New insights into the bioactivity of peptides from probiotics.

Probiotics are unique bacteria that offer several therapeutic benefits to human beings when administered in optimum amounts. Probiotics are able to produce antimicrobial substances, which stimulate the body's immune responses. Here, we review in detail the anti-infective peptides derived from probiotics and their potential immunomodulatory and anti-inflammatory activities, including a major role in cross-talk between probiotics and gut microbiota under adverse conditions. Insights from the engineered cell surface of probiotics may provide novel anti-infective therapy by heterologous expression of receptor peptides of bacterial toxins. It may be possible to use antigenic peptides from viral pathogens as live vaccines. Another possibility is to generate antiviral peptides that bind directly to virus particles, while some peptides exert anti-inflammatory and anticancer effects. Some extracellular polymeric substances might serve as anti-infective peptides. These avenues of treatment have remained largely unexplored to date, despite their potential in generating powerful anti-inflammatory and anti-infective products.

Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC-MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis.

Dissemination of antibiotic resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant S aureus strains isolated from hospital effluents.

Vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S aureus (MRSA) strains were examined in hospital effluents. Most S aureus strains are resistant to methicillin (MRSA), followed by tetracycline. Approximately 15% of MRSA strains are also resistant to vancomycin (VRSA). All VRSA strains developed a VanR/VanS-regulated 2-component system of VanA-type resistance in their genome. Results indicate that there is a possibility of developing resistance to aminoglycosides by VRSA strains in the near future.

Enzymatic hydrolyzed feather peptide, a welcoming drug for multiple-antibiotic-resistant Staphylococcus aureus: structural analysis and characterization.

This study aimed to explore the bactericidal activity of a feather-degraded active peptide against multiple-antibiotic-resistant (MAR) Staphylococcus aureus. An antibacterial peptide (ABP) was isolated from the chicken feathers containing fermented media of Paenibacillus woosongensis TKB2, a keratinolytic soil isolate. It was purified by HPLC, and its mass was found to be 4666.87 Da using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectroscopy. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this peptide were 22.5 and 90 µg/ml, respectively. SEM study revealed the distorted cell wall of the test strain along with pore formation. The possible reason for bactericidal activity of the peptide is due to generation of reactive oxygen species (ROS), resulting in membrane damage and leakage of intracellular protein. Complete sequence of the peptide was predicted and retrieved from the sequence database of chicken feather keratin after in silico trypsin digestion using ExPASy tools. Further, net charge, hydrophobicity (77.7 %) and molecular modelling of the peptide were evaluated for better understanding of its mode of action. The hydrophobic region (17 to 27) of the peptide may facilitate for initial attachment on the bacterial membrane. The ABP exhibited no adverse effects on RBC membrane and HT-29 human cell line. This cytosafe peptide can be exploited as an effective therapeutic agent to combat Staphylococcal infections.

Role of probiotic Lactobacillus fermentum KKL1 in the preparation of a rice based fermented beverage.

A dominant lactic acid bacteria, Lactobacillus fermentum KKL1 was isolated from an Indian rice based fermented beverage and its fermentative behavior on rice was evaluated. The isolate grown well in rice and decreased the pH, with an increase of total titratable acidity on account of high yield in lactic acid and acetic acid. The production of α-amylase and glucoamylase by the strain reached plateau on 1st and 2nd day of fermentation respectively. The accumulation of malto-oligosaccharides of different degrees of polymerization was also found highest on 4th day. Besides, phytase activity along with accumulation of free minerals also unremittingly increased throughout the fermentation. The fermented materials showed free radical scavenging activity against DPPH radicals. In-vitro characteristics revealed the suitability of the isolate as probiotic organism. The above profiling revealed that probiotic L. fermentum KKL1 have the significant impact in preparation of rice beer and improves its functional characteristics.

Microbial, saccharifying and antioxidant properties of an Indian rice based fermented beverage.

Haria, a popular rice based ethnic fermented beverage, is consumed as a staple food and refreshing drink by the vast number of Indian tribal people. In this study, the composition of microbial consortia and the occurrence of some important nutraceuticals during haria preparation were investigated. The quantities of moulds and yeasts were highest at 2nd day, and then declined, but, on the contrary, the quantity of Lactic Acid Bacteria and Bifidobacterium sp. increased concurrently during the course of fermentation. Accumulation of starch hydrolytic enzymes along with different types of malto-oligosaccharides like maltotetrose (26.18µg/gm), maltotriose (28.16µg/gm), and maltose (26.94µg/gm) were also noted. Furthermore, GC-MS analysis indicated the occurrence of pyranose derivatives in the fermented products. The fermented materials showed higher free radicals scavenging activity (82.54%, 4th day) against DPPH radicals. These studies clearly demonstrated that the microbial interaction during fermentation of rice makes it more nutritious, and most likely more beneficial for health.

Hypobaric-hypoxia induces alteration in microbes and microbes-associated enzyme profile in rat colonic samples.

Present study deals with the straight impact of hypobaric hypoxia on the quantity and composition of some predominant fecal microflora and its functional aspects. For that, isolated fecal contents of rat were exposed to two different simulated air pressures (70 kPa and 40 kPa) for different time durations (1, 3, and 5 h) and the bacterial community composition was compared with normobaric groups (101.3 kPa). It was found that the total anaerobes, Escherichia coli, Enterbacters spp., Bifidobacterium spp., Clostridium spp. were increased whereas total aerobes were decreased at both hypobaric treatments. The increased number of amplicon was detected in the pressure-treated groups than the control that clearly mentioned the disruption of microbiota structure at different simulated hypobaric-hypoxia. The amylase, protease, tannase, ß-glucuronidase, and alkaline phosphatase activities were increased at these atmospheric pressures. Thus, the present investigation demonstrates that the hypobaric hypoxia is an important environmental factor which can strongly modulate the composition of intestinal flora as well as microflora-derived functional aspects.

Low cost single-step purification of endoglucanase from Aspergillus fumigatus ABK-9.

Low cost agro-waste was used as adsorption support for single-step purification of endoglucanase from the culture filtrate of A. fumigatus ABK-9. Among various agro-waste substrates, 1% NaOH pretreated rice bran was proved to be the best for adsorbing about 74.8 and 71.1% of endoglucanase at 4 degrees C and 10 degrees C respectively. Langmuir type adsorption isotherm at 4 degrees C showed maximum adsorption of enzyme at pH 5.0, which was in the range of optimum pH of the enzyme. The rice bran column bound enzyme was maximally eluted by a mixture of acetate buffer (0.05 M, pH 5.5) and ethanol (40%, v/v) at a ratio of 3:2 and a flow rate of 1 mL/min. A 5.52-fold purification of the enzyme was achieved from culture supernatant. The specific activity and recovery yield after purification were 294.0 U/mg and 40.15%, respectively, which were comparable with other contemporary protocols. The homogeneity of the enzyme was tested through sodium dodecyl sulphate polyacrylamide gel electrophoresis and a single band of 56.3 kDa was observed. Zymogram analysis finally confirmed the occurrence of endoglucanase in the single band.

Production of cellulolytic enzymes by Aspergillus fumigatus ABK9 in wheat bran-rice straw mixed substrate and use of cocktail enzymes for deinking of waste office paper pulp.

Response surface methodology was employed to optimize mixed substrate solid state fermentation for the production of cellulases and xylanase by Aspergillus fumigatus ABK9. Among 11 different parameters, fermentation time (86-88 h), medium pH (6.1-6.2), substrate amount (10.0-10.5 g) and substrate ratio (wheat bran:rice straw) (1.1) had significantly influences on enzyme production. Under these conditions endoglucanase, ß-glucosidase, FPase (filter paper degrading activity) and xylanase activities of 826.2, 255.16, 102.5 and 1130.4 U/g, respectively were obtained. The enzyme cocktail extracted (solid to water ratio of 1:10) from the ferments increased brightness of waste office paper pulp by 82.8% ISO, Ink(D) value by 82.1%, removed chromophores (2.53 OD; A(237)nm) and hydrophobic compounds (1.15 OD; A(465)nm) and also decreased the kappa number to 13.5 from 16.8.

Biotransformation of p-coumaric acid and 2,4-dichlorophenoxy acetic acid by Azotobacter sp. strain SSB81.

A comprehensive study was made on biotransformation of p-coumaric acid and 2,4-dichlorophenoxyacetic acid by an Azotobacter sp. strain SSB81. The strain was able to tolerate a high amount of both the phenolic acids and p-coumaric acid degraded maximum (50%) than 2,4-D (29%) after five days of incubation. The intermediate products during transformation have been identified and quantified using UV-Vis and LC-MS/MS analysis. Para-coumaric acid was degraded via p-hydroxybenzoic acid and protocatechuic acid, a non-oxidative pathway whereas 2,4-D via 4-chlorophenoxyacetic acid, 4-chlorophenol and 4-chlorocatechol, an oxidative pathway. The results suggest that SSB81 developed both the oxidative and non-oxidative pathway to degrade the soil accumulated phenolic acids. Thus, Azotobacter provides an advantage to reduce the toxic level of soil accumulated phenolic acids in addition to increase the soil fertility.

Impact of Azotobacter exopolysaccharides on sustainable agriculture.

Recently, increasing attention have lead to search other avenue of biofertilizers with multipurpose activities as a manner of sustainable soil health to improve the plant productivity. Azotobacter have been universally accepted as a major inoculum used in biofertilizer to restore the nitrogen level into cultivated field. Azotobacter is well characterized for their profuse production of exopolysaccharides (EPS). Several reviews on biogenesis and multifunctional role of Azotobacter EPS have been documented with special emphasis on industrial applications. But the impact of Azotobacter EPS in plant growth promotion has not received adequate attention. This review outlines the evidence that demonstrates not only the contribution of Azotobacter EPS in global nutrient cycle but also help to compete successfully in different adverse ecological and edaphic conditions. This also focuses on new insights and concepts of Azotobacter EPS which have positive effects caused by the biofilm formation on overall plant growth promotion with other PGPRs. In addition, their potentials in agricultural improvement are also discussed. Recent data realized that Azotobacter EPS have an immense agro-economical importance including the survivability and maintenance of microbial community in their habitat. This leads us to confirm that the next generation Azotobacter inoculum with high yielding EPS and high nitrogen fixing ability can be utilized to satisfy the future demand of augmented crop production attributed to increase plant growth promoting agents.

Xylanase isozymes from the newly isolated Bacillus sp. CKBx1D and optimization of its deinking potentiality.

Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480 U/ml) at 36 h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94 kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2 h of continuous shaking at constant temperature of 35 °C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.

Removal of arsenic from aqueous solution using pottery granules coated with cyst of Azotobacter and portland cement: characterization, kinetics and modeling.

A new low cost adsorbents, pottery granules coated with cyst of Azotobacter and portland cement has been developed for aqueous arsenic removal. The developed granule is solid and porous structure forms a stable complex of Fe-Al-Si-O(2) allied with cyst biomass. Batch experiments were revealed that As removal was up to 96% using PGAC beads, whereas 65% by cyst biomass. Immobilization of cyst biomass to pottery granules through portland cement improved the stability of granules and adsorption capacity. Kinetics studies revealed that Langmuir isotherm was followed with a better correlation than the Freundlich isotherm and adsorption was first order diffusion controlled. Presence of Fe-Al-Si-O(2) and polysaccharide complex on the granule surface may be responsible for the adsorption of arsenic and preferentially binds to biomass containing composite than only biomass. Thus, this recently developed cost-effective novel biocomposite, PGAC granule can be used as household level to mitigate the arsenic problem.

Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis.

Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 µg/ml and 16 µg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.

Effect of Arsenic on Nodulation and Nitrogen Fixation of Blackgram (Vigna mungo).

Rhizobium-legume symbiotic interaction is an efficient model system for soil remediation and reclamation. We earlier isolated an arsenic (As) (2.8 mM arsenate) tolerant and symbiotically effective Rhizobium strain, VMA301 from Vigna mungo and in this study we further characterized its efficacy for arsenic removal from the soil and its nitrogen fixation capacity. Although nodule formation is delayed in plants with As-treated composite when the inoculum was prepared without arsenic in culture medium, whereas it attains the significant number of nodules compare to plant grown in As-free soil when the inoculum was prepared with arsenic supplemented medium. Arsenic accumulation was higher in roots than root nodules. Nitrogenase activity is reduced to almost 2 fold in plants with As-treated soil but not abolished. These results suggest that this strain, VMA301, has been able to establish an effective symbiotic interaction in V. mungo in As-contaminated soil and can perform dual role of arsenic bioremediation as well as soil nitrogen improvement.

Effect of amino acids on tannase biosynthesis by Bacillus licheniformis KBR6.

BACKGROUND AND PURPOSE: Microbial tannase (tannin acyl hydrolase, EC 3.1.1.20), a hydrolysable tannin-degrading enzyme, has gained importance in various industrial processes, and is used extensively in the manufacture of instant tea, beer, wine, and gallic acid. Tannase is an inducible enzyme, and hydrolysable tannin, especially tannic acid, is the sole inducer. This study is of the effect of various amino acids and their analogues on tannase biosynthesis by Bacillus licheniformis KBR6 to ascertain the mode of action of these growth factors on tannase biosynthesis from microbial origin. METHODS: Enzyme production was carried out in enriched tannic acid medium through submerged fermentation for 20 h at 35 degrees C. Different amino acids at a concentration of 0.05 g% (w/v) were added to the culture medium immediately after sterilization. Culture supernatant was used as the source of the enzyme and the quantity of tannase was estimated by the colorimetric assay method. Growth of the organism was estimated according to biomass dry weight. RESULTS: Maximum tannase (2.87-fold that of the control) was synthesized by B. licheniformis KBR6 when alanine was added to the culture medium. Other amino acids, such as DL-serine, L-cystine, glycine, L-ornithine, aspartic acid, L-glutamic acid, DL-valine, L-leucine and L-lysine, also induced tannase synthesis. L-Cysteine monohydrochloride and DL-threonine were the most potent inhibitors. CONCLUSIONS: Regulation of tannase biosynthesis by B. licheniformis in the presence of various amino acids is shown. This information will be helpful for formulating an enriched culture medium for industrial-scale tannase production.

Proteomics view of a Rhizobium isolate response to arsenite [As(III)] stress.

The genus Rhizobium colonizes in leguminous plants by symbiotic relationship and enriches soil nitrogen through the formation of the root nodule. Legumes are the appropriate crops for the recovery of marginal lands and they can easily grow in adverse climatic condition. Rhizobium sp. VMA301 was isolated from the root nodules of V. mungo, grown in arsenic contaminated field. The LC50 value of arsenite for VMA301 was found to be 1.8 mM. Sixteen differentially expressed proteins were identified using RP-HPLC and MALDI ToF mass spectrometry from arsenite induced whole cell lysate soluble proteins. It is also found that nine proteins were up-regulated and seven proteins were down-regulated in comparison to the control group (cells grown without arsenite). These differential protein expressions mitigate the toxic effect of arsenite and stimulate the detoxification process.

Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81.

A strain was selected by its highest extracellular polysaccharide (EPS) production ability compare to other isolates from the same rhizospheric soil. The selected strain was identified by 16S rDNA sequencing and designated as SSB81. Phylogenetic analysis of the gene sequence showed its close relatedness with Azotobacter vinelandii and Azotobacter salinestris. Maximum EPS (2.52 g l(-1)) was recovered when the basal medium was supplemented with glucose (2.0%), riboflavin (1 mg l(-1)) and casamino acid (0.2%). The EPS showed a stable viscosity level at acidic pH (3.0-6.5) and the pyrolysis temperature was found to be at 116.73 degrees C with an enthalpy (DeltaH) of 1330.72 J g(-1). MALDI TOF mass spectrometric result suggests that polymer contained Hex(5)Pent(3) as oligomeric building subunit. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. This novel EPS may find possible application as a polymer for environmental bioremediation and biotechnological processes.

Stimulation of indoleacetic acid production in a Rhizobium isolate of Vigna mungo by root nodule phenolic acids.

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in L-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 microg ml(-1)) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium).

Characterization of a symbiotically effective Rhizobium resistant to arsenic: Isolated from the root nodules of Vigna mungo (L.) Hepper grown in an arsenic-contaminated field.

Bacteria were isolated from the root nodules of Vigna mungo (L.) Hepper, grown in an arsenic-contaminated field and the strain was selected by its nodulation ability as well as better arsenic tolerant capacity compared to others. The selected strain was identified as Rhizobium by 16S rDNA sequencing and designated as VMA301. Phylogenetic analysis of the gene sequences showed its close relatedness with Sinorhizobium fredii. LC(50) value of arsenate for the bacteria as determined by flow cytometry was found to be 2.8 mM and arsenic uptake was measured by atomic absorption spectrometry as 0.048 mg g(-1) biomass. The high amount of arsenic was toxic to the cell, which changed the morphology of the bacteria to an elongated shape. Presence of a transcriptional regulatory gene (ArsR) of the ars genetic system was confirmed by amplification and sequencing. The symbiotic property of the isolate was also confirmed by amplification and sequencing of the NodC gene. These results indicate that the isolated Rhizobium bacteria may exert dual roles in the environment, arsenic bioremediation from the soil as well as increase of soil fertility through nitrogen fixation.