Amperometric bienzymatic biosensor in flow injection analysis system for determination of aspartame in foods.

An amperometric bienzymatic biosensor was developed for the determination of aspartame in a flow injection analysis (FIA) system, consisting of two enzyme reactor columns packed with immobilized α-chymotrypsin (CHY) and alcohol oxidase (AOX) beads and a hydrogen peroxide electrode, connected in series. The CHY and AOX were separately immobilized on glutaraldehyde (GA)-activated beads through covalent bonding. The biosensor fabrication and operational conditions were optimized. The optimal fabrication conditions were: 2% GA with 120 min activation time; and 250 U/mL CHY and 100 U/mL AOX, with 180 min enzyme immobilization time. The optimal operational conditions were a flow rate of 0.5 mL/min and pH 8.0 at room temperature. The developed biosensor showed linearity over the aspartame concentration range 0.01-1.2 mM, with a detection limit of 0.005 mM. The developed biosensor was satisfactorily applied for detecting aspartame in beverage samples without any excessive pretreatments.

Proteolytic activity from chicken intestine and pancreas: extraction, partial characterization and application for hyaluronic acid separation from chicken comb.

BACKGROUND: The potential utilization of viscera and combs, by-products from the chicken-processing industry, was investigated. Chicken combs have been reported to consist of hyaluronic acid (HA) bound to protein. Thus proteases extracted from broiler viscera were applied to digest combs to separate HA, a high-value ingredient widely used in pharmaceutical and medical products. RESULTS: The highest activity of crude protease extracted from chicken intestine and pancreas was 0.35 U mg(-1) enzyme at 60 °C and pH 7.5. pH stability of the enzyme was attained from pH 6 to 8, while its thermal stability declined from 30 to 50 °C, with complete activity loss occurring after 30 min at temperatures above 60 °C. Therefore the optimal conditions for broiler comb digestion by crude protease in this study were chosen as pH 7.5 and 50 °C. The results showed that the yield of crude enzyme-extracted HA was lower (P < 0.05) than that obtained by commercial papain digestion. Similar identity of extracted HA and HA standard was verified by cellulose acetate electrophoresis and Fourier transform infrared spectroscopy. CONCLUSION: The crude enzyme extract from chicken intestine and pancreas had high proteolytic activity and could be used successfully to separate HA from broiler combs. The method provides an appropriate means to add value to poultry-processing waste.

Effects of fabrication parameters on the enzyme loading and sensor response of enzyme-carrying conductive polymer electrodes.

Hydrogen peroxide sensors where horseradish peroxidase (HRP) is incorporated into pyrrole/3-alkylsulfonate pyrrole copolymer films deposited on an SnO2 electrode (HRP/Py-PS electrode) were investigated with regard to the effects of the fabrication parameters (electropolymerization charge, deposition current density, and electrodeposition solution pH) on the amount of surface-immobilized enzyme and the sensor response. The amount of incorporated enzyme was determined with a method recently developed by ourselves. The results suggest that the amount of entrapped enzyme increases almost linearly with the total charge passed, and strongly depends on the polymer film growth rate and the electropolymerization pH. These findings open up a way to control the amount of enzyme and the resultant response of the biosensor by modifying the preparation conditions.

Novel hydrogen peroxide sensors based on peroxidase-carrying poly[pyrrole-co-[4-(3-pyrrolyl)butanesulfonate]] copolymer films.

Amperometric hydrogen peroxide biosensors were fabricated by incorporating horseradish peroxidase (HRP) into poly[pyrrole-co-[4-(3-pyrrolyl)butanesulfonate]] (Py-PS) copolymer films deposited on an SnO2 electrode surface by electropolymerization. The HRP/Py-PS electrodes exhibited an extended dynamic range and a markedly improved operational and storage stability, compared with HRP-incorporated polypyrrole (PPy) electrodes prepared under similar conditions. The linear range was expanded from 10(-7)-10(-4) M to 10(-7)-10(-3) M H2O2. In about 80 measurements over three weeks, the HRP/Py-PS electrode retained 60% of its initial response, while the HRP/PPy electrode almost completely lost activity. The influence of the electrodeposition solution pH on the sensor response was also investigated. Our results suggest that an expansion of the linear range and an enhancement of lifetime are due to electrostatic interactions of HRP with a negatively-charged Py-PS copolymer.