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.

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.

Isolation and structural characterization of levan produced by probiotic Bacillus tequilensis-GM from Tunisian fermented goat milk.

The probiotic features of strain GM newly isolated from Tunisian spontaneously fermented goat milk and identified as Bacillus tequilensis-GM were assessed. Strain GM showed high resistance to saliva (90.64%), gastric juice (88.55%), intestinal juice (72.83%) and resistance to bile salts (65.22%), was able to act against Listeria monocytogenes ATCC 15313, Escherichia coli ATCC 25922 and Enterococcus feacalis ATCC 25912, showed high surface hydrophobicity (77.3%) and was sensitive to most of the studied antibiotics. Strain GM did not exhibit any hemolytic activity whereas it was able to produce protease, amylase and ß-galactosidase. Moreover, results showed that strain GM produced high molecular weight ß-(2 → 6)-levan with high ability to inhibit and to disrupt pathogenic biofilms and with high ability to reduce syneresis of sucrose-supplemented skimmed milk. B. tequilensis-GM can therefore be suitable to be used as starter culture in fermented dairy products, since it possesses desirable probiotic properties in addition to its ability to produce levan.

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.

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.

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.

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.