Hypoxanthine-based enzymatic sensor for determination of pork meat freshness

An enzyme sensor employing xanthine oxidase (XO), soluble or immobilised, in combination of an oxygen electrode has been developed and optimised to determinate the hypoxanthine (Hx) content in pork meat at different post-mortem times as measure of meat freshness. The amperometric signal obtained due to the oxygen depletion during the Hx oxidation was related with the consumed oxygen at 190 s in the soluble enzyme sensor or enzymatic rate at 10 s in the immobilised enzyme sensor. In both cases a linear relationship between the signal and the Hx concentration in the range 8.68–26.05 μM (R² = 0.999) and 15.63–127 μM. (R² = 0.995), respectively, was found. Both enzyme sensors exhibited very good working conditions and storage stability. A study of Hx oxidation was carried out in order to compare the Hx content measured by both sensors and those measured by high performance liquid chromatography (HPLC) obtaining a good agreement between both techniques. Therefore, the easy preparation and operation of both enzyme sensors suggests a reliable, rapid and an economical alternative for simple or multiple Hx measurements constituting a useful tool as quality control of meat freshness.     

Determination of Ge(IV) in rice in a mercury-coated glassy carbon electrode in the presence of catechol

Electrochemical adsorptive cathodic stripping voltammetry determination of Ge(IV) using the catechol complex on a mercury-coated (MC) electrode was prepared using a glassy carbon electrode (GCE), the peak potential of which was −0.5 V vs. Ag/AgCl on MCGCE. The various parameters of the catechol concentration, its pH, and others were optimized. The linear working ranges were obtained in the concentration of 2–700 μg L⁻¹ Ge(IV). The relative standard deviation at the Ge(IV) concentration of 50 μg L⁻¹ was 1.37% (n = 15) using the optimum condition, and the detection limit was found to be 0.6 μg L⁻¹ (8.26 × 10⁻⁹ M) (S/N = 3), with an adsorption time of 180 s. The Ge(IV) response was highly linear. This developed method was applied to Ge(IV) to determine the presence of rice grains.     

Detection of ethanol in food: A new biosensor based on bacteria

A microbial biosensor for determination of ethanol has been developed. The microbial ethanol biosensor comprises a Methylobacterium organophilium-immobilized eggshell membrane and an oxygen (O₂) electrode. The microbial biosensor responds linearly to ethanol in the range 0.050–7.5 mM with a detection limit of 0.025 mM (S/N = 3) and the response time is 100 s. The optimal loading of bacterial cells on the biosensor membrane is 40 mg (wet weight). The optimal working conditions for the microbial biosensor are pH 7.0 phosphate buffer (50 mM) at 20–25 °C. The interference test, operational and storage stability of the biosensor are studied in detail. Finally, the biosensor is applied to determine the ethanol contents in various alcohol samples and the results are comparable to that obtained by a gas chromatographic method. Our work demonstrates that the proposed microbial biosensor is a reliable method to determine the ethanol content in wine samples.     

Pico molar assay of riboflavin in human urine using voltammetry

Using a new type of DNA and carbon nano tube (CNT) mixed paste electrode using cyclic and square wave anodic stripping voltammetric (SWASV) methods, this study presents an assay of riboflavin (RF) under optimum conditions. Results of the experiment yielded a low working concentration range of nanograms with 1–10 and 10–170 ng L⁻¹ and 5–105 μg L⁻¹, at an accumulation time of 80 s in a 0.1 M H₃PO₄ electrolyte solution. A relative standard deviation of 30 μg L⁻¹ was observed at an accuracy level of 0.1164% (n = 15) under optimum conditions. The detection limit (S/N) was pegged at 0.2 ng L⁻¹ (5.31 × 10⁻¹³ mol L⁻¹ RF). The proposed method was successfully applied to an actual human urine and drug sample, and can be applied to assays of other biological samples.     

Antioxidant properties and chemical composition of technical Cashew Nut Shell Liquid (tCNSL)

The present study analysed the antioxidant activity of the technical Cashew Nut Shell Liquid (tCNSL) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay and the xanthine oxidase assay, as well as in vivo evaluation by Saccharomycescerevisiae assay. Also, the chemical composition of tCNSL was determined by gas chromatography–mass spectrometry (GC–MS), revealing the presence of cardanols (40.26%), cardols (29.95%), phytosterol (10.68%), triacontanes (4.66%) and anacardic acid (1.79%). The DPPH-based assay results showed that tCNSL 1000 μg/ml reduced the radical level by 88.9%, and the scavenging of hydroxyl radicals in the xanthine oxidase assay indicated significant antioxidant activity (IC₅₀ = 702 μg/ml). In addition, tCNSL exerts an important protective effect against oxidative stress in yeast when used in 100–500 μg/ml concentration range, when exposed to paraquat or H₂O₂, indicating an in vivo antioxidant effect.     

Inhibition of xanthine oxidase activity by an oxathiolanone derivative of quercetin

An oxathiolanone derivative of rutin could be produced in the stomach after the ingestion of rutin containing foods, and the oxathiolanone derivative could be hydrolysed to an oxathiolanone derivative of quercetin (quercetin-oxathiolanone) in the intestine. Quercetin-oxathiolanone as well as quercetin inhibited xanthine oxidase. Approximately 0.05 μM quercetin-oxathiolanone inhibited the activity by 50%, whereas 50% inhibition by quercetin was observed at approximately 0.4 μM. The results suggested that quercetin-oxathiolanone can be used as an effective inhibitor of xanthine oxidase and that the ingestion of rutin-rich foods may be useful to prevent the increase in the blood concentration of uric acid.     

Stabilizing effects of sucrose–polymer formulations on the activities of freeze-dried enzyme mixtures of alkaline phosphatase,nucleoside phosphorylase and xanthine oxidase

To stabilize two freeze-dried enzyme mixtures, consisting of alkaline phosphatase, nucleoside phosphorylase and xanthine oxidase, the effects of sucrose–polymer (bovine serum albumin, gelatin, dextran, polyethylene glycol and polyvinylpyrrolidone) formulations on the remaining activity of the enzyme mixtures were investigated. The enzyme mixtures were freeze-dried with the additives, and then stored at 25, 40 and 55 °C. The glass transition temperatures (T_g) of the freeze-dried samples were assessed in order to determine their physical stability. The T_g values of sucrose–polymer formulations, with the exception of sucrose–polyethylene glycol, were higher than that of sucrose alone. Comparison of the remaining activities of freeze-dried samples showed that sucrose–bovine serum albumin and/or –gelatin prevented activity loss more effectively than did sucrose. Sucrose–polyethylene glycol showed protective ability equivalent to that of sucrose. On the other hand, sucrose–dextran and/or –polyvinylpyrrolidone diminished the stabilizing effect of sucrose. During storage, sucrose–gelatin prevented gradual activity loss to a much greater degree than did sucrose alone.     

Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films

Lactoperoxidase (LPS), purified directly from bovine rennet whey by Toyopearl-SP cation-exchange chromatography and lyophilized by using dextran as supporting material, maintained almost 70 and 60% of its activity after almost 2 and 5 months storage at −18 °C, respectively. Incorporation of the prepared LPS into alginate films between 0.08 and 0.69 mg/cm² (516–4325 U/cm²) caused the immobilization of most of the enzyme and gave films with LPS activity between 0.05 and 2.8 U/cm², determined in the presence of 8 μM H₂O₂. Between 2 and 24 μM H₂O₂ concentrations, a two-fold increase in H₂O₂ concentration caused 1.5–2.5-fold increase in LPS activity of films incorporated with 0.24–0.28 mg/cm² (1200 U/cm²) LPS. The Q₁₀ and E_a of immobilized enzyme activity between 4 and 16 °C were 1.69 and 34.6 kJ/mol, respectively. However, in the 16–30 °C range, the temperature change had almost no effect on LPS activity of films. The optimal activity of immobilized LPS was observed at pH 6.0, but the enzyme maintained 30–85% of its activity between pH 3.0 and 7.0. The immobilized LPS also had a high stability between pH 4.0 and 6.0. The results of this study showed the good potential of LPS-incorporated alginate films in forming a natural antimicrobial mechanism in different foods.     

Physicochemical,thermal and pasting properties of starch separated from γ-irradiated and stored potatoes

The morphological, thermal and pasting properties of starch separated from potatoes of three varieties (Kufri Chandramukhi, Kufri Jyoti and Kufri Chipsona-2), treated either with CIPC (isopropyl N-(3 chlorophenyl) carbamate) or γ-irradiation (Co⁶⁰, 0.1 and 0.5 kGy) and subsequently stored at 8, 12 and 16 °C for 90 days, were studied. Scanning electron microscopy (SEM) showed the presence of oval and irregular shaped starch granules with a diameter range of 15–16 μm. Mean granule size of starch separated from potatoes stored at 12 °C ranged from 18–25 μm and irradiation treatment resulted in an increase in the proportion of small size granules. The irradiation of potatoes with 0.5 kGy resulted in starch with significantly lower peak-, trough- and breakdown-viscosity as compared to starch from potatoes treated with either CIPC or 0.1 kGy irradiation. The irradiation of potatoes with 0.5 kGy caused a significant increase in setback and pasting temperature. Pasting temperature of starch was observed to vary with the storage temperature. Starch separated from potatoes stored at higher temperature showed lower pasting temperature and vice versa. The starch from potatoes stored at 8 °C showed higher peak-, trough- and breakdown-viscosity and lower setback. Peak viscosity increased and swelling volume decreased with increase in storage temperature. FTIR spectra showed that the starch from irradiated potatoes displayed a significant decrease in the intensity of the C–H stretch region between 2800 and 3000 cm⁻¹, which was observed to be irradiation dose-dependent, and higher with 0.5 than 0.1 kGy. However, a slight broadening of O–H stretch (3000–3600 cm⁻¹) in starches from irradiated potatoes was observed. The spectral changes caused by γ-irradiation were apparent in the O–H stretch (3000–3600 cm⁻¹), C–H stretch (2800–3000 cm⁻¹) and bending mode of water (1600–1800 cm⁻¹).     

Amperometric assessment of glucose electrode behaviour in mixed solvents and determination of glucose in dairy products

An amperometric biosensor based on a ruthenium(III), nickel(II) and iron(II) hexacyanometallate (HCM)-modified graphite electrode and immobilized glucose oxidase has been used for the determination of glucose in water-miscible organic solvent/aqueous buffer mixtures. Although the specific activity of biochemically active molecule such as enzyme is reduced in organic environment, it was established that the presence of water soluble organic solvents such as methanol, ethanol and acetonitrile (φ = 10%) enhance the biosensor response. Hydrogen peroxide, produced by enzyme-catalysed reduction of glucose, was measured in phosphate buffer solution (pH = 6.86) at −50 mV against a reference Hg|Hg₂Cl₂|3 M KCl electrode to determine the concentration of glucose. The influence of the addition of different volume fractions (φ = 10–60%) of methanol, ethanol, acetone, acetonitrile and isopropanol on biosensor response was investigated. The obtained amperometric signals were fast, reproducible and linearly proportional to glucose concentrations in the range of 0.1–0.8 mM, with a squared correlation coefficient of 0.9994 for buffer solution. With the addition of ethanol (φ = 10% and 40%) the plateau on I/c curve was obtained for concentrations of glucose higher than 0.8 and 1.1 mM, respectively. The biosensor proved to be stable for several months. The recoveries of added glucose (0.200 and 0.300 mM) from aqueous solution and from solution with ethanol φ = 10% ranged from 96.0% to 108.0%. The biosensor was used for the determination of glucose in some food samples of dairy industry, and the results were consistent with those obtained with the commercially available glucose enzyme photometric kit.     

Monitoring of beef aging using a two-line flow injection analysis biosensor consisting of putrescine and xanthine electrodes

A two-line flow injection analysis (FIA) biosensor was developed which can simultaneously detect bacterial spoilage and the progress of aging. This FIA biosensor was composed of a putrescine oxidase immobilized electrode and a xanthine oxidase immobilized electrode as detectors. The putrescine electrode measures putrescine and cadaverine which are produced by bacteria, and the xanthine electrode measures hypoxanthine and xanthine which accumulate in meat with aging.

The analytical conditions for this system were set as follows; flow rate, 1 ml/ min; water bath temperature, 30 °C; flow buffer, 0.1 M phosphate buffer (pH 7.0); injection volume for putrescine electrode, 200 μl; injection volume for xanthine electrode, 40 μl; and measurement cycle, 2 min.

The linear relationship for standard solution was between 20 and 800 nmol/ml in the putrescine electrode and between 0.1 and 3.0 μmol/ml in the xanthine electrode. The coefficients of variation in standard solutions were 2.14% with the putrescine electrode and 2.83% with the xanthine electrode. The coefficients of variation values in the specimen solution were 3.22% and 3.76%, respectively.

This two-line FIA biosensor was applied to the vacuum-packed beef stored at 0, 5 and 10 °C. The progress of aging could be monitored at all temperatures, and the bacterial spoilage could be detected before the appearance of putrid odor at 5 and 10 °C. However, at 0 °C the putrid odor did not appear during storage, and neither putrescine nor cadaverine was detected. Thus, this FIA biosensor was confirmed to be useful for the quality control of beef aging at 5 and 10 °C, but not at 0 °C.     

Rapid and direct determination of fructose in food: A new osmium-polymer mediated biosensor

This paper describes the development and performance of a new rapid amperometric biosensor for fructose monitoring in food analysis. The biosensor is based on the activity of fructose dehydrogenase (FDH) immobilised into a carbon nanotube paste electrode according to two different procedures. The direct wiring of the FDH in a highly original osmium-polymer hydrogel was found to offer a better enzyme entrapment compared to the immobilisation of the enzyme in an albumin hydrogel. The optimised biosensor required only 5 U of FDH and kept the 80% of its initial sensitivity after 4 months. During this time, the biosensor showed a detection limit for fructose of 1 μM, a large linear range between 0.1 and 5 mM, a high sensitivity (1.95 μA cm⁻² mM), good reproducibility (RSD = 2.1%) and a fast response time (4 s).     

Antioxidant activity of anacardic acids

Anacardic acids, 6-pentadec(en)ylsalicylic acids isolated from the cashew Anacardium occidentale L. (Anacardiaceae) nut and apple, were found to possess preventive antioxidant activity while salicylic acid did not show this activity. These anacardic acids prevent generation of superoxide radicals by inhibiting xanthine oxidase (EC 1.1.3.22, Grade IV) without radical-scavenging activity. Notably, the inhibition kinetics of anacardic acids do not follow hyperbolic dependence of enzyme inhibition on inhibitor contents (Michaelis–Menten equation) but follow the Hill equation instead. Anacardic acid (C_15:1) inhibited the soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) catalyzed oxidation of linoleic acid with an IC₅₀ of 6.8 μM. The inhibition is a slow and reversible reaction without residual enzyme activity. The inhibition kinetics indicate that anacardic acid (C_15:1) is a competitive inhibitor and the inhibition constant, K_I, was 2.8 μM. Anacardic acids act as antioxidants in a variety ways, including inhibition of various prooxidant enzymes involved in the production of the reactive oxygen species and chelate divalent metal ions such as Fe²⁺ or Cu²⁺, but do not quench reactive oxygen species. The C₁₅-alkenyl side chain is largely associated with the activity.     

Construction of an amperometric polyphenol biosensor based on PVA membrane

A method is described for construction of an amperometric biosensor for determination of polyphenolic content using polyvinyl alcohol membrane bound polyphenol oxidase mounted on a gold coated Ag wire as a working electrode, a silver/silver chloride (Ag/AgCl) reference electrode and Pt wire as auxiliary electrode. The biosensor showed optimum response within 30 s, when operated at 30 °C. A linear relationship was obtained between L-DOPA concentration (0.5–20 μM) and current (μA). Limit of detection of the method was 0.5 μM. The biosensor measured polyphenols in tea leaves, alcoholic beverages and water. The enzyme electrode was used 280 times over 6 months, when stored at 4 °C. The biosensor is better than earlier membrane based biosensors in terms of detection limit (0.5 μM) and stability (6 months).     

Antioxidant triterpenoids from the stems of Momordica charantia

A new multiflorane triterpenoid and two new cucurbitane triterpenoids were isolated from the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods. These three new compounds, 1, 2 and 3 displayed ABTS radical cation scavenging activity with IC₅₀ values of 268.5 ± 7.9, 352.1 ± 11.5 and 458.9 ± 13.0 μM, respectively and an inhibitory effect on xanthine oxidase (XO) activity with IC₅₀ values of 142.3 ± 30.2, 36.8 ± 20.5 and 124.9 ± 8.3 μM, respectively. This finding indicated that 1–3 can be used as antioxidants.     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

Effect of luteolin on xanthine oxidase: Inhibition kinetics and interaction mechanism merging with docking simulation

Xanthine oxidase (XO) catalyses hypoxanthine and xanthine to uric acid in human metabolism. Overproduction of uric acid will lead to hyperuricemia and finally cause gout and other diseases. Luteolin is one of the major components of celery and green peppers, its inhibitory activity on XO and their interaction mechanism were evaluated by multispectroscopic methods, coupled with molecular simulation. It was found that luteolin reversibly inhibited XO in a competitive manner with inhibition constant (K_i) value of (2.38 ± 0.05) × 10⁻⁶ mol l⁻¹. Luteolin could bind to XO at a single binding site and the binding was driven mainly by hydrophobic interactions. Analysis of synchronous fluorescence and circular dichroism spectra demonstrated that the microenvironment and secondary structure of XO were altered upon interaction with luteolin. The molecular docking results revealed luteolin actually interacted with the primary amino acid residues located within the active site pocket of XO.     

Effectiveness of a polyamide film releasing lactic acid on the growth of E. coli O157:H7, Enterobacteriaceae and Total Aerobic Count on vacuum-packed beef

The suitability of a polyamide 6 monolayer film containing lactic acid for use as an antimicrobial package for fresh beef cuts was studied. The release of lactic acid in an aqueous environment was immediate (within 1 h) and was from approx. 55 μg lactic acid/cm² film at 0–8 °C to approx. 67 μg lactic acid/cm² film at 12–20 °C. Beef was contaminated with an Escherichia coli O157:H7 isolate with known minimum inhibitory concentration against lactic acid (0.09% v/v), then wrapped with the lactic-acid polyamide film and vacuum packaged. During storage at 12 °C, the numbers of E. coli were 1 log unit lower than that of a control (untreated polyamide film) and decreased by an additional 1 log during storage for 14 days.     

Kinetics of the formation of radicals in meat during high pressure processing

The kinetics of the formation of radicals in meat by high pressure processing (HPP) has been described for the first time. A threshold for the radicals to form at 400 MPa at 25 °C and at 500 MPa at 5 °C has been found. Above this threshold, an increased formation of radicals was observed with increasing pressure (400–800 MPa), temperature (5–40 °C) and time (0–60 min). The volume of activation (ΔV^#) was found to have the value −17 ml mol⁻¹. The energy of activation (E_a) was calculated to be 25–29 kJ mol⁻¹ within the pressure range (500–800 MPa) indicating high independence on the temperature at high pressures whereas the reaction was strongly dependent at atmospheric pressure (E_a = 181 kJ mol⁻¹). According to the effect of the processing conditions on the reaction rate, three groups of increasing order of radical formation were established: (1) 55 °C at 0.1 MPa, (2) 500 and 600 MPa at 25 °C and 65 °C at 0.1 MPa, and (3) 700 MPa at 25 °C and 75 °C at 0.1 MPa. The implication of the formation of radicals as initiators of lipid oxidation under HPP is discussed.     

Preparation of molecular imprinted film based on chitosan/nafion/nano-silver/poly quercetin for clenbuterol sensing

A useful molecular imprinted film (MIP-CNSQ/WGE) based on chitosan/nafion/nano-silver/poly quercetin compound was prepared by a compound electrochemical method at paraffin-impregnated graphite electrode for clenbuterol (CLB) sensing, which was characterised by means of field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy spectra (XPS), infrared spectra (IR) and electrochemical techniques to confirm the formation. The molecular imprinted film modified electrode was successfully applied to the determination of CLB with a reliable result. In optimum conditions, CLB at concentrations of 0.3–50.0 μM could be determined with a detection limit of 0.01 μM (3σ). Determination of CLB in practical samples of pork liver showed good recovery.