Anti-oxidative property of xylolipid produced by Lactococcus lactis LNH70 and its potential use as fruit juice preservative.

In the present study, 20 lactic acid bacteria (LAB) were isolated from different fruit juices, milk, and milk products. Based on preliminary screening methods like emulsification index, oil displacement method, hemolysis, and reduction in surface tension, strain LNH70 was selected for further studies. Further, it was evaluated for preliminary probiotic characteristics, identified by 16 s rRNA sequencing as Lactococcus lactis, submitted to NCBI, and an accession number was obtained (MH174454). In addition, LNH70 was found to tolerate over wide range of temperatures (10-45 °C), pH (3-10), NaCl (up to 9%), bile (0.7%), and phenol (0.1%) concentrations. Further, optimization studies at flask level revealed that lactose as carbon source, peptone as organic nitrogen, and inorganic nitrogen (ammonium sulfate) enhanced biosurfactant production. Chemical composition of purified biosurfactant obtained from LNH70 was characterized by various physico-chemical analytical techniques and identified as xylolipid. Xylolipid biosurfactant exhibited anti-adhesion activity against food borne pathogens in in vitro conditions. Its anti-oxidative property by 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP) radical scavenging activity was found in range of 60.76 ± 0.5 to 83.50 ± 0.73%. Furthermore, xylolipid (0.05, 0.1, 0.3 mg/mL) when used for its potential as orange and pineapple juices preservation revealed miniature changes in the physico-chemical parameters evaluated in this study. However, the microbial population slightly lowered when xylolipid was used at 0.3 mg/mL after 5th day. Hence, this study supports the potential use of biosurfactant from L. lactis for its application as food preservative.

Induction of Systemic Resistance in Maize and Antibiofilm Activity of Surfactin From Bacillus velezensis MS20.

Surfactin lipopeptide is an eco-friendly microbially synthesized bioproduct that holds considerable potential in therapeutics (antibiofilm) as well as in agriculture (antifungal). In the present study, production of surfactin by a marine strain Bacillus velezensis MS20 was carried out, followed by physico-chemical characterization, anti-biofilm activity, plant growth promotion, and quantitative Reverse Transcriptase-Polymerase Chain Reaction (q RT-PCR) studies. From the results, it was inferred that MS20 was found to produce biosurfactant (3,300 mg L-1) under optimized conditions. From the physicochemical characterization [Thin layer chromatography (TLC), Fourier Transform Infrared (FTIR) Spectroscopy, Liquid Chromatography/Mass Spectroscopy (LC/MS), and Polymerase Chain Reaction (PCR) amplification] it was revealed to be surfactin. From bio-assay and scanning electron microscope (SEM) images, it was observed that surfactin (MIC 50 µg Ml-1) has appreciable bacterial aggregation against clinical pathogens Pseudomonas aeruginosa MTCC424, Escherichia coli MTCC43, Klebsiella pneumoniae MTCC9751, and Methicillin resistant Staphylococcus aureus (MRSA) and mycelial condensation property against a fungal phytopathogen Rhizoctonia solani. In addition, the q-RTPCR studies revealed 8-fold upregulation (9.34 ± 0.11-fold) of srfA-A gene compared to controls. Further, treatment of maize crop (infected with R. solani) with surfactin and MS20 led to the production of defense enzymes. In conclusion, concentration and synergy of a carbon source with inorganic/mineral salts can ameliorate surfactin yield and, application wise, it has antibiofilm and antifungal activities. In addition, it induced systemic resistance in maize crop, which makes it a good candidate to be employed in sustainable agricultural practices.

Melanin pigment of Streptomyces puniceus RHPR9 exhibits antibacterial, antioxidant and anticancer activities.

The present study reveals the production of dark, extracellular melanin pigment (386 mg/L) on peptone yeast extract iron agar medium by Streptomyces puniceus RHPR9 using the gravimetric method. UV-Visible, Fourier Transform Infrared (FTIR), and Nuclear Magnetic Resonance (1H) (NMR) spectroscopy confirmed the presence of melanin. Extracted melanin showed antibacterial activity against human pathogens such as Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli except for Klebsiella pneumoniae. A potent free radical scavenging activity was observed at 100 µg/mL of melanin by the DPPH method with a concentration of 89.01±0.05% compared with ascorbic acid 96.16±0.01%. Antitumor activity of melanin was evaluated by MTT assay against HEK 293, HeLa, and SK-MEL-28 cell lines with IC50 values of 64.11±0.00, 14.43±0.02, and 13.31±0.01 µg/mL respectively. Melanin showed maximum anti-inflammatory activity with human red blood cells (hRBC) (78.63 ± 0.01%) and minimum hemolysis of 21.37±0.2%. The wound healing potential of the pigment was confirmed on HeLa cells, cell migration was calculated, and it was observed that cell migration efficiency decreased with an increase in the concentration of melanin. To our knowledge, this is the first evidence of melanin produced from S. puniceus RHPR9 that exhibited profound scavenging, anti-inflammatory and cytotoxic activities.

Biosurfactant producing multifarious Streptomyces puniceus RHPR9 of Coscinium fenestratum rhizosphere promotes plant growth in chilli.

The present study involves isolation of Streptomyces spp. from rhizosphere of Coscinium fenestratum Gaertn, an endangered medicinal plant from Western Ghats of Karnataka, India. Four potential isolates were identified by 16S rRNA sequencing as Streptomyces sp. RHPR3, Streptomyces puniceus RHPR9, Streptomyces sp. RHPR14 and Streptomyces mediolani RHPR25. An enrichment culture method was used for the isolation of Streptomyces spp. for biosurfactant activity. Among four potential Streptomyces spp., S. puniceus RHPR9 showed highest Emulsification index (EI) (78±0.2%) and Emulsification assay (EA) (223±0.2 EU mL-1). Thin layer chromatography, Fourier transform infrared spectroscopy (FTIR) and mass spectrometric analysis revealed that as glycolipid. Further confirmed by presence of fatty acids like hexanoic acid methyl ester, decanoic acid by Gas chromatography mass spectroscopy (GC-MS) analysis. S. puniceus RHPR9 showed a significant IAA production (41µg mL-1), solubilized P (749.1 µg mL-1), growth promotion of chilli (Capsicum annuum L.) was evaluated using paper towel method and greenhouse conditions. S. puniceus RHPR9 showed a significant increase in seed vigor index (2047) and increase in plant biomass (65%) when compared to uninoculated control. To our knowledge, this is the first report on epiphytic S. puniceus RHPR9 isolated from an endangered medicinal plant C. fenestratum Gaertn, for biosurfactant production and plant growth promotion activities.

Characterization of rhizobia isolated from leguminous plants and their impact on the growth of ICCV 2 variety of chickpea (Cicer arietinum L.).

Six rhizobia-like-bacterial strains in total, secluded from the root and stem nodules of various leguminous plants were characterized for growth promoting ability on ICCV 2 variety of chickpea. Bacterial strains showed production of IAA, NH3, siderophore, HCN, ACC deaminase, hydrolytic enzyme production such as chitinase, amylase, protease, lipase, ß-1, 3-glucanase and solubilization of nutrients such as phosphate, zinc and potassium. However the performance of PGP traits characterized in-vitro varied among the six bacterial strains. The sequences of 16S rRNA gene of bacterial strains IHSR, IHRG, IHAA, IHGN-3, IHCP-1 and IHCP-2 showed maximum identity with Rhizobium sp., Rhizobium tropici, Rhizobium multihospitium, Mesorhizobium sp., Burkholderia cepacia and Rhizobium pusense. In plate culture conditions the bacterial strains changed the colour of media (NFB) from green to blue and showed amplification of nifH gene by PCR, and also enhanced nodule formation in chickpea under greenhouse conditions, which explains their nitrogen fixing ability. Scanning electron microscopy studies of chickpea roots showed colonization by all the six bacterial strains in solo and by consortium (IHRG + IHGN-3). Under greenhouse conditions, chickpea plants inoculated with different strains showed improvement in plant height, number of branches, total chlorophyll, nodule number, nodule weight, shoot weight, root weight, root volume and root surface area at 30 and 45 days after sowing (DAS) over the uninoculated control plants. It was also observed at the crop maturity stage all the bacterial strains inoculated separately enhanced pod number, seed number and total NPK compared to uninoculated control plants. This study suggests that bacteria associated with root and stem nodules can be a promising resource to enhance nodulation, PGP and crop yields in chickpea.

Correction: Silver nanoparticles from insect wing extract: Biosynthesis and evaluation for antioxidant and antimicrobial potential.

[This corrects the article DOI: 10.1371/journal.pone.0241729.].

Silver nanoparticles from insect wing extract: Biosynthesis and evaluation for antioxidant and antimicrobial potential.

Silver nanoparticles (AgNPs) are among the most widely synthesized and used nanoparticles (NPs). AgNPs have been traditionally synthesized from plant extracts, cobwebs, microorganisms, etc. However, their synthesis from wing extracts of common insect; Mang mao which is abundantly available in most of the Asian countries has not been explored yet. We report the synthesis of AgNPs from M. mao wings extract and its antioxidant and antimicrobial activity. The synthesized AgNPs were spherical, 40-60 nm in size and revealed strong absorption plasmon band around at 430 nm. Highly crystalline nature of these particles as determined by Energy-dispersive X-ray analysis and X-ray diffraction further confirmed the presence of AgNPs. Hydrodynamic size and zeta potential of AgNPs were observed to be 43.9 nm and -7.12 mV, respectively. Fourier-transform infrared spectroscopy analysis revealed the presence of characteristic amide proteins and aromatic functional groups. Thin-layer chromatography (TLC) and Gas chromatography-mass spectroscopy (GC-MS) analysis revealed the presence of fatty acids in the wings extract that may be responsible for biosynthesis and stabilization of AgNPs. Further, SDS-PAGE of the insect wing extract protein showed the molecular weight of 49 kDa. M. mao silver nanoparticles (MMAgNPs) exhibit strong antioxidant, broad-range antibacterial and antifungal activities, (66.8 to 87.0%), broad-range antibacterial and antifungal activities was found with maximum zone of inhibition against Staphylococcus aureus MTCC 96 (35±0.4 mm) and Fusarium oxysporum f. sp. ricini (86.6±0.4) which signifies their biomedical and agricultural potential.

Multifarious Indigenous Diazotrophic Rhizobacteria of Rice (Oryza sativa L.) Rhizosphere and Their Effect on Plant Growth Promotion.

A diverse group of rhizobacteria persists in the rhizospheric soil, on the surface of roots, or in association with rice plants. These bacteria colonize plant root systems, enhance plant growth and crop yield. Indigenous rhizobacteria are known to promote soil health, grain production quality and serve as sustainable bioinoculant. The present study was aimed to isolate, identify and characterize indigenous plant growth promoting (PGP) diazotrophic bacteria associated with the rhizosphere of rice fields from different areas of Jammu and Kashmir, India. A total of 15 bacteria were isolated and evaluated for various PGP traits, antagonistic activity against phytopathogens, production of hydrolytic enzymes and biofilm formation under in-vitro conditions. The majority of the isolated bacteria were Gram-negative. Out of 15 bacterial isolates, nine isolates produced IAA (12.24 ± 2.86 to 250.3 ± 1.15 µg/ml), 6 isolates exhibited phosphate solubilization activity (36.69 ± 1.63 to 312.4 ± 1.15 µg/ml), 7 isolates exhibited rock phosphate solubilization while 5 isolates solubilized zinc (10-18 mm), 7 isolates showed siderophore production, 8 isolates exhibited HCN production, 6 isolates exhibited aminocyclopropane-1-carboxylate (ACC) deaminase activity, 13 isolates exhibited cellulase activity, nine isolates exhibited amylase and lipase activity and six isolates exhibited chitinase activity. In addition, 5 isolates showed amplification with the nifH gene and showed a significant amount of nitrogenase activity in a range of 0.127-4.39 µmol C2H4/mg protein/h. Five isolates viz., IHK-1, IHK-3, IHK-13, IHK-15 and IHK-25 exhibited most PGP attributes and successfully limited the mycelial growth of Rhizoctonia solani and Fusarium oxysporum in-vitro. All the five bacterial isolates were identified based on morphological, biochemical and 16S rDNA gene sequencing study, as Stenotrophomonas maltophilia, Enterobacter sp., Bacillus sp., Ochrobactrum haematophilum and Pseudomonas aeruginosa. Rice plants developed from seeds inoculated with these PGP strains individually had considerably higher germination percentage, seed vigor index and total dry biomass when compared to control. These findings strongly imply that the PGP diazotrophic bacteria identified in this work could be employed as plant growth stimulators in rice.

Enhancement of atrazine biodegradation by marine isolate Bacillus velezensis MHNK1 in presence of surfactin lipopeptide.

Atrazine is one of the widely used toxic herbicide and considered as serious environmental contaminant worldwide due to its long term use in crop production. In this study, the effect of surfactin lipopeptide produced by Bacillus velezensis MHNK1 on atrazine biodegradation was investigated. B. velezensis MHNK1 produced 0.83 ±â€¯0.07 g/L of anionic biosurfactant that reduced surface tension from 72.12 ±â€¯0.02 to 33.2 ±â€¯0.61 mN/m and CMC was 40 mg/L with 85.21 ±â€¯1.60% emulsification index. Further, biosurfactant was characterized as surfactin by TLC, HPLC, FTIR, 1H and 13C NMR and LCMS-ESI. B. velezensis MHNK1 showed 87.10 ±â€¯3.10% atrazine biodegradation within 5 days which was revealed by HPLC and MS analysis. Atrazine biodegradation using a combination of B. velezensis MHNK1 (2%) and surfactin (2 CMC) resulted in 100 ±â€¯1.20% degradation within 4 days. Presence of atrazine degrading genes in B. velezensis MHNK1 was also confirmed by PCR. To the best of our knowledge, there are no previous reports available on atrazine degradation using B. velezensis strain and also in combination with surfactin. The results of this study reveal that strain B. velezensis MHNK1 and surfactin can be potential source of ecofriendly application for removal of atrazine from contaminated sites.

Statistical optimization of antifungal iturin A production from Bacillus amyloliquefaciens RHNK22 using agro-industrial wastes.

Biosurfactants are secondary metabolites with surface active properties and have wide application in agriculture, industrial and therapeutic products. The present study was aimed to screen bacteria for the production of biosurfactant, its characterization and development of a cost effective media formulation for iturin A production. A total of 100 bacterial isolates were isolated from different rhizosphere soil samples by enrichment culture method and screened for biosurfactant activity. Twenty isolates were selected for further studies based on their biosurfactant activity [emulsification index (EI%), emulsification assay (EA), surface tension (ST) reduction] and antagonistic activity. Among them one potential isolate Bacillus sp. RHNK22 showed good EI% and EA with different hydrocarbons tested in this study. Using biochemical methods and 16S rRNA gene sequence, it was identified as Bacillus amyloliquefaciens. Presence of iturin A in RHNK22 was identified by gene specific primers and confirmed as iturin A by FTIR and HPLC. B. amyloliquefaciens RHNK22 exhibited good surface active properties and antifungal activity against Sclerotium rolfsii and Macrophomina phaseolina. For cost-effective production of iturin A, 16 different agro-industrial wastes were screened as substrates, and Sunflower oil cake (SOC) was favouring high iturin A production. Further, using response surface methodology (RSM) model, there was a 3-fold increase in iturin A production (using SOC 4%, inoculum size 1%, at pH 6.0 and 37 °C temperature in 48 h). This is the first report on using SOC as a substrate for iturin A production.

Molecular dynamics and protein interaction studies of lipopeptide (Iturin A) on α- amylase of Spodoptera litura.

The small mottled willow moth (Spodoptera litura) is one of the best-known agricultural pest insects. To understand the insecticidal activity, we have selected iturin A compound produced by Bacillus amyloliquefaciens RHNK22 which showed the strongest and most common inhibitory effect on the Spodoptera litura protein. In this work we have identified the action of iturin A on α- amylase is a major digestive enzyme of Spodoptera litura using docking studies. A 3D model of α- amylase from Spodoptera litura was generated using 2HPH as a template with the help of Modeller7v7. With the aid of the molecular mechanics and molecular dynamics methods, the final model is obtained and is further checked by Procheck and Verify 3D graph programs, which showed that the final refined model is reliable. With this model, a adjustable docking study was performed with iturin A using GOLD software. The results indicated that ARG 18, THR15, LEU42 in α- amylase are important determinant residues in binding as they have strong hydrogen bonding interactions with iturin A. These hydrogen binding interactions play an important role for the stability of the complex.

Utilization of mango kernel oil for the rhamnolipid production by Pseudomonas aeruginosa DR1 towards its application as biocontrol agent.

Mango kernel oil (MKO), derived from mango kernels, considered to be one of the highly generated agro-industrial waste, is assessed for its use as substrate for sustainable production of rhamnolipids. In the present study, MKO in combination with glucose gave maximum rhamnolipid yield of 2.8g/l which reduced the surface tension of water from 72 to 30mN/m, holding a CMC of 80mg/l and also showed high emulsification activity (73%) with diesel. Cell free broth was found to be stable even at high temperature (autoclaved at 121°C for 30min), pH value (up to pH 12) and salinity (up to 20% NaCl). The LC-MS data showed mono-rhamnolipid to be predominant congener followed by di-rhamnolipid in presence of MKO. Whereas, di-rhamnolipid was abundant when a combination of MKO with glucose was used. The produced rhamnolipid mixture showed good antifungal activity against various phytopathogens.

Draft Genome Sequence of Bacillus amyloliquefaciens Strain RHNK22, Isolated from Rhizosphere with Biosurfactant (Surfactin, Iturin, and Fengycin) and Antifungal Activity.

Bacillus amyloliquefaciens strain RHNK22 isolated from groundnut rhizosphere showed direct and indirect plant growth-promoting traits along with biosurfactant activity and reduction in surface tension of water. Biosurfactants were identified as lipopeptides (surfactin, iturin, and fengycin) by molecular and biochemical analysis in our studies.