Applications of Nanotechnology in Sensor-Based Detection of Foodborne Pathogens.

The intake of microbial-contaminated food poses severe health issues due to the outbreaks of stern food-borne diseases. Therefore, there is a need for precise detection and identification of pathogenic microbes and toxins in food to prevent these concerns. Thus, understanding the concept of biosensing has enabled researchers to develop nanobiosensors with different nanomaterials and composites to improve the sensitivity as well as the specificity of pathogen detection. The application of nanomaterials has enabled researchers to use advanced technologies in biosensors for the transfer of signals to enhance their efficiency and sensitivity. Nanomaterials like carbon nanotubes, magnetic and gold, dendrimers, graphene nanomaterials and quantum dots are predominantly used for developing biosensors with improved specificity and sensitivity of detection due to their exclusive chemical, magnetic, mechanical, optical and physical properties. All nanoparticles and new composites used in biosensors need to be classified and categorized for their enhanced performance, quick detection, and unobtrusive and effective use in foodborne analysis. Hence, this review intends to summarize the different sensing methods used in foodborne pathogen detection, their design, working principle and advances in sensing systems.

Xanthine oxidase of Acinetobacter calcoaceticus RL2-M4: Production, purification and characterization.

Xanthine oxidase (EC 1.17.3.2) is a key enzyme of purine metabolism and has potential applications in food and pharmaceutical industries. In the present study, a new bacterial source of xanthine oxidase i.e. Acinetobacter calcoaceticus RL2-M4 with high oxidase activity was isolated from soil. The culture conditions were optimized with one variable at a time (OVAT) and response surface methodology (RSM) approaches included: a minimal salt medium (MSM) of pH 7.0 supplemented with 0.8% yeast extract, 8.5 mM xanthine and incubation at 30 °C for 24 h. Under these culture conditions 11.57 fold increase in the production of this enzyme was achieved. The enzyme was purified from A. calcoaceticus RL2-M4 using anion exchange chromatography to 8.18 fold with 31% yield and specific activity of 4.58 U/mg protein. SDS-PAGE analysis of the purified enzyme revealed that it was homodimer of 95 kDa and its native molecular mass was estimated to be 190 kDa. This enzyme was found to be stable at 35 °C for 5 h. The purified xanthine oxidase of A. calcoaceticus RL2-M4 had Km 0.3 mM and Vmax 5.8 U/mg protein using xanthine as substrate. The activity and stability characteristic of xanthine oxidase of A. calcoaceticus RL2-M4 makes it a potentially good enzyme for industrial applications.

Molecular insights into the activity and mechanism of cyanide hydratase enzyme associated with cyanide biodegradation by Serratia marcescens.

The present study provides molecular insights into the activity and mechanism of cyanide hydratase enzyme associated with degradation of cyanide compounds, using Serratia marcescens RL2b as a model organism. Resting cells harvested after 20 h achieved complete degradation of 12 mmol l- 1 cyanide in approximately 10 h. High-performance liquid chromatography analysis of reaction samples revealed formation of formamide as the only end product, which confirmed the presence of cyanide hydratase activity in S. marcescens RL2b. Comparative structural analysis with the other nitrilase family proteins, which was carried out using a sequence of cyanide hydratase from a phylogenetically related strain S. marcescens WW4, also revealed subtle but significant differences in amino acid residues of the substrate-binding pocket and catalytic triad (Cys-Lys-Glu).

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase.

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters KM and Vmax for free and immobilized cutinase were found to be 0.55mM and 1410µmolmin-1mg-1 protein, 2.99mM and 996µmolmin-1mg-1 protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.

Isolation and characterization of lactic acid bacteria from traditional pickles of Himachal Pradesh, India.

A total of 15 isolates of lactic acid bacteria (LAB) were isolated from traditional pickles of rural and urban areas of Himachal Pradesh. These isolates were identified as Enterococcus faecalis (7), Lactobacillus plantarum (3), Pediococcus pentosaceus (2), Leuconostoc mesenteroides (1), Lactococcus lactis (1) and Enterococcus sp. (1) on the basis of morphological, biochemical and 16S rDNA sequence analysis. All the LABs produced exopolysaccharides, have antibiotic susceptibility, and possess activity of ß-galactosidase, ß-glucosidase, protease and amylase, while none of the isolates showed haemolytic activity. Out of 15 isolates tested, only 6 isolates showed the phytase activity. These LAB exhibited antimicrobial activity against food borne pathogenic bacteria i.e. Bacillus cereus, Escherichia coli, Staphylococcus aureus and Shigella dysenteriae. These results suggested that LAB isolates from traditional pickles of Himachal Pradesh have very good potential to be used as probiotics.

Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae.

The resting cells of Gordonia terrae mutant E9 having enhanced nitrilase activity were used for biotransformation of 4-hydroxy-3-methoxybenzonitrile into vanillic acid. The maximum conversion was observed in 0.1 M phosphate buffer (pH 8.0), using 60 mM substrate and 0.75 mgDCW resting cells in 1 mL reaction at 40 °C. Km of the whole cell nitrilase of wild and mutant strains of G. terrae for this substrate were 20 and 16.6 mM, and Vmax were 0.19 and 0.95 Umg(-1)(DCW), respectively. Fed batch reaction for transformation of 4-hydroxy-3-methoxybenzonitrile using whole cell nitrilase of wild G. terrae resulted in 2.36 g of vanillic acid in 5 h with a catalytic and volumetric productivity of 0.78 gg(-1)(DCW) h(-1) and 4.72 gL(-1)h(-1), respectively. The whole cell nitrilase of G. terrae mutant E9 resulted in higher catalytic and volumetric productivity, i.e., 1.68 gg(-1)DCW h(-1) and 10 gL(-1)h(-1). A total 5.04 g of vanillic acid with 99% purity were accumulated in 100 mL of reaction after 5 h.

Thermostable Xanthine Oxidase Activity from Bacillus pumilus RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization.

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg(2+), Ag(+) and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.

Probiotic attributes of indigenous Lactobacillus spp. isolated from traditional fermented foods and beverages of north-western Himalayas using in vitro screening and principal component analysis.

The present research was designed to explore indigenous probiotic Lactic acid bacteria from traditional fermented foods and beverages of North-western Himalayas for their probiotic potential. It was achieved through a step-by step approach focused on the technological characterization, evaluation of the probiotic traits and adherence ability. Fifty one LAB isolates from traditional fermented foods and beverages were initially screened for their technological properties and among them twenty isolates were selected. These isolates were further characterized and identified using 16S rRNA gene sequencing as Lactobacillus brevis (7 isolates), Lactobacillus casei (5), Lactobacillus paracasei (2), Lactobacillus buchneri (1), Lactobacillus plantarum (1) and Lactobacillus sp. (3). Identified isolates were evaluated by in vitro methods including survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, exopolysacharride production and haemolytic activity. The results of these experiments were used as input data for Principal Component Analysis; thus, to select the most promising probiotic isolates. Three isolates (L. brevis PLA2, L. paracasei PLA8 and L. brevis PLA16) were found to be most technological relevant and promising probiotic candidates in comparison to commercial probiotic strains. L. brevis PLA2 was selected as best isolate with probiotic potential by in vitro adherence to the human intestinal HT-29 cell line.

Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9.

Mutants of Gordonia terrae were generated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50 mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1 M potassium phosphate buffer (pH 8.0) at 40 °C. Total six feedings each at an interval of 45 min resulted in accumulation of 360 mM (21.6 g) of p-HBA with a purity of 99%. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8 g h(-1) g DCW (-1) and 43.2 g L(-1), respectively, from 0.78 g h(-1) g DCW (-1) and 28.8 g L(-1) using resting cells of wild strain. K m and V max of purified nitrilase from mutant E9 were 55 U mg(-1) and 1.8 mM for p-HBN with a higher turnover number of 36 s(-1) × 10(-3).

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a.

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg(-1). The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5-11.5) and temperature (40-90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K m and V max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 µmol min(-1) mg(-1) protein by analyzing Michaelis-Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.

Bench scale production of benzohydroxamic acid using acyl transfer activity of amidase from Alcaligenes sp. MTCC 10674.

The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 has been applied to the conversion of benzamide and hydroxylamine to benzohydroxamic acid. The unique features of the acyl transfer activity of this organism include its optimal activity at 50 °C and very high substrate (100 mM benzamide) and product (90 mM benzohydroxamic acid) tolerance among the hitherto reported enzymes. The bench scale production of benzohydroxamic acid was carried out in a fed-batch reaction (final volume 1 l) by adding 50 mM benzamide and 250 mM of hydroxylamine after every 20 min for 80 min in 0.1 M potassium phosphate buffer (pH 7.0) at 50 °C, using resting cells equal to 4.0 mg dcm/ml of reaction mixture. From 1 l of reaction mixture 33 g of benzohydroxamic acid was recovered with 24.6 g l(-1) h(-1) productivity. The acyl transfer activity of the amidase of Alcaligenes sp. MTCC 10674 and the process developed in the present study are of industrial significance for the enzyme-mediated production of benzohydroxamic acid.

Arylacetonitrilases: Potential Biocatalysts for Green Chemistry.

Nitrilases are the enzymes that catalyze the hydrolysis of nitriles to corresponding carboxylic acid and ammonia. They are broadly categorized into aromatic, aliphatic, and arylacetonitrilases based on their substrate specificity. Most of the studies pertaining to these enzymes in the literature have focused on aromatic and aliphatic nitrilases. However, arylacetonitrilases have attracted the attention of academia and industry in the last several years due to their aryl specificity and enantioselectivity. They have emerged as interesting biocatalytic tools in green chemistry to synthesize useful aryl acids such as mandelic acid and derivatives of phenylacetic acid. The aim of the present review is to collate information on the arylacetonitrilases and their catalytic properties including enantioselectivity and potential applications in organic synthesis.

Biotransformation of benzonitrile herbicides via the nitrile hydratase-amidase pathway in rhodococci.

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.

Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of Geobacillus pallidus BTP-5x MTCC 9225.

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90-95% (mol mol(-1)) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.

An improved process for synthesis of nicotinic acid using hyper induced whole cell nitrilase of Gordonia terrae MTCC8139.

Nitrilase mediated synthesis of nicotinic acid (vitamin B3) from 3-cyanopyridine has emerged as promising viable alternative to its chemical synthesis. In the present investigation, the nitrilase production in Gordonia terrae MTCC8139 has been increased by two fold with inducer feeding approach [i.e. the addition of 0.5% (v/v) isobutyronitrile as inducer at 0, 16 and 24 h of incubation of the culture]. The use of hyper induced whole cell nitrilase of G. terrae as biocatalyst (10 U per ml reaction) to synthesize nicotinic acid from 3-cyanopyridine in a fed batch reaction at one litre scale resulted in accumulation of 1.65 M (202 g) nicotinic acid in 330 min. The catalytic productivity of hyper induced whole cell nitrilase was increased from 8.95 to 15.3 g/h/g dcw and the reaction time was reduced to half. This is the highest productivity of nicotinic acid in a nitrilase mediated process so far reported. The achievements of the present investigation will lead to mitigate the cost of nitrilase vis-a-vis nicotinic acid production at large scale.

An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2.

Nitrilase of Nocardia globerula NHB-2 was induced by short-chain aliphatic nitriles (valeronitrile > isobutyronitrile > butyronitrile > propionitrile) and exhibited activity towards aromatic nitriles (benzonitrile > 3-cyanopyridine > 4-cyanopyridine > m-tolunitrile > p-tolunitrile). Hyperinduction of nitrilase (6.67 U mg (DCW) (-1), 18.7 U mL(-1)) was achieved in short incubation time (30 h, 30°C) through multiple feeding of isobutyronitrile in the growth medium. The nitrilase of this organism exhibits both substrate and product inhibition effects. In a fed batch reaction at 1 L scale using hyperinduced resting cells corresponding to 10 U mL(-1) nitrilase activity (1.5 mg(DCW) mL(-1)), a total of 123.11 g nicotinic acid was produced at a rate of 24 g h(-1) g (DCW) (-1).

Nanohybrid electrochemical enzyme sensor for xanthine determination in fish samples.

An amperometric biosensor for xanthine was designed, based on covalent immobilization of xanthine oxidase (XO) of Bacillus pumilus RL-2d onto a screen-printed multi-walled carbon nanotubes gold nanoparticle-based electrodes (Nano-Au/c-MWCNT). The carboxyl groups at the electrode surface were activated by the use of 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC) and N-hydroxysuccinimide (NHS). The working electrode was then coated with 6 µL of xanthine oxidase (0.273 U/mg protein). The cyclic voltammetry (CV) study was done for the characterization of the sensor using [K3Fe(CN)6] as an artificial electron donor. The sensitivity (S) and the limit of detection (LOD) of the biosensor were 2388.88 µA/cm2/nM (2.388 µA/cm2/µM) and 1.14 nM, respectively. The developed biosensor was used for determination of fish meat freshness.

Detection of Bacterial Pathogens and Antibiotic Residues in Chicken Meat: A Review.

Detection of pathogenic microbes as well as antibiotic residues in food animals, especially in chicken, has become a matter of food security worldwide. The association of various pathogenic bacteria in different diseases and selective pressure induced by accumulated antibiotic residue to develop antibiotic resistance is also emerging as the threat to human health. These challenges have made the containment of pathogenic bacteria and early detection of antibiotic residue highly crucial for robust and precise detection. However, the traditional culture-based approaches are well-comprehended for identifying microbes. Nevertheless, because they are inadequate, time-consuming and laborious, these conventional methods are not predominantly used. Therefore, it has become essential to explore alternatives for the easy and robust detection of pathogenic microbes and antibiotic residue in the food source. Presently, different monitoring, as well as detection techniques like PCR-based, assay (nucleic acid)-based, enzyme-linked immunosorbent assays (ELISA)-based, aptamer-based, biosensor-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based and electronic nose-based methods, have been developed for detecting the presence of bacterial contaminants and antibiotic residues. The current review intends to summarize the different techniques and underline the potential of every method used for the detection of bacterial pathogens and antibiotic residue in chicken meat.

Classifying nitrilases as aliphatic and aromatic using machine learning technique.

ProCos (Protein Composition Server, script version), one of the machine learning techniques, was used to classify nitrilases as aliphatic and aromatic nitrilases. Some important feature vectors were used to train the algorithm, which included pseudo-amino acid composition (PAAC) and five-factor solution score (5FSS). This clearly differentiated into two groups of nitrilases, i.e., aliphatic and aromatic, achieving maximum sensitivity of 100.00%, specificity of 90.00%, accuracy of 95.00% and Mathew Correlation Coefficient (MCC) of about 0.90 for the pseudo-amino acid composition. On the other hand, five-factor solution score achieved a sensitivity of 96.00%, specificity of 84.00%, accuracy of 90.00% and Mathew Correlation Coefficient (MCC) of about 0.81. The total count of aliphatic amino acids, Ala (A), Gly (G), Leu (L), Ile (I), Val (V), Met (M) and Pro (P), was found to be higher, i.e., 42.7 in case of aliphatic nitrilases, whereas it was 40.1 in aromatic nitrilases. On the other hand, aromatic amino acids, Tyr (Y), Trp (W), His (H) and Phe (F) number, were found to be higher, i.e., 12.7 in aromatic nitrilases as compared to aliphatic nitrilases which was 10.7. This approach will help in predicting a nitrilase as aromatic or aliphatic nitrilase based on its amino acid sequence. Access to the scripts can be done logging onto GitHub using keyword 'Nitrilase' or 'https://github.com/rover2380/Nitrilase.git'.

Alkaline active cyanide dihydratase of Flavobacterium indicum MTCC 6936: Growth optimization, purification, characterization and in silico analysis.

The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. Km and Vmax for cyanide dihydratase of F. indicum was measured to be 4.76 mM and 45 U mg-1 with kcat calculated to be 27.3 s-1 and specificity constant (kcat/Km) to be around 5.67 mM-1 s-1. MALDI-TOF analysis of purified protein revealed that the amino acid sequence has 50% and 43% sequence identity with putative amino acid sequence of F. indicum and earlier reported cyanide dihydratase of Bacillus pumilus respectively. Homology modeling studies of cyanide dihydratase of F. indicum predicted the catalytic triad of the enzyme indicating Cys at 164, Glu at 46 and Lys at 130th position. The purified enzyme has potential applications in bioremediation and analytical sector.