Mitigation of acrylamide in fried food systems using a combination of zein-pectin hydrocolloid complex and a food-grade l-asparaginase.

Acrylamide, a Maillard reaction product, formed in fried food poses a serious concern to food safety due to its neurotoxic and carcinogenic nature. A "Green Approach" using L-Asparaginase enzyme from GRAS-status bacteria synergized with hydrocolloid protective coating could be effective in inhibiting acrylamide formation. To fill this void, the present study reports a new variant of type-II L-asparaginase (AsnLb) from Levilactobacillus brevis NKN55, a food-grade bacterium isolated using a unique metabolite profiling approach. The recombinant AsnLb enzyme was characterized to study acrylamide inhibition ability and showed excellent specificity towards L-asparagine (157.2 U/mg) with Km, Vmax of 0.833 mM, 4.12 mM/min respectively. Pretreatment of potato slices with AsnLb (60 IU/mL) followed by zein-pectin nanocomplex led to >70% reduction of acrylamide formation suggesting synergistic effect of this dual component system. The developed strategy can be employed as a sustainable treatment method by food industries for alleviating acrylamide formation and associated health hazard in fried foods.

Production and characterization of an organic solvent activated protease from haloalkaliphilic bacterium Halobiforma sp. strain BNMIITR.

An unusual haloalkaliphilic bacterium known as Halobiforma sp. strain BNMIITR, which was noticed to produce an extracellular alkaline protease, was found in a soil sample from Northern India's Sambhar Lake. On the generation of protease, the effects of dietary elements including nitrogen and carbon sources, amino acids, and growth conditions like temperature and pH were investigated. When low-cost agricultural by-products were employed as nitrogen sources, the manufacturing of enzymes was significantly boosted. In the present study, protease production was enhanced by 2.94 fold and 2.17 fold. By solvent precipitation and Hydrophobic interaction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow matrix, the enzyme was purified 31.67 fold. It was determined that the apparent molecular mass was 21 kDa. The pH range where the enzyme was most stable was 6.0-12.0, with a temperature of 50 °C as optimum. When there was alkaline earth metals and heavy metals, protease was discovered to be active. It was evident that the enzyme was a serine type of protease because it was active in the presence of a variety of surfactants, oxidizing and reducing chemicals, and phenylmethylsulfonyl fluoride (PMSF) completely inhibited activity. Enzyme exhibited a wide range of substrate specificity. Amazingly, enzyme remained stable both in polar and nonpolar solvents. The most interesting aspect of this enzyme is enhanced activity in polar solvents like dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). It was discovered that the protease was stable and compatible with a number of widely available detergents.

Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Draft Genome Sequence of Alkalihalobacillus clausii Strain AXA-BCL3, Isolated from the Sahastradhara Springs of Uttarakhand, India.

Here, we report the draft genome sequence of Alkalihalobacillus clausii strain AXA-BCL3, which was isolated from a soil sample from the Sahastradhara springs of Uttarakhand, India. The genome was assembled in 125 contigs with a total length of 4,428,477 bp and a GC content of 44.5%. Genome annotation predicted 4,278 protein-coding genes and 75 tRNA genes.

Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Genetic regulatory element based whole-cell biosensors for the detection of metabolic disorders.

Clinicians require simple, and cost-effective diagnostic tools for the quantitative determination of amino acids in physiological fluids for the detection of metabolic disorder diseases. Besides, amino acids also act as biological markers for different types of cancers and cardiovascular diseases. Herein, we applied an in-silico based approach to identify potential amino acid-responsive genetic regulatory elements for the detection of metabolic disorders in humans. Identified sequences were further transcriptionally fused with GFP, thus generating an optical readout in response to their cognate targets. Screening of genetic regulatory elements led us to discover two promoter elements (pmetE::GFP and ptrpL::GFP) that showed a significant change in the fluorescence response to homocysteine and tryptophan, respectively. The developed biosensors respond specifically and sensitively with a limit of detection of 3.8 µM and 3 µM for homocysteine and tryptophan, respectively. Furthermore, the clinical utility of this assay was demonstrated by employing it to identify homocystinuria and tryptophanuria diseases through the quantification of homocysteine and tryptophan in plasma and urine samples within 5 h. The precision and accuracy of the biosensors for disease diagnosis were well within an acceptable range. The general strategy used in this system can be expanded to screen different genetic regulatory elements present in other gram-negative and gram-positive bacteria for the detection of metabolic disorders.

A chemical genetic approach using genetically encoded reporters to detect and assess the toxicity of plant secondary metabolites against bacterial pathogens.

Plant secondary metabolites are emerging as attractive alternatives in the development of therapeutics against infectious and chronic diseases. Due to the present pandemic, therapeutics showing toxicity against bacterial pathogens and viruses are gaining interest. Plant metabolites of terpenoid and phenylpropanoid categories have known antibacterial and antiviral properties. These metabolites have also been associated with toxicity to eukaryotic cells in terms of carcinogenicity, hepatotoxicity, and neurotoxicity. Sensing methods that can report the exact antibacterial dosage, formation, and accumulation of these antibacterial compounds are needed. The whole-cell reporters for such antibacterial metabolites are cost-effective and easy to maintain. In the present study, battery of toxicity sensors containing fluorescent transcriptional bioreporters was constructed, followed by fine-tuning the response using gene-debilitated E. coli mutants. This study shows that by combining regulatory switches with chemical genetics strategy, it may be possible to detect and elucidate the mode of action of effective antibacterial plant secondary metabolites - thymol, cinnamaldehyde, eugenol, and carvacrol in both pure and complex formats. Apart from the detection of adulteration of pure compounds present in complex mixture of essential oils, this approach will be useful to detect authenticity of essential oils and thus reduce unintended harmful effects on human and animal health.

Draft Genome Sequence of Limosilactobacillus fermentum Strain NKN-51, Isolated from Fermented Yak Milk in the Western Himalayas of India.

Here, we report the draft genome sequence of Limosilactobacillus fermentum strain NKN-51, which was isolated from naturally processed yak cheese from the western Himalayas of India. The genome was assembled in 101 contigs with a total length of 1,879,705 bp and a GC content of 53.5%. Genome annotation predicted 1,730 protein-coding genes and 50 tRNA genes.

Homogentisic Acid-Based Whole-Cell Biosensor for Detection of Alkaptonuria Disease.

Clinicians require simple quantitative tools for the detection of homogentisic acid in alkaptonuria patients, a rare inherited disorder of amino acid metabolism. In this study, we report a whole-cell biosensor for homogentisic acid to detect alkaptonuria disease through the expression of green fluorescence protein. The assay system utilizes a promoter sequence (hmgA) isolated from the Pseudomonas aeruginosa genome. To increase the sensitivity, the sensor module harboring phmgA::GFP was further transformed into various transposon mutants debilitated in steps involved in the metabolism of phenylalanine and tyrosine via homogentisic acid as a central intermediate. The proposed biosensor was further checked for analytical features such as sensitivity, selectivity, linearity, and precision for the quantification of homogentisic acid in spiked urine samples. The limit of detection for the developed biosensor was calculated to be 3.9 µM, which is comparable to that of the various analytical techniques currently in use. The sensor construct showed no interference from all of the amino acids and its homolog molecules. The accuracy and precision of the proposed biosensor were validated using high-performance liquid chromatography (HPLC) with satisfactory results.

Transcription factor based whole-cell biosensor for specific and sensitive detection of sodium dodecyl sulfate.

Extensive use of Sodium Dodecyl Sulfate (SDS) in households, agricultural operations, and industries is leading to its subsequent disposal in waterways. There is an apprehension of the adverse effect of such detergents on various living organisms. Thus, an efficient, specific, and simple detection method to monitor SDS reliably in the environment is needed. We used sdsB1 activator protein and SDS-responsive promoter of sdsA1 gene along with Green Fluorescent Protein (GFP) to construct a novel SDS biosensor in Pseudomonas aeruginosa chassis. The GFP intensity of the biosensor showed a linear relationship (R2 = 0.99) from 0.4 to 62.5 ppm of SDS with a detection limit of 0.1 ppm. This biosensor is highly specific for SDS and has minimal interference from other detergents, metals, and inorganic ions. The biosensor showed a satisfactory and reproducible recovery rate for the detection of SDS in real samples. Overall, this is a low cost, easy-to-use, selective, and reliable biosensor for monitoring SDS in the environment.

Magnetically recyclable catalytic nanoparticles grafted with Bacillus subtilis ß-glucosidase for efficient cellobiose hydrolysis.

This study reports covalent immobilization of ß-glucosidase (BGL) from Bacillus subtilis PS on magnetically recyclable iron nanoparticles for enhancing robustness, facile recovery and reuse of enzyme. Immobilized BGL iron nanoparticles (BGL-INPs) were characterized by various biophysical techniques viz. TEM, DLS, FTIR and CD spectroscopy. The efficiency and yield of immobilization were 89.78 and 84.80%, respectively. After immobilization, optimum pH remained 6.0 whereas optimum temperature upraised to 70 °C whereas apparent Km and Vmax shifted from 0.819 mM to 0.941 mM and 54.46 to 57.67 µmole/min/mg, respectively. Immobilization conferred lower activation energy and improved pH and thermal stabilities. The BGL-INPs retained 85% activity up to 10th cycle of reuse and hydrolyzed more than 90% of cellobiose to glucose within 30 min. Conclusively, improved pH, thermal stability and excellent reusability over free enzyme make BGL-INPs a promising candidate for sustainable bioethanol production and other industrial applications.

Electrostatic Surface Potential of Macrophages Correlates with Their Functional Phenotype.

Macrophages exist in various functional phenotypes, which could be identified by specific surface molecules. Previous studies have shown that modulation of surface charges could alter the phagocytic function of macrophages. In this study, we show that activation of both human peripheral blood monocyte and THP-1-derived macrophages with lipopolysaccharide (LPS) or IL-1ß resulted in a significant decrease in the zeta potential compared to freshly isolated monocytes and unstimulated macrophages. Interestingly, interaction with bacteria significantly increased the zeta potential of such cells irrespective of activation conditions. Similarly, IFNγ-treated pro-inflammatory macrophages showed lesser negative zeta potential compared to untreated control. A moderate reduction was also seen in IL-4-treated anti-inflammatory subtype. Additionally, in an LPS-induced systemic inflammation model, bone marrow cells isolated after 2 h of LPS injection showed significant reduction in zeta potential compared to naïve cells. Furthermore, electrostatic potential measurement of surface proteins associated with pro-inflammatory and anti-inflammatory macrophages, using in silico modeling under physiological and protonation conditions, showed that the average electrostatic potential of pro-inflammatory type surface proteins was less negative than anti-inflammatory subtype. These data suggest that the expression of different protein molecules on macrophages under different environments may contribute to the zeta potential and that this quick and low-cost technique could be used in monitoring macrophage functional phenotypes.

Nucleic Acid Aptamers as Emerging Tools for Diagnostics and Theranostics.

Aptamers are ssDNA or RNA sequences (20-80 nucleotides) generated in vitro by SELEX (Systematic Evolution of Ligands using EXponential enrichment) against diverse range of targets from small molecules to bacteria, viruses, and even eukaryotic cells. Aptamers, also known as chemical bodies, bind to their respective targets with tunable affinity and specificity, making aptamers as potent probes for diagnostics and excellent ligands for drug delivery in therapeutics. In this chapter, we have described the methods for generating DNA aptamers against proteins and their use in theranostics.

Mechanistic Insights Into Probiotic Properties of Lactic Acid Bacteria Associated With Ethnic Fermented Dairy Products.

Gut microbes and their metabolites maintain the health and homeostasis of the host by communicating with the host via various biochemical and physical factors. Changing lifestyle, chronic intake of foods rich in refined carbohydrates and fats have caused intestinal dysbiosis and other lifestyle-based diseases. Thus, supplementation with probiotics has gained popularity as biotherapies for improving gut health and treating disorders. Research shows that probiotic organisms enhance gastrointestinal health, immunomodulation, generation of essential micronutrients, and prevention of cancer. Ethnically fermented milk and dairy products are hotspots for novel probiotic organisms and bioactive compounds. These ethnic fermented foods have been traditionally prepared by indigenous populations, and have preserved unique microflora for ages. To apply these unique microflora for amelioration of human health, it is important that probiotic properties of the bacterial species are well studied. Majority of the published research and reviews focus on the probiotic organisms and their properties, fermented food products, isolation techniques, and animal studies with their health pathologies. As a consequence, there is a dearth of information about the underlying molecular mechanism behind probiotics associated with ethnically prepared dairy foods. This review is targeted at stimulating research on understanding these mechanisms of bacterial species and beneficial attributes of ethnically fermented dairy products.

A Combination of Linalool, Vitamin C, and Copper Synergistically Triggers Reactive Oxygen Species and DNA Damage and Inhibits Salmonella enterica subsp. enterica Serovar Typhi and Vibrio fluvialis.

Inappropriate and disproportionate use of antibiotics is contributing immensely to the development of antibiotic resistance in bacterial species associated with food contamination. The use of natural products in combination can be a potent alternative hurdle strategy to inactivate foodborne pathogens. Here, we explored the pro-oxidant properties of essential oil linalool and vitamin C in combination with copper (LVC) in combating the foodborne pathogens Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi using a three-dimensional (3D) checkerboard microdilution assay. Antibacterial activity in terms of the MIC revealed that the triple combination exerted a synergistic effect compared to the effects of the individual constituents. The bactericidal effect of the triple combination was confirmed by a live/dead staining assay. Reactive oxygen species (ROS) measurements with the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay and scanning electron microscopy imaging strongly suggested that the increase in ROS production is the underlying mechanism of the enhanced antibacterial potency of the LVC combination (linalool [1.298 mM], vitamin C [8 mM], copper [16.3 µM]). In addition, the hypersensitivity of oxidative stress regulator mutants (oxyR, katG, ahpC, and sodA mutants) toward LVC corroborated the involvement of ROS in cell death. Live/dead staining and changes in cellular morphology revealed that oxidative stress did not transform the cells into the viable but nonculturable (VBNC) state; rather, killing was associated with intracellular and extracellular oxidative burst. Furthermore, the LVC combination did not display toxicity to human cells, while it effectively reduced the pathogen levels in acidic fruit juices by 3 to 4 log CFU/ml without adversely altering the organoleptic properties. This study opens a new outlook for combinatorial antimicrobial therapy.IMPORTANCE There is a need to develop effective antibacterial therapies for mitigating bacterial pathogens in food systems. We used a 3D checkerboard assay to ascertain a safe synergistic combination of food-grade components: vitamin C, copper, and the essential oil linalool. Individually, these constituents have to be added in large amounts to exert their antibacterial effect, which leads to unwanted organoleptic properties. The triple combination could exceptionally inhibit foodborne Gram-negative pathogens like Vibrio fluvialis and Salmonella enterica subsp. enterica serovar Typhi at low concentrations (linalool, 1.298 mM; vitamin C, 8 mM; copper, 16.3 µM) and displayed potent microbial inhibition in acidic beverages. We found increased susceptibility in deletion mutants of oxidative stress regulators (oxyR, katG, ahpC, and sodA mutants) due to ROS generation by Fenton's chemistry. The results of this study show that it may be possible to use plant-based antimicrobials in synergistic combinations to control microbial contaminants.

Retracted: Bacteria diversity overview and endoglucanase assessment from Himalayan Tapovan geothermal spring.

The above article from the Journal of Basic Microbiology, published online on 25 August 2015 in Wiley Online Library as Early View (http://onlinelibrary.wiley.com/doi/10.1002/jobm.201500135/pdf), has been retracted by agreement between Naveen Kumar Navani and Ranjana Pathania, the Editor-in-Chief and Wiley-VCH GmbH & Co. KGaA. The retraction has been agreed because the article has been submitted and approved for publication by Jitendra Kumar Sahoo without consent in any form by the named co-authors Naveen Kumar Navani and Ranjana Pathania.

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach.

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell-cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.

Secretory expression, characterization and docking study of glucose-tolerant ß-glucosidase from B. subtilis.

The thermostable, glucose tolerant ß-glucosidase gene (bgl) of Glycoside hydrolase family 1, isolated from Bacillus subtilis, was cloned and overexpressed in Escherichia coli. The bgl has open reading frame of 1,407 bp, encoding 469 amino acids with predicted molecular weight of 53 kDa. The recombinant protein (BGL) was purified 10.76 fold to homogeneity with specific activity of 54.04U/mg and recovery of 38.67%. The purified BGL was optimally active at pH 6.0 and temperature 60°C. The enzyme retained more than 85% of maximum activity after 1h preincubation at 60°C. The kinetic analysis indicated that BGL has highest catalytic efficiency (Kcat/Km) against p-nitrophenyl-ß-d-xylopyranoside (654.58 mM(-1)s(-1)) followed by p-nitrophenyl-ß-d-glucopyranoside (292.53 mM(-1)s(-1)) and p-nitrophenyl-ß-d-galactopyranoside (61.17 mM(-1)s(-1)). The Ki value for glucose and δ-gluconolactone was determined to be 1.9 mM and 0.018 mM, respectively. The BGL exhibited high tolerance against detergents and organic solvents. The homology modeling revealed that protein has 19 α-helices and 4 ß-sheets and adopted (α/ß)8 TIM barrel structure. Substrate docking and LigPlot analysis depicted the amino acids of active site involved in hydrogen bonding and hydrophobic interactions with substrates. The efficient BGL secretion with exploration of structural and functional relationship offer vistas for large scale production and various industrial applications.

Antibacterial potential of a small peptide from Bacillus sp. RPT-0001 and its capping for green synthesis of silver nanoparticles.

Infirmity and death from diseases caused by unsafe food are a continual hazard to communal health safety and socio-economic growth throughout the world. Chemical preservatives are associated with health hazards and toxicity issues. In the study reported here, 200 soil isolates from Western Himalayan region in India were screened for potential antibacterial activity against food-borne pathogens. This study led to the isolation of a bacterial strain belonging to the Genus Bacillus and was designated as RPT-0001. The associated antibacterial activity was sensitive to pronase E treatment. Bioassay-guided fractionation using reverse phase high performance liquid chromatography (RP-HPLC) led to isolation of the antibacterial peptide designated as RPT-0001. The molecular weight of RPT-0001 was determined by electro-spray ionization mass spectroscopy (ESI-MS) as 276.9 Da. RPT-0001 was inhibitory to both Gram-negative and Grampositive food-borne bacteria tested. The characteristics of RPT-0001 do not match with that of any other known antibacterial peptides produced by Bacillus sp. or related genera. Purified RPT-0001 was successfully used in synthesis of silver nanoparticles effective against food-borne pathogenic bacteria. The antibacterial peptide and silver nanoparticles synthesized utilizing it as a capping and reducing agent hold promising potential in food preservation, in packaging material and as a therapeutic agent in the treatment of foodborne infections.

Non-enzymatic detection of urea using unmodified gold nanoparticles based aptasensor.

Biosensing nitrogenous compounds like urea is required to control the incidents of Economically Motivated Adulteration (EMA). In this study, we report the FluMag Systematic Evolution of Ligands by Exponential Enrichment (FluMag-SELEX) method to isolate a urea specific DNA aptamer with a dissociation constant (Kd) of 232 nM. The interaction of DNA aptamer with urea has been confirmed by affinity assay, CD analysis, melting curve analysis and truncation studies. Unlike other urea sensing methods reported so far, using this urea aptamer, we demonstrate a simple, 'non-enzymatic' easy-to-use, dual readout aptasensor that exploits unmodified gold nanoparticles (AuNPs) to transduce the signals of aptamer binding to urea in terms of intrinsic fluorescence differences and color changes simultaneously. This method is free from complicated sample processing and labeling steps. The urea aptasensor displays high selectivity for urea and is free from interference from common milk adulterants. The developed aptasensor reliably detects urea adulteration in milk. The response signals linearly correlate with the increasing concentrations of urea in milk ranging from 20mM to 150 mM with detection limit of 20mM. We also show that this aptasensor can also be used as a simple fluorescence based "turn-on" sensor. The results obtained in this study are comparable to the commercial urease based detection methods.