Bacillus methylotrophicus DCS1: Production of Different Lipopeptide Families, In Vitro Antifungal Activity and Suppression of Fusarium Wilt in Tomato Plants.

The present work aims to quantitatively and qualitatively monitor the production of lipopeptide mixtures by Bacillus methylotrophicus DCS1 strain in Landy medium and to investigate the antifungal activities of DCS1 strain and its produced lipopeptides. The in vitro activities were tested by the direct confrontation and agar well diffusion methods, while the in vivo study was carried out in order to test the efficiency of DCS1 bacterial suspension in the control of Fusarium wilt in tomato plants. Identification of lipopeptides by mass spectrometry (LC/MSD-TOF) showed that lipopeptide isoforms produced during the first 24 h and 48 h of fermentation are identical, belonging to bacillomycin D and fengycins A and B homologues with a difference in the yield of production. After 72 h of fermentation corresponding to the end of incubation period, B. methylotrophicus DCS1 is able to produce a mixture of surfactin, pumilacidin, iturin A/mycosubtilin, iturin C1, bacillomycin D and fengycins A and B isoforms. The results of in vitro antifungal experiments suggest that B. methylotrophicus DCS1 has a significant potential as a biocontrol agent, owing to lipopeptides produced, endowed with antifungal activity against several phytopathogenic fungi. The curative treatment of tomato plants with DCS1 bacterial suspension was more effective in the protection against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) than the preventive treatment by comparing the average number of leaves remaining healthy after 30 days of each treatment and the appearance of tomato plants roots. The results indicate that B. methylotrophicus DCS1 exhibit a significant suppression of Fusarium wilt symptoms in tomato plants comparable to that of commercial fungicides and could be an alternative to chemically synthesized pesticides.

Bacillus Swezeyi B2 Strain: A Novel Alkaliphilic Bacterium Producer of Alkaline-, Thermal, Oxidant-, and Surfactant-Stable Protease, Extremely Efficient in Detergency.

Proteolytic enzymes that are currently used to meet industrial demand are usually derived from Bacillus species. They find multiple technical applications, particularly they have been increasingly used as a key bio-additive in detergents. In this study, a novel alkalophilic bacterium was isolated from contaminated soil, exhibiting 1400 U/ml proteolytic activity, and identified as Bacillus swezeyi B2. The crude enzyme likely contained a single extracellular protease. This enzyme revealed optimum activity at pH 10 and 70 °C and was highly alkaline thermostable (7-12.5) and up to 70 °C. The protease activity was completely inhibited by Phenylmethylsulfonyl fluoride (PMSF) suggesting that it belongs to the serine protease group. It was highly stable in the presence of the strong anionic surfactant (SDS) and oxidizing agents (H2O2). The supernatant was lyophilized and showed high storage stability retaining 100% of its original activity after one year of conservation at 4 °C. The lyophilized product was evaluated for its detergent efficacy, it revealed excellent compatibility with various laundry detergents keeping its full original activity after incubation for 1 h with seven solid and liquid commercial detergents and it effectively removed chocolate stains at low washing temperature (40 °C) and low supplementation level (125 U/ml). The features of this single alkaline and thermotolerant protease, stable toward surfactants, oxidizing agents, and commercial detergents with stain removal efficacy support its ideal choice for supplementation in detergent formulations.

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour.

This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunussegnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg-1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL-1 and 2000 ± 23 µmol min-1 mg-1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

The conservative effects of lipopeptides from Bacillus methylotrophicus DCS1 on sunflower oil-in-water emulsion and raw beef patties quality.

Lipid oxidation was considered as a problem in food conservation. The present study aims to investigate the effect of lipopeptides DCS1 on the conservation of food models against lipid oxidation by determining the primary and the secondary oxidation products. Lipopeptides DCS1 are able to preserve the nutritional properties of the emulsion during 23 days of storage, at a concentration of 0.0125% (w/w of emulsion), by slowing down the formation of hydroperoxides and malondialdehyde (MDA) compounds. The direct incorporation of lipopeptides in ground beef patties at a concentration of 0.5% (w/w of meat) was found to be more effective than gelatin film enriched with lipopeptides (2.5%, w/w of gelatin) as a coating, in inhibiting lipid oxidation. Furthermore, lipopeptides DCS1 are not toxic to human kidney cells HEK293 up to a concentration of 250 µg/ml. The results indicate that lipopeptides DCS1 are effective for the preservation of fatty foods against lipid oxidation.

Simultaneous production of alkaline amylase and biosurfactant by Bacillus methylotrophicus DCS1: application as detergent additive.

This study investigated the coproduction of alkaline amylase and lipopeptides by Bacillus methylotrophicus DCS1 strain, as well as their biochemical characterisation. The best production of both amylase and biosurfactant was obtained when potato starch (10 g/L) and glutamic acid (5 g/L) were used as carbon and nitrogen sources, respectively. The bacterial strain was incubated for 48 h at 25 °C and 150 rpm. This strain produced a unique amylase as showed by zymography technique. The optima pH and temperature were 60-65 °C and 8.0, respectively. Amylase activity was partially inhibited by EDTA (5 mM). The main hydrolysis products of potato starch were maltose and maltotriose. The alkaline amylase showed excellent stability and compatibility with various solid and liquid detergents. Furthermore, the biosurfactant, produced simultaneously with alkaline amylase, demonstrated high stability at different ranges of salinity, pH, and temperature. Considering its promising properties, B. methylotrophicus DCS1 crude extract containing both biosurfactants and amylase activity may be considered as a potential candidate for future use in detergent processing industries and environmental remediation processes.

Economic production and biocontrol efficiency of lipopeptide biosurfactants from Bacillus mojavenis A21.

In the wake of an increased attention on the eco-friendly biopesticidal products and the rising market requirements for organic agents, lipopeptides compounds have been described as biological control agents which improve the overall health growth and development of plants. Nevertheless, their high production cost constitue the major flaw in their wide use to control plant diseases. The present article aims to formulate an economic media for lipopeptides production by Bacillus mojavensis A21 for application as natural fungicides for plant disease treatment. We herein demonstrated the suitability of the potato waste, as low cost substrate, for lipopeptides production. Moreover, sea water  was found to be a good mineral salts sources. In the second part of this study, we investigate the inhibitory activity of A21 lipopeptides against the phtopathogenic Fusarium sp. The in vitro test showed a minimal inhibitory concentration of about 0.3 mg/ml. The microscopic examination, of the treated Fusarium revealed an excessive lysis of the mycelia ultrastructure with destructed spores. The in vivo antagonist activity was confirmed towards the infected potato tubers. A21 lipopeptides are effective in decreasing by about 78.26% and 60.68% when applied as preventive and curative treatments, respectively, as compared to the untreated tubers.

Isolation and characterization of kurstakin and surfactin isoforms produced by Enterobacter cloacae C3 strain.

In this work, the extraction, structural analysis, and identification as well as antimicrobial, anti-adhesive, and antibiofilm activities of lipopeptides produced by Enterobacter cloacae C3 strain were studied. A combination of chromatographic and spectroscopic techniques offers opportunities for a better characterization of the biosurfactant structure. Thin layer chromatography (TLC) and HPLC for amino acid composition determination are used. Efficient spectroscopic techniques have been utilized for investigations on the biochemical structure of biosurfactants, such as Fourier transform infrared (FT-IR) spectroscopy and mass spectrometry analysis. This is the first work describing the production of different isoforms belonging to kurstakin and surfactin families by E cloacae strain. Three kurstakin homologues differing by the fatty acid chain length from C10 to C12 were detected. The spectrum of lipopeptides belonging to surfactin family contains various isoforms differing by the fatty acid chain length as well as the amino acids at positions four and seven. Lipopeptide C3 extract exhibited important antibacterial activity against Gram-positive and Gram-negative bacteria, antifungal activity, and interesting anti-adhesive and disruptive properties against biofilm formation by human pathogenic bacterial strains: Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, and Candida albicans.

Enhancement of Surfactin and Fengycin Production by Bacillus mojavensis A21: Application for Diesel Biodegradation.

This work concerns the study of the enhancement of surfactin and fengycin production by B. mojavensis A21 and application of the produced product in diesel biodegradation. The influences of the culture medium and cells immobilization were studied. The highest lipopeptides production was achieved after 72 hours of incubation in a culture medium containing 30 g/L glucose as carbon source and a combination of yeast extract (1 g/L) and glutamic acid (5 g/L) as nitrogen sources with initial pH 7.0 at 30°C and 90% volumetric aeration. The study of primary metabolites production showed mainly the production of acetoin, with a maximum production after 24 h of strain growth. The use of immobilized cells seemed to be a promising method for improving lipopeptides productivity. In fact, the synthesis of both lipopeptides, mainly fengycin, was greatly enhanced by the immobilization of A21 cells. An increase of diesel degradation capacity of approximately 20, 27, and 40% in the presence of 0.5, 1, and 2 g/L of produced lipopeptides, respectively, was observed. Considering these properties, B. mojavensis A21 strain producing a lipopeptide mixture, containing both surfactin and fengycin, may be considered as a potential candidate for future use in bioremediation and crop protection.

Structural characterization and identification of cyclic lipopeptides produced by Bacillus methylotrophicus DCS1 strain.

Bacillus methylotrophicus DCS1 strain was isolated from diesel contaminated soil and screened for its ability to produce biosurfactants; it was found effective for the production of surface active molecules. The structural characterization of the isolated lipopeptides was studied by a variety of analytical techniques. The organic extract of DCS1 lipopeptides was fractionated by silica gel column chromatography (60Mesh). Fractions containing lipopeptides were collected and identified by tandem mass spectrometry MALDI-TOF-MS and MALDI-TOF MS2. The crude biosurfactants contains a mixture of homologous lipopeptides with molecular weights between 1016 and 1556Da. Mass spectrometry analysis of partially purified lipopeptides revealed that it contains different isoforms belonging to three families: surfactin, iturin and fengycin. To identify lipopeptides isoforms, MALDI-TOF MS2 was used and ions representing characteristic fragmentations were detected. The mass spectrometry characterization revealed the presence of four variants of surfactin lipopeptides, four variants of pumilacidin that differ according to the ß-hydroxy fatty acid chain length as well as the type of amino acid at position 7, five variants of iturin A/mycosubtilin varying in the ß-amino fatty acid chain length from C12 to C16, C16 iturin C1, five isoforms of bacillomycin D varying in the ß-amino fatty acid chain length from C14 to C18, and six fengycin isoforms that differ according to the length of the ß-hydroxy fatty acid side chain as well as the amino acid at position 6. The capacity of B. methylotrohicus DCS1 strain to produce many lipopeptides isoforms belonging to different families and having a structural diversity is a very interesting characteristic that allows them to be used in various fields of biotechnological applications.

Antioxidant properties, antimicrobial and anti-adhesive activities of DCS1 lipopeptides from Bacillus methylotrophicus DCS1.

BACKGROUND: The present work aims to investigate the antioxidant and antimicrobial activities as well as the potential of DCS1 lipopeptides produced by Bacillus methylotrophicus DCS1 strain at inhibition and disruption of biofilm formation. RESULTS: The produced biosurfactants were characterized as lipopeptides molecules by using thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). The DCS1 lipopeptides were assayed for their antioxidant activity through five different tests. The scavenging effect on DPPH radicals at a concentration of 1 mg mL-1 was 80.6%. The reducing power reached a maximum value of 3.0 (OD700 nm) at 2 mg mL-1. Moreover, the DCS1 lipopeptides exhibited a strong inhibition of ß-carotene bleaching by linoleic acid assay with 80.8% at 1 mg mL-1 and showed good chelating ability and lipid peroxidation inhibition. The in vitro antimicrobial activity of DCS1 lipopeptides showed that they display significant antibacterial and antifungal activities. The anti-adhesive activity of DCS1 lipopeptides was evaluated against several pathogenic microorganisms. The lipopeptides showed excellent anti-adhesive activity, even at low concentrations, in a polystyrene surface pre-treatment against all the microorganisms tested. Further, they can disrupt performed biofilms. CONCLUSION: This study shows the potentiality of DCS1 lipopeptides as natural antioxidants, antimicrobial and/or anti-adhesive agent for several biomedical and industrial applications.

Depolymerization of Pseudomonas stutzeri exopolysaccharide upon fermentation as a promising production process of antibacterial compounds.

Many researchers have focused on high molecular weight (Mw) exopolysaccharides (EPS) as a source of potentially bioactive lower Mw derivatives. Therefore, it is of interest to find means for efficient and safe production of depolymerized-polymer derivatives. Exopolysaccharide-depolymerization products (EDP) varying in molecular weight were recovered from fermentative depolymerization of a native EPS produced by Pseudomonas stutzeri AS22. Following their purification and physicochemical characterization, the antibacterial activity of EDP on food spoilage and food poisoning microorganisms was evaluated through the measurement of the inhibition zone diameter, the half maximal (IC50) and the minimal (MIC) inhibitory concentrations. Our results indicate that the lower the Mw, the higher will be the effectiveness of EDP on reducing Gram-negative bacteria growth and the opposite trend was observed in the case of Gram-positive bacteria. EDP bioactivities may provide novel insights into the potentiality of P. stutzeri EPS and its derivatives to be used as functional-food components.

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6.

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI-TOF-MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration-dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of ß-carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram-positive, Gram-negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional-food components.

Characterization and properties of biosurfactants produced by a newly isolated strain Bacillus methylotrophicus DCS1 and their applications in enhancing solubility of hydrocarbon.

Six biosurfactant-producing bacteria were isolated from hydrocarbon contaminated soils in Sfax, Tunisia. Isolates were screened for biosurfactant production by different conventional methods including hemolytic activity, surface tension reduction, drop-collapsing and oil displacement tests. All these screening tests show that all the isolates behave differently. Among the isolated bacteria, DCS1 strain was selected for further studies based on its highest activities and it was identified as Bacillus methylotrophicus DCS1. This strain was found to be a potent producer of biosurfactant when cultivated in mineral-salts medium supplemented with diesel oil (2 %, v/v) as a sole carbon source. Physicochemical properties and stability of biosurfactants synthesized by B. methylotrophicus DCS1 were investigated. The produced biosurfactants DCS1, from Landy medium, possess high surface activity that could lower the surface tension of water to a value of 31 from 72 mN m(-1) and have a critical micelle concentration (CMC) of 100 mg L(-1). Compared with SDS and Tween 80, biosurfactants showed excellent emulsification activities against different hydrocarbon substrates and high solubilization efficiency towards diesel oil. Biosurfactants DCS1 showed good stability in a wide range of temperature, pH and salinity. These results suggested that biosurfactants produced by B. methylotrophicus DCS1 could be an alternative to chemically synthesized surfactants for use in bioremediation processes to enhance the solubility of hydrophobic compounds.

Acute and sub-chronic oral toxicity profiles of lipopeptides from Bacillus mojavensis A21 and evaluation of their in vitro anticoagulant activity.

The aim of the present study was to evaluate the acute and sub-chronic toxicity of lipopeptides mixture produced by Bacillus mojavensis A21 as well as their in vitro anticoagulant activity. A21 lipopeptides was given to mice at single dose from 75 mg to 1000 mg/kg body weight (bw). The median lethal dose (LD50) of A21 lipopeptides was about 550 mg/kg bw. Sub-chronic toxicity study for 28 days was done by daily oral administration of A21 lipopeptides at doses of 40 and 400 mg/kg bw in rats. Results showed that A21 lipopeptides did not cause any change in body weights and they did not produce any marked alterations in the hematological blood parameters including hematocrit concentration, hemoglobin level, white and red cells count. However, the platelets level decreased significantly compared to control value. Moreover, no significant differences in the serum biochemical characteristics were observed for rats treated by the lowest dose. In contrast, a little enhancement of alanine-aminotransferase (ALT) activity and decrease in total cholesterol were observed with the highest dose. A21 lipopeptides were also found to cause a prolongation of the thrombin time (TT), the prothrombin time (PT) and the activated partial thromboplastin time (APTT). Overall, A21 lipopeptides may be very promising compounds for therapeutic purposes.

Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus amyloliquefaciens An6.

This study focuses on the isolation and characterisation of a peptide with bacteriocin-like properties from Bacillus amyloliquefaciens An6. Incubation conditions were optimised, and the effects of the incubation period and of carbon and nitrogen sources were investigated. The produced bacteriocin was partially purified with ammonium sulphate precipitation, dialysis and ultrafiltration and was then biochemically characterised. Maximum bacteriocin production was achieved after 48h of incubation in a culture medium containing 20g/L starch and 10g/L yeast extract, with an initial pH 8.0 at 30°C under continuous agitation at 200rpm. The bacteriocin was sequentially purified and its molecular weight was determined to be 11kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The bacteriocin was relatively heat-resistant and was not sensitive to acid and alkaline conditions (pH 4.0-10.0). Its inhibitory activity was sensitive to proteinase K but was resistant to the proteolytic action of alcalase, trypsin, chymotrypsin and pepsin. In conclusion, bacteriocin An6, owing its wide spectrum of activity as well as its high tolerance to acidic and alkaline pH values, temperature and proteases shows great potential for use as a food biopreservative.

Rhelogical, dermal wound healing and in vitro antioxidant properties of exopolysaccharide hydrogel from Pseudomonas stutzeri AS22.

The in vitro antioxidant activity and the in vivo wound healing performance of the exopolysaccharide EPS22, produced by Pseudomonas stutzeri AS22, were investigated. Antioxidant activity was evaluated by three different tests. The scavenging effect on DPPH radicals at a concentration of 1mg/ml was 80±1.41%. The reducing power reached a maximum of 1.26±0.02 at 2 mg/ml. Moreover, EPS22 showed good chelating ability and chelated almost 88.5±0.7% of ferrous ions at 0.75 mg/ml. The rheological characterization of EPS22 gel (0.5%) showed a pseudoplastic behavior, high elasticity, good mechanical strength and stability with high water-absorption ability. The application of the EPS22 gel on dermal full-thickness excision wounds in a rat model every two days, enhanced significantly wound healing activity and a total closure was achieved after 12 days of wound induction. Further, histological examination of biopsies showed advanced tissue regeneration, characterized by the presence of well-organized stratum of both derma and epidermis.

Purification and structural data of a highly substituted exopolysaccharide from Pseudomonas stutzeri AS22.

Pseudomonas stutzeri AS22, when grown on media containing starch and yeast extract and incubated at 30 °C and 200 rpm for 24h, was found to produce an acidic and high-molecular mass exopolysaccharide (EPS22). The EPS22 was purified and a yield of 1.3g/l was achieved. The average molecular mass of the EPS22 was determined by high-performance size-exclusion chromatography (HPSEC) and showed an average molecular mass of 9.9 × 10(5)Da and a polydispersity index Mw/Mn (Mw, weight-average and Mn, number-average) of 1.197 ± 0.015. Structural data of this EPS22 were determined using a combination approach including monosaccharide composition (HPAEC-PAD and GLC), methylation analysis (GC-MS) and NMR spectroscopy analysis. EPS22 was found to be a complex heteropolysaccharide with a repeating unit mainly composed of glucose, mannose and lactyl rhamnose in a molar ratio of 1:1.1:0.7. The acidic nature of the polysaccharide is due to the presence of three non-osidic substituents consisting of a lactyl, acetyl, and pyruvyl groups.

Production and biochemical characterization of a high maltotetraose (G4) producing amylase from Pseudomonas stutzeri AS22.

Amylase production and biochemical characterization of the crude enzyme preparation from Pseudomonas stutzeri AS22 were evaluated. The highest α-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crude α -amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that both α -amylase production and activity were Ca(2+)-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups.

Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5-11) and temperature (25-70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca(2+) and Mg(2+). EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.