Determination of Hypoxanthine in Fish Meat with an Enzyme Sensor

An enzyme sensor specific for hypoxanthine (Hx) was developed using immobilized xanthine oxidase-membrane and an oxygen probe. Xanthine oxidase (E.C. 1.2.3.2.) was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Hx is oxidized to uric acid by the immobilized enzyme, the output current of the oxygen probe decreasing due to oxygen consumption. A linear relationship was obtained between current decrease and Hx concentration in the range 0.06–1.5 mM. The enzyme sensor could be used for more than 100 assays without decrease of output current After 30-day-storage at 5°C, no remarkable decrease of output current was observed. The enzyme sensor system was applicable to the simple, rapid, and economical determination of Hx in several fish meats including sea bass, saurel, mackerel, yellowfish, and flounder.     

Hypoxanthine Ratio Determination in Fish Extract Using Capillary Electrophoresis and Immobilized Enzymes

Fish freshness was assessed using capillary electrophoresis and an immobilized enzyme procedure to monitor degradation of inosine-5′-monophosphate (IMP), inosine (HxR) and hypoxanthine (Hx). The enzymatic method used an amperometric probe at + 0.7 V (platinum vs silver/silver chloride) with immobilized xanthine oxidase, catalase, nucleoside phosphorylase, and nucleotidase for converting Hx, HxR or IMP to uric acid. Capillary electrophoresis resolved IMP, inosine and Hx by migration rates resulting from an applied electric field (416 V/cm, 50 μA). Components were detected at 250 nm. The H ratio of Hx/[IMP + HxR + Hx] and simplified K value of [HxR + Hx]/ [IMP + HxR + Hx] were determined in cod, salmon and trout stored on ice (0-4°C) and at 20°C. The two procedures agreed and for all species H ratio and K values increased with storage time.     

Selective determination of lactic acid in dry-fermented sausages using a sensor based on immobilized lactate oxidase

Selective determination of lactic acid in dry-fermented sausages is an indicator of quality and its presence prevents the growth of pathogenic bacteria. The analysis of lactic acid represents a high cost for the relevant food industries. The use of an enzymatic sensor would allow to reduce the time and cost of this analysis. An enzymatic sensor employing lactate oxidase (LacOx) with the immobilized enzyme system in combination with an oxygen electrode was optimized to determine the lactic acid content in dry-fermented sausages. In this study using LacOx from Pediococcus sp., a voltage of ?600 mV, low volumes of reaction and the amperometric signal obtained due to the oxygen depletion (consumed oxygen) during the lactic acid oxidation was recorded at 15 s in the immobilized enzyme sensor so that the reaction rates (slope) were related to the lactic acid content. A positive linear relationship between the consumed oxygen as a function of time (mg O2/L*s^?1) and the lactic acid concentration in the range of 250–600 μM, with a coefficient ofR² = 0.9936 for the immobilized enzyme system, was determined. The immobilized enzyme sensor showed a high specificity K_M = 0.865 and sensitivity of 0.25 mM and was stable enough to allow the reutilization of the membranes up to 20 times without loss of activity, where 90% of its initial activity remained after 45 days. The analysis of lactic acid with the immobilized enzyme system in dry-fermented sausages revealed very good agreement with the determination performed through standard HPLC methodology using the same linear range in both methods, which validated the use of this sensor as an alternative technique to evaluate cured meat quality.     

Influence of temperature on continuous high gravity brewing with yeasts immobilized on spent grains

Flavor compounds’ formation and fermentative parameters of continuous high gravity brewing with yeasts immobilized on spent grains were evaluated at three different temperatures (7, 10 and 15 °C). The assays were performed in a bubble column reactor at constant dilution rate (0.05 h⁻¹) and total gas flow rate (240 ml/min of CO₂ and 10 ml/min of air), with high-gravity all-malt wort (15°Plato). The results revealed that as the fermentation temperature was increased from 7 to 15 °C, the apparent and real degrees of fermentation, rate of extract consumption, ethanol volumetric productivity and consumption of free amino nitrogen (FAN) increased. In addition, beer produced at 15 °C presented a higher alcohols to esters ratio (2.2–2.4:1) similar to the optimum values described in the literature. It was thus concluded that primary high-gravity (15°Plato) all-malt wort fermentation by continuous process with yeasts immobilized on spent grains, can be carried out with a good performance at 15 °C.     

Optimization of Covalent Immobilization of Extracellular Ice Nucleators from Erwinia herbicola on Magnetic Fe3O4/Chitosan Nanoparticles for Potential Application in Freeze Concentration

A magnetically separable extracellular ice nucleators (ECINs) immobilized system was constructed, ECINs from Erwinia herbicola were covalently immobilized on magnetic Fe₃O₄/chitosan nanoparticles by coupling via glutaraldehyde (GA) or epichlorohydrin (ECH). The immobilization process parameters were optimized, and under optimized conditions, ice nucleation activity (INA) of GA and ECH immobilized ECINs reached at 1.56?×?10⁶?±?1.54?×?10⁵ Units/mg and 1.62?×?10⁶?±?1.91?×?10⁵ Units/mg, respectively. The stability, reusability and cytotoxicity of GA and ECH immobilized ECINs were characterized and compared; both GA and ECH immobilized ECINs demonstrated good reusability performance after 15 freeze–thaw cycles and low cytotoxicity, while GA immobilized ECINs were recovered more easily and more stable at different pH compared to ECH immobilized ECINs. Application of the immobilized ECINs in freeze concentration was investigated, and an increase of ice nucleation temperature for water, seawater and apple juice (6.83?±?0.18 °C, 8.08?±?0.50 °C and 9.38?±?1.12 °C) was achieved by using GA immobilized ECINs. The results collected so far show that there are extensively promising applications for GA immobilized ECINs in freeze concentration industry fields.     

Tetrathiafulvalene-mediated Biosensor for L-lactate in Dairy Products

A tetrathiafulvalene (TTF)-mediated, amperometric biosensor was developed for the specific determination of L-lactate. L-lactate oxidase was immobilized by adsorption and retained on the surface of a carbon-paste electrode using an electro-polymerized film of 1,3-phenylenediamine-resorcinol. The response of the biosensor was maximum at pH 7.0 and 30°C. Response was linear up to 0.6 mM lactate and had a minimum detection limit of 56 PM. Response time was less than 90 sec. When stored at 4°C response was stable for > 36 days and subsequently decreased. The sensor was unresponsive after 96 days, This enzyme-based biosensor was suitable for determining lactate concentrations in milk and yogurt.     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

Sensory Changes in Umami Taste of Inosine 5'-Monophosphate Solution after Heating

Discrimination in umami taste of inosine 5′-monophosphate (IMP) solution caused by thermal degradation was investigated by sensory evaluation. The difference threshold of umami taste of 0.005% IMP solution in the presence of 0.05% monosodium glutamate (MSG) was 0.002%. The difference threshold of a 0.005% IMP solution decreased by about one half when heated at 95°C for 15 h. Inosine, one of the main products of the thermal degradation of IMP, had a bitter taste. The detection threshold of inosine varied widely among panelists. Heating a 0.005% IMP solution at 95°C for 15 h formed inosine at about one tenth of its lowest detection threshold.     

Inactivation ofEscherichia coliO157:H7 by combinations of GRAS chemicals and temperature

Reported outbreaks of foodborne illness involvingEscherichia coliO157:H7 have increased in the United States during the last decade, with a variety of food products being implicated as vehicles of infection. Studies were carried out to determine the efficacy of combinations of various GRAS chemicals and moderate temperatures to killE. coliO157:H7. A five-strain mixture ofE. coliO157:H7 of approximately 10⁸cfu ml⁻¹was inoculated into 0·1% peptone solutions containing 1·0 or 1·5% lactic acid plus 0·1% hydrogen peroxide, 0·1% sodium benzoate or 0·005% glycerol monolaurate. The solutions were incubated at 8°C for 0, 15 and 30 min; at 22°C for 0, 10 and 20 min; or at 40°C for 0, 10 and 15 min; populations ofE. coliO157:H7 were determined at each sampling time. At 40°C, the pathogen was inactivated to undetectable levels within 10 min of incubation in the presence of 1·0 or 1·5% lactic acid plus hydrogen peroxide, and within 15 min of incubation in the presence of 1·5% lactic acid plus sodium benzoate or glycerol monolaurate. At 22°C, complete inactivation ofE. coliO157:H7 was observed after 20 min of exposure to 1·5% lactic acid plus 0·1% hydrogen peroxide, whereas a reduction of 5 log₁₀cfu ml⁻¹was observed with a treatment of 1·5% lactic acid plus glycerol monolaurate. None of the treatments resulted in total inactivation of the pathogen at 8°C. The aforementioned treatments could potentially be used to inactivate or reduceE. coliO157:H7 populations on raw produce.     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Determination of kinetic parameters of pectolytic enzymes at low pectin concentrations by a simple method

A simple and sensitive method for determination of the pectolytic enzyme activity was improved for soluble and immobilized forms of the enzyme. During enzymatic hydrolyzation of pectin, samples were collected from substrate solution at certain time intervals. Pectin in the samples was precipitated by alcohol and then pectin concentration was determined by measuring the refractive index of a prepared aqueous solution of these precipitates. The validity of this technique was evaluated using the kinetic behavior of soluble and immobilized enzymes. The kinetics of free and Duolite A568-immobilized pectinase was investigated. Michaelis-Menten constants and maximal reaction rates were found as K _m=20.71 g l ^-1 and V _max=81.30 g l ^-1s ^-1 for free enzyme and K _m=1.02 g l ^-1 and V _max=0.035 g s ^-1 g ^-1 particles for immobilized pectinase at 20 °C.     

Characterization of Quality Deterioration in Yellowfin Tuna

The spoilage pattern of thawed yellowfin tuna carcasses was examined. After five days storage at 10° and 18°C, bacterial counts were low (< 10³) although visible softening of the flesh was apparent. Direct measurement of ethanol as an indicator of pre-process spoilage also failed to reflect differences among carcasses which were aged for up to five days. Analysis of nucleotides by high performance liquid chro-matography suggested that the ratio of inosine monophosphate (IMP) to other nucleotides may be a valuable indication of spoilage time where the IMP ratio decreased linearly with time after thawing. Average recoveries of adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine were 50%, 75%, 64% and 92%, respectively, from spiked tuna which had been cooked at 122°C for 67 min.     

Analytical performance and characterization of antibody immobilized magnetoelastic biosensors

This article presents the results of an investigation into the enhancement of sensitivity and thermal stability of polyclonal antibody immobilized magnetoelastic biosensors. The Langmuir–Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors. Biosensor performance was investigated by exposing to graded concentrations (5 × 10¹–5 × 10⁸ cfu mL⁻¹) of Salmonella typhimurium solutions in a flow through mode. Bacterial binding to the antibody on the sensor surfaces changed the resonance parameters, and these changes were quantified by the sensor’s resonance frequency shift. An increase in the sensitivity from 159 Hz decade⁻¹ for a 2 mm sensor to 246 Hz decade⁻¹ for a 1 mm sensor was observed during the dose–response measurements. The stability of the biosensor was also investigated by storing the biosensor at 25, 45 and 65 °C. The binding activity of the stored biosensor was estimated by measuring the changes in resonance frequency after exposure to the bacterial solutions (10⁹ cfu mL⁻¹). Binding activity was also confirmed by counting bound S. typhimurium cells on the sensor surface using Scanning Electron Microscopy (SEM) micrographs. The results show that at each temperature, the binding activity of the biosensor gradually decreased over the testing period. Degradation of biosensor accelerated at higher storage temperatures. The activation energy of biosensor system degradation was determined to be 7.7 kcal mol⁻¹.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97?a_w . Mancozeb at 40?mg?l^?1 inhibited fungal growth and provided lag phases >24 days at 10–20?mg?l^?1 and 15°C. OTA was observed only at 25°C and doses <10?mg?l^?1. At 15°C, copper oxychloride proved inhibitory at 800?mg?l^?1, while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5–8?g?l^?1 (at 15°C) while at 25°C growth took place even at 8?g?l^?1, although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb?>?copper oxychloride?>?sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

Storage Stability of Intermediate Moisture Mullet Roe

A storage stability study was performed on intermediate moisture roe (a_w= 0.84, salt content = 4%). Samples were stored at various temperatures for up to 1 month. Microbial analyses indicated that bacteria could grow from 5–25°C. Fungi grew at 15° and 25°C while their growth was inhibited at 5°C; however, a lag phase was detected at 15°C. TBA values increased linearly during storage. Microbial analyses, chemical determination of rancidity and sensory evaluations showed that the product was still acceptable after 30 days storage at 5°, 15° or 25°C.     

Effect of storage temperature and duration on glucosinolate,total vitamin C and nitrate contents in rocket salad (Eruca sativa Mill.)

The effect of storage temperature and duration on nitrate, total vitamin C and glucosinolate (GSL) contents was investigated in rocket salad (Eruca sativa Mill.) grown in soilless culture. Harvested plants were either maintained intact as leaves with roots (RL) or separated as leaves only (LO) before being placed in oriented polypropylene packages and immediately stored at 4 °C (RL and LO) or 15 °C (LO only) in darkness. Packages were sampled after 1, 3, 6 and 10 days of storage (DOS) and, along with fresh samples (0 DOS), submitted to chemical analysis. For RL (4 °C), leaf moisture content decreased gradually with increasing storage duration, from 98.7% at 1 DOS to 93.8% at 10 DOS. For LO (15 °C), leaf colour at 10 DOS had shifted from an initial greenish hue to a yellowish one. Nitrate contents were highest at 10 DOS in all treatments but did not show any significant differences between LO (15 °C) and RL (4 °C). Total vitamin C content ranged from 0.87 to 1.81 mg g^?1 fresh weight and was higher in both LO (4 °C) and RL (4 °C) leaves than in those of LO (15 °C) regardless of storage duration. Total GSL content in the leaves of both LO (4 °C) and LO (15 °C) increased up to 3 DOS and decreased thereafter. The correlation between total vitamin C and GSL contents only existed at 0 DOS before storage and at 3 DOS of LO (15 °C) during storage in all treatments. Storage temperature generally had a significant effect on GSLs individually or collectively, except for 4‐methoxyglucobrassicin. Copyright © 2007 Society of Chemical Industry     

Hydrolysis of sucrose by invertase immobilized onto novel magnetic polyvinylalcohol microspheres

The magnetic polyvinylalcohol (PVAL) microspheres were prepared by crosslinking glutaraldehyde. 1,1′-Carbonyldiimidazole (CDI), a carbonylating agent was used for the activation of hydroxyl groups of polyvinylalcohol, and invertase immobilized onto the magnetic PVAL microspheres by covalent bonding through the amino group. The retained activity of the immobilized invertase was 74%. Kinetic parameters were determined for immobilized invertase, as well as for the free enzyme. The K_m values for immobilized invertase (55 mM sucrose) were higher than that of the free enzyme (24 mM sucrose), whereas V_max values were smaller for the immobilized invertase. The optimum operational temperature was 5°C higher for immobilized enzyme than that of the free enzyme. The operational inactivation rate constant (k_opi) of the immobilized invertase at 35°C with 200 mM sucrose was 5.83×10⁻⁵ min⁻¹. Thermal and storage stabilities were found to increase with immobilization.     

Prevention of Clostridium botulinum Type A, Proteolytic B and E Toxin Formation in Refrigerated Pea Soup by Lactobacillus plantarum ATCC 8014

The ability of Lactobacillus plantarum ATCC 8014 to inhibit Clostridium botulinum toxin production in pea soup was investigated. Soup containing C. botulinum spores (10³/g) with and without L. plantarum (10⁶/g) were evaluated. Soup containing only type A spores was toxic on days 1 and 2 when incubated at 35°C and 25°C, respectively. Soup containing only proteolytic type B spores was toxic on days 2 and 5 at 35°C and 25°C, respectively. Soup containing only type E spores was toxic at 25°C, 15°C, and 5°C in 7, 7, and 63 days respectively. No toxin was found in soup containing C. botulinum spores plus L. plantarum at any temperature studied.     

Amperometric determination of tyramine in sauce and beer by epoxy resin biocomposite membrane bound tyramine oxidase

A method is described for construction of an amperometric biosensor for specific determination of tyramine, using black gram tyramine oxidase immobilized covalently on an epoxy resin membrane. The biosensor had optimum response within 10 s at pH 8.5 and 35 °C. A linear relationship was observed between tyramine concentrations and current (mA) in the range of 0.24 to 3.47 mg/dL. The biosensor was employed for determination of tyramine in beer and sauce. The detection limit of sensor was 0.24 mg/dL. The mean analytical recovery of added tyramine (0.5 and 1.0 mg/dL) was 97.3 ± 2.3 and 95.9 ± 3.4%. Within and between batch coefficient of variation were 5.1 and 5.34%, respectively. Enzyme electrode showed 35% loss in its initial activity after its regular use over a period of 2 months. The biosensor has the advantage that it does not suffer from leaching of enzyme and measures tyramine specifically.     

USE OF IMMOBILIZED LACTASE IN PROCESSING CHEESE WHEY ULTRAFILTRATE

Enzymatic hydrolysis of lactose in cottage cheese whey ultrafiltrate was investigated. Lactase of A. niger was immobilized on an alumina-silica catalyst support by the linking agent tolylene-2, 4-diisocyanate. The resulting immobilized enzyme preparation had an activity of 3 standard international units of lactase per gram at pH 4 and 37 °C. The optimum pH and temperature for hydrolysis of lactose by immobilized lactase were 3.5 and 50°C, respectively. Immobilization of the enzyme resulted in reductions of 1.1 pH units and 15°C in optimum pH and temperature, respectively. The two constants of the simple Michaelis-Menten rate expression were obtained from Lineweaver-Burk plots of the initial reaction rate data obtained at 37°C. Estimated values for V_max and the apparent K_m were 7.8 (μmoles/min-g) and 0.26 (M), respectively. Inhibition by the product galactose was measured by studying the hydrolysis reaction in a batch reactor. The inhibition constant K_i was estimated from batch reactor data to be 0.005 and 0.053 (M) at 35 and 50°C, respectively. Activation energies of 8.1 and 6.4 (kcal/gmole) were obtained for the immobilized and soluble enzyme reactions, respectively. The behavior of the batch reactor as measured in terms of a plot of conversion versus time was essentially the same for both conventional and deionized whey ultrafiltrate.