A new amperometric biosensor for fructose determination based on epoxy-graphite-TTF-TCNQ-FDH-biocomposite

In this work a novel amperometric biosensor for fructose determination in solutions was developed. The device was constructed by the incorporation of a tetrathiofulvalene-tetracyanoquinodimethane organic conducting salt and fructose dehydrogenase enzyme, include in a polymeric matrix of epoxy resin and graphite powder. Because of the electrocatalytic function of the salt, the direct transfer of the electron between the reduced prosthetic group (PQQH₂) of the enzyme and the transducing material, was verified at a low working potential (150 mV vs. Ag/AgCl), where the interfering reactions were minimized. The response time at 90% of the steady state value was less than 20 s. The current response was directly proportional to the D-fructose concentration from 0.01 to 0.3 mmol/l with a detection limit of 0.005 mmol/l (signal/noise of 3) and a sensitivity of 1.9985 μA/mmol. The biosensor sensitivity diminishes when its surface is not polished between successive determinations, and remains constant (rsd=1.85, n=10) when the surface is polished between determinations. The effects of temperature and pH on the biosensor response were studied and analyzed; also the properties of the enzyme (Km _ap, I _max, Q₁₀) were determinate in this work. The biosensor was used to determine fructose in high fructose syrups and there were not significant differences between these results and those obtained by HPLC (p≤0.05). During 4 months, in intermittent determinations the biosensor kept 100% of its original sensitivity and after 18 months stored at 4°C, it only lost 32% of its sensitivity. The simplicity, low working potential, high stability and good performance of this biosensor shows a great potential for its use in the fructose determination.     

Electrochemical biosensor based on gold coated iron nanoparticles/chitosan composite bound xanthine oxidase for detection of xanthine in fish meat

A xanthine oxidase was immobilized covalently onto chitosan bound gold coated iron nanoparticles (CHIT/Fe-NPs@Au) electrodeposited on the surface of pencil graphite electrode (PGE). A xanthine biosensor was fabricated using XOD/CHIT/Fe-NPs@Au/PGE as working, Ag/AgCl as reference and Pt as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The biosensor exhibited optimum current response within 3 s at pH 7.4, 35 °C and working range 0.1–300 μM, when polarized at 0.5 V vs Ag/AgCl. The sensitivity of the biosensor was 0.001169 mAμ M^–1 cm^–2 with detection limit of 0.1 μM (S/N = 3). The biosensor showed only 25% loss in its initial activity after its 100 uses over 100 days, when stored at 4 °C.     

Determination of calcium in milk and water samples by using catalase enzyme electrode

A biosensor based on catalase enzyme was developed for the investigation of the effect of calcium ions on the activity of the enzyme. Calcium plays an activator role for the catalase enzyme that catalyses the degradation of hydrogen peroxide to O₂ and H₂O. Determination method of the effect of calcium ion on the activity of the enzyme was based on the assay of the differences on the responses of the biosensor in the absence and the presence of calcium in the reaction medium. The biosensor had a linear relation to calcium concentrations and good measurement correlation between 1 and 10 mM with 1 min response time. Tris–HCl buffer (pH 7.0; 50 mM) and 37 °C were obtained as the optimum working conditions. In the application studies, the biosensor was used determination of calcium level of real samples such as milk, spring and mineral water.     

Purification and characterization of soluble invertases from suspension-cultured bamboo (Bambusa edulis) cells

An alkaline invertase (IT I) and an acid invertase (IT II) were purified from the soluble fraction of suspension cultured bamboo cells. Both purified invertases were homogeneous as examined by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and were identified as β-fructofuranosidases able to attack the β-fructofuranoside from the fructose end. With respect to sucrose hydrolysis, the optimal pHs were 7.0 and 4.5 for IT I and IT II, respectively. The Km’s were 10.9 and 3.7 mM. The IT I and IT II molecular masses were 240 and 68 kDa, respectively, as estimated by gel filtration. The isoelectric points were 4.8 and 7.4. IT I was a homotetrameric enzyme activated by bovine serum albumin (BSA). IT II was a monomeric enzyme activated by BSA, concanavalin A (ConA) and urease. Both isoforms were significantly inhibited by heavy metal ions Ag⁺ (5 mM) and Hg²⁺ (1 mM), and mercaptide forming agent ρ-chloromercuribenzoic acid (PCMB; 0.5 mM).     

An amperometric biosensor for the rapid assessment of histamine level in tiger prawn (Penaeus monodon) spoilage

Histamine could accumulate in seafood when bacteria spoilage commenced and caused histamine poisoning without altering the fish normal appearance and odor. Therefore, a histamine biosensor using immobilized enzyme diamine oxidase (DAO) has been developed for the rapid monitoring of the histamine levels in tiger prawn (Penaeus monodon). The histamine biosensor had a response time of <1 min and optimum pH of operation was 7.4 with reproducibility and repeatability (n = 5) of 4.87% and 5.26% relative standard deviations (RSD) respectively. Recoveries ranging from 93.11% to 100.58% were obtained for histamine spiked at levels from 5 to 20 ppm. The variation in histamine levels of some tiger prawn samples after a 5-h exposure at temperature of 30 °C ± 2 were studied using the histamine biosensor and the results were comparable to histamine levels determined by an HPLC method. The two methods showed a linear correlation with R² = 0.9612 (Y = 0.9164x + 5.58). The limit of detection was 0.65 ppm of histamine, which is below the indicator level of 50 ppm established by USA FDA. The reusable biosensor is simple and can be used for direct histamine determination without further sample pretreatment, and is suitable for the routine analysis of histamine in tiger prawns to monitor spoilage.     

Determination of phenolic acids in coffee by micellar electrokinetic chromatography

A seven-minute micellar electrokinetic chromatography analytical procedure capable of resolving the five principal phenolic acids in coffee, including the isomers of 5-caffeoylquinic acid (5-CQA), 4-CQA and 3-CQA, caffeic acid and ferulic acid is reported. The electrophoretic conditions consisted of an SDS (70 mM)-phosphate (17.6 mM)-methanol (5% v/v) buffer system, pH 2.5, 22.1 °C, −17 kV and detection at 325 nm. The Joule effect and the possible interactions between the buffer components and temperature with respect to peak quality, resolution and selectivity were assessed in the concentration range of 25–900 μg/mL. Performance evaluation of the system used a 3^3–7 factorial design at the 95% confidence level. The lowest correlation coefficient for linearity was 0.99888 for the 4-CQA. Limits of detection and quantification were 0.98 and 4 μg/mL. The method was tested with both green and roasted coffee beans using four systems of extraction.     

A Biosensor Utilizing l-Glutamate Dehydrogenase and Diaphorase Immobilized on Nanocomposite Electrode for Determination of l-Glutamate in Food Samples

Amperometric biosensor utilizing bienzymatic composition consisting of l-glutamate dehydrogenase and diaphorase for the determination of l-glutamate has been developed. Enzymes were immobilized between chitosan layers onto the surface of planar nanocomposite electrodes consisting of multi-walled carbon nanotubes (diameter?=?60–100 nm; length?=?5–15 μm, 95+?% purity). Linear response was obtained from 10 to 3,495 μM in phosphate buffer solution of pH 9.0 and in the presence of enzyme cofactor NAD⁺ (2 mM) and mediator ferricyanide (5 mM). The limit of detection was 5.4 μM, and sensitivity was found to be 28 nA μM^?1 cm^?2. The biosensor showed a short response time (within 60 s), good storage (no loss of activity for at least 3 months), and operational (response ability above 90 % after 7 days since its first use) stability. Finally, the results obtained from measurements of the food samples were compared with those obtained with an enzymatic–spectrophotometric method and correlated well. Analytical performance of the biosensor indicated that the bienzyme system utilizing diaphorase as a secondary enzyme could be a general basis for other biosensors based on NAD⁺-dependent dehydrogenases.     

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.     

Transglutaminase cross-linking to enhance elastic properties of soy protein hydrogels with intercalated montmorillonite nanoclay

To strengthen the network of bio-nanocomposite hydrogels, layered montmorillonite (MMT) nanoclay was intercalated by surface-coating with soy protein (SP) before mixing with 6% w/v SP for cross-linking by microbial transglutaminase (mTGase). Dynamic rheology was performed to study variables of NaCl and mTGase concentrations, with and without 1% w/v MMT. Without mTGase, the highest storage modulus (G′) was observed at 100 mM for samples without MMT, which was twice of the highest G′ for samples with MMT, at 200 mM NaCl. With mTGase, a shorter gelation time and a stronger hydrogel were observed at a higher enzyme level. Overall, the non-gelling 6% w/v SP dispersion was transformed to a hydrogel with G′ of 1099 Pa after addition of 100 mM NaCl and 1% SP-coated MMT and treatments by 6.25 U/g-protein mTGase for 2 h and heating/cooling steps. The integration of surface-coating and mTGase cross-linking is promising to improve properties of the nanocomposite system.     

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.     

Determination of diquat and paraquat in olive oil by ion-pair liquid chromatography–electrospray ionization mass spectrometry (MRM)

For the first time a method for determination of herbicides diquat (DQ) and paraquat (PQ) in olive oil was developed utilising liquid chromatography–electrospray ionization mass spectrometry (MRM). n-Hexane/10 mM HFBA aqueous solution partitioning was used as the extraction method. Separation was carried out in an Xterra C8 column (100 × 21 mm, 3 μm), using the gradient mode. Solvent A was a HFBA aqueous solution (5 mM, pH 2) and solvent B acetonitrile/methanol 75/25 (v/v). Peaks used for quantification were m/z = 157 (diquat) and m/z = 158 (paraquat). Detection limit found for both diquat and paraquat was 4 μg kg⁻¹. The method can also be applied for determination of chlormequat (CQ, quantification peak m/z = 58), the detection limit being 0.3 μg kg⁻¹. Such limits are clearly lower than the MCLs commonly applied to olive oil as reference criteria (5 times MCLs in olives). Good reproducibilities (day to day and run to run) were obtained.     

Effect of reactant concentrations on the Maillard reaction in a fructose–glycine model system and the inhibition of black tiger shrimp polyphenoloxidase

The characteristics and the inhibitory activity towards black tiger shrimp polyphenoloxidase of Maillard reaction products (MRPs), prepared by heating an equimolar mixture of fructose and glycine at various concentrations (0.75–30 mM) at 100 °C for 12 h, were investigated. Increase in the intermediate products was observed with increasing reactant concentration, as evidenced by the increase in A₂₉₄ and fluorescence intensity. Furthermore, development of browning (A₄₂₀) was noticeable when the reactant concentration increased. The inhibitory activity of MRPs towards PPO gradually increased when the concentration of each reactant increased and reached a maximum at 30 mM (80% inhibition). The increase in the inhibitory activity of MRPs was coincidental with the increase in the reducing power, A₂₉₄, fluorescence intensity, as well as browning intensity. Generally, the development of Maillard reaction products was associated with decrease in pH and loss of reducing sugar and free amino groups, with coincidental increase in reducing power and copper-chelating property. MRPs with the reactant concentration of 4.5–30 mM were able to chelate the copper ion. Therefore, the inhibitory activity of MRPs towards browning, induced by PPO, was most likely due to their copper-chelating property as well as reducing power.     

Effects of grain,fructose, and histidine feeding on endotoxin and oxidative stress measures in dairy heifers

Ruminal endotoxin and plasma oxidative stress biomarker concentrations were studied in dairy heifers challenged with grain, fructose, and histidine in a partial factorial study. Holstein-Friesian heifers [n = 30; average body weight (BW) of 359.3 ± 47.3 kg] were randomly allocated to 5 treatment groups: (1) control (no grain); (2) grain [crushed triticale at 1.2% of BW dry matter intake (DMI)]; (3) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI); (4) grain (1.2% of BW DMI) + histidine (6 g/head); and (5) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI) + histidine (6 g/head). Rumen samples were collected by stomach tube 5, 65, 115, 165, and 215 min after diet consumption and blood samples at 5 and 215 min after consumption. Rumen fluid was analyzed for endotoxin concentrations. Plasma was analyzed for concentrations of the following oxidative stress biomarkers: reactive oxygen metabolites (dROM), biological antioxidant potential (BAP), advanced oxidation protein products, and ceruloplasmin, and activity of glutathione peroxidase. Dietary treatment had no effect on concentrations of endotoxin or oxidative stress biomarkers. We observed no interactions of treatment by time. Ruminal concentrations of endotoxin decreased during the sampling period from 1.12 × 10⁵ ± 0.06 to 0.92 × 10⁵ endotoxin units/mL ± 0.05 (5 and 215 min after diet consumption, respectively). Concentrations of dROM and the oxidative stress index (dROM/BAP × 100) increased over the sampling period, from 108.7 to 123.5 Carratelli units (Carr U), and from 4.1 to 4.8, respectively. Ceruloplasmin concentrations markedly declined 5 min after the consumption of diets, from 190 to 90 mg/L over the 215-min sampling period. Overall, a single feeding challenge for dairy cattle with grain, fructose, and histidine, and combinations thereof, may not be sufficient to induce marked changes in endotoxin or oxidative stress biomarker concentrations.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Ultrasound-assisted solid liquid extraction (USLE) of olive fruit (Olea europaea) phenolic compounds

A new method of ultrasound-assisted solid liquid extraction (USLE) of olive fruit phenols is described. Phenolics were extracted using high intensity probe ultrasonication and analysed by HPLC-DAD-FLD-MS/MS. Four USLE parameters – sonication time (4, 15, 20, 30 min), temperature (25, 45 °C), solvent composition (80%, 100% methanol) and extraction steps (1–5) were studied and optimised on the basis of nine major olive fruit phenols. A three-step extraction of 20 min with pure methanol (25 mL) at 45 °C was needed for sufficient phenol recoveries (94.1–98.7%) from 1.5 g of freeze-dried olive fruits. The proposed USLE method was more efficient in comparison to US bath and agitation, with up to 33% and 80% enhancement in the case of oleuropein, respectively. In addition, the overall method provided high selectivity, precision and sensitivity with LODs/LOQs ranging from 0.66–4.92 μg g⁻¹ and 2.00–14.77 μg g⁻¹ of olives DW, respectively.     

Inhibitory effects of α-cyano-4-hydroxycinnamic acid on the activity of mushroom tyrosinase

The effects of α-cyano-4-hydroxycinnamic acid (HCCA) on the activity of mushroom tyrosinase have been studied. Results showed that HCCA could inhibit both the monophenolase activity and diphenolase activity of mushroom tyrosinase. For the monophenolase activity, the lag phase was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. When the concentration of HCCA reached to 80 μM, the lag time was lengthened from 20 s to 150 s and the steady-state activity was lost by about 75%. The IC₅₀ value was estimated to be 48 μM. For the diphenolase activity, the inhibitory effect of HCCA was also dose-dependent and the IC₅₀ value was estimated to be 2.17 mM. The kinetic analyses showed that the inhibition of HCCA on the diphenolase activity was reversible and competitive with the inhibition constants (K_I) determined to be 1.24 mM.     

Development of a simple capillary electrophoretic determination of glucosamine in nutritional supplements using in-capillary derivatisation with o-phthalaldehyde

A simple capillary electrophoretic method was developed for the determination of glucosamine using in-capillary derivatisation. Glucosamine in commercial products was extracted with purified water. The CE separation was achieved on an uncoated fused-silica capillary using a 20 mM borate buffer (pH 9.2) containing 5 mM o-phthalaldehyde (OPA) and 5 mM 3-mercaptopropionic acid (MPA) at 25 kV, followed by UV detection at 340 nm. The detector response was linear (r² > 0.999) in the concentration range 10–1000 μg/mL. The limit of detection (LOD) was 1.3 mg/g. Spiked glucosamine recoveries at 50 and 100 mg/g level were 95.1% and 104.3%, respectively. The method was applied to 16 commercial products. The concentrations of glucosamine were 109–705 mg/g, and the ratios of detected glucosamine content to the labelled value were 88.8–124%. No significant bias was observed (r² = 0.989, p < 0.01), between results obtained by the proposed CE method and an official colorimetric method (Japanese Health Food & Nutrition Food Association).     

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.