soil. Soil samples were collected from three different locations such as fruit garden, dairy farm and agricultural land in Peshawar Khyber Pakhtunkhwa, Pakistan. The isolates were screened to produce L-asparaginase in growth medium supplemented with 1% L-asparagine using a phenol red indicator. Among 30 bacterial isolates, only two strains initially coded as A5 and FG7 showed L-asparaginase activity. Based on biochemical and 16S rRNA sequencing analysis, the isolate A5 and FG7 were identified as Bacillus amyloliquefaciens and Bacillus proteolyticus respectively. Different factors like pH and time were optimized for maximum L-asparaginase activity. Bacillus amyloliquefaciens showed maximum asparaginase activity at pH 7 after 24 hours incubation at 30oC, while Bacillus proteolyticus showed optimum activity at pH 7 after 48 hours of incubation at 30oC. The present study first time reported the production of L-asparginase enzyme from Bacillus amyloliquefaciens and Bacillus proteolyticus. Keywords: L-asparaginase, Bacillusamyloliquefaciens, Bacillus proteolyticus, 16sRNA.

Download Full-text

A Novel Microbial Zearalenone Transformation through Phosphorylation

Toxins ◽

10.3390/toxins13050294 ◽

2021 ◽

Vol 13 (5) ◽

pp. 294

Author(s):

Yan Zhu ◽

Pascal Drouin ◽

Dion Lepp ◽

Xiu-Zhen Li ◽

Honghui Zhu ◽

...

Keyword(s):

Microbial Transformation ◽

Acid Tolerance ◽

Transformation Product ◽

The United States ◽

Corn Silage ◽

Rrna Gene ◽

Bacillus Spp ◽

High Concentrations ◽

The 16S Rrna Gene ◽

Bacillus Strains

Zearalenone (ZEA) is a mycotoxin widely occurring in many agricultural commodities. In this study, a purified bacterial isolate, Bacillus sp. S62-W, obtained from one of 104 corn silage samples from various silos located in the United States, exhibited activity to transform the mycotoxin ZEA. A novel microbial transformation product, ZEA-14-phosphate, was detected, purified, and identified by HPLC, LC-MS, and NMR analyses. The isolate has been identified as belonging to the genus Bacillus according to phylogenetic analysis of the 16S rRNA gene and whole genome alignments. The isolate showed high efficacy in transforming ZEA to ZEA-14-phosphate (100% transformation within 24 h) and possessed advantages of acid tolerance (work at pH = 4.0), working under a broad range of temperatures (22–42 °C), and a capability of transforming ZEA at high concentrations (up to 200 µg/mL). In addition, 23 Bacillus strains of various species were tested for their ZEA phosphorylation activity. Thirteen of the Bacillus strains showed phosphorylation functionality at an efficacy of between 20.3% and 99.4% after 24 h incubation, suggesting the metabolism pathway is widely conserved in Bacillus spp. This study established a new transformation system for potential application of controlling ZEA although the metabolism and toxicity of ZEA-14-phosphate requires further investigation.

Download Full-text

Isolation and Characterization of Lactic Acid Bacteria and Yeasts from Typical Bulgarian Sourdoughs

Microorganisms ◽

10.3390/microorganisms9071346 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1346

Author(s):

Mariana Petkova ◽

Petya Stefanova ◽

Velitchka Gotcheva ◽

Angel Angelov

Keyword(s):

Lactic Acid ◽

Sequence Analysis ◽

Lactic Acid Bacteria ◽

Antimicrobial Properties ◽

Matter Content ◽

Starter Cultures ◽

Rrna Gene ◽

Dry Matter Content ◽

Isolation And Characterization ◽

Selection Of

Traditional sourdoughs in Bulgaria were almost extinct during the centralized food production system. However, a rapidly developing trend of sourdough revival in the country is setting the demand for increased production and use of commercial starter cultures. The selection of strains for such cultures is based on geographical specificity and beneficial technological properties. In this connection, the aim of this study was to isolate, identify and characterize lactic acid bacteria (LAB) and yeasts from typical Bulgarian sourdoughs for the selection of strains for commercial sourdough starter cultures. Twelve samples of typical Bulgarian sourdoughs were collected from different geographical locations. All samples were analyzed for pH, total titratable acidity and dry matter content. Enumeration of LAB and yeast was also carried out. Molecular identification by 16S rDNA sequence analysis was performed for 167 LAB isolates, and 106 yeast strains were identified by ITS1-5.8S-ITS2 rRNA gene partial sequence analysis. The LAB strains were characterized according to their amylolytic and proteolytic activity and acidification capacity, and 11 strains were selected for further testing of their antimicrobial properties. The strains with the most pronounced antibacterial and antifungal activity are listed as recommended candidates for the development of starter cultures for sourdoughs or other food products.

Download Full-text