A novel biosensor based on Lactobacillus acidophilus for determination of phenolic compounds in milk products and wastewater

Different branches of industry need to use phenolic compounds (PCs) in their production, so determination of PCs sensitively, accurately, rapidly, and economically is very important. For the sensitive determination of PCs, some biosensors based on pure polyphenol oxidase, plant tissue and microorganisms were developed before. But there has been no study to develop a microbial phenolic compounds biosensor based on Lactobacillus species, which contain polyphenol oxidase enzyme. In this study, we used different forms of Lactobacillus species as enzyme sources of biosensor and compared biosensor performances of these forms for determination of PCs. For this purpose, we used lyophilized Lactobacillus cells (containing L. bulgaricus, L. acidophilus, Streptococcus thermophilus), pure L. acidophilus, pure L. bulgaricus, and L. acidophilus- and L. bulgaricus adapted to catechol in Lactobacilli MRS Broth. The most suitable form was determined and optimization studies of the biosensor were carried out by using this form. For preparing the bioactive layer of the biosensor, the Lactobacillus cells were immobilized in gelatin by using glutaraldehyde. In the study, we used catechol as a substrate. Phenolic compound determination is based on the assay of the differences on the respiration activity of the cells on the oxygen meter in the absence and the presence of catechol. The microbial biosensor response depends directly on catechol concentration between 0.5 and 5.0 mM with 18 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount was found to be 10 mg, and also phosphate buffer (pH 8.0; 50 mM) and 37.5 °C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity on the biosensor response and operational and storage stability were examine. Furthermore, the determination of PC levels in synthetic wastewater, industrial wastewater, and milk products was investigated by using the developed biosensor under optimum conditions.     

Optimization of catechol production by membrane-immobilized polyphenol oxidase: a modeling approach

Although previous research has focused on phenol removal efficiencies using polyphenol oxidase in nonimmobilized and immobilized forms, there has been little consideration of the use of polyphenol oxidase in a biotransformation system for the production of catechols. In this study, polyphenol oxidase was successfully immobilized on various synthetic membranes and used to convert phenolic substrates to catechol products. A neural network model was developed and used to model the rates of substrate utilization and catechol production for both nonimmobilized and immobilized polyphenol oxidase. The results indicate that the biotransformation of the phenols to their corresponding catechols was strongly influenced by the immobilization support, resulting in differing yields of catechols. Hydrophilic membranes were found to be the most suitable immobilization supports for catechol production. The successful biocatalytic production of 3-methylcatechol, 4-methylcatechol, catechol, and 4-chlorocatechol is demonstrated.     

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans-p-coumaric acid, trans-ferulic acid and trans-caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.     

Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine

An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.     

Solanum melongena polyphenol oxidase biosensor for the electrochemical analysis of paracetamol

A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50?mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200?µM, with a detection limit of 5?µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.     

Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).     

Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial waste-water samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.     

Evaluation of a new biosensor-based mushroom (Agaricus bisporus) tissue homogenate: investigation of certain phenolic compounds and some inhibitor effects

A biosensor based on mushroom tissue homogenate for detecting some phenolic compounds (PCs) and usage of the biosensor for quantifying certain substances that inhibit the polyphenol oxidase activity in mushroom (Agaricus bisporus) tissue homogenate is described. The mushroom tissue homogenate was immobilized to the top of a Clark-type oxygen electrode with gelatin and glutaraldehyde. Optimization of the experimental parameters was done by buffer system, pH, buffer concentration, and temperature. Besides, the detection range of eight phenolic compounds were obtained with the help of the calibration graphs. Thermal stability, storage stability, and repeatability of the biosensor were also investigated. A linear response was observed from 20 x 10(-3) to 200 x 10(-3) mM phenol. The biosensor retained approximately 74% of its original activity after 25 days of storage at 4 degrees C. In repeatability studies, variation coefficient (C.V.) and standard deviation (S.D.) were calculated as 2.44% and +/-0.002, respectively. Inhibition studies revealed that the proposed biosensor was applicable for monitoring benzoic acid and thiourea in soft drinks and fruit juices.     

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.     

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea.

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands.     

Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

Amperometric biosensor for lactate analysis in wines and grape must during fermentation

The amperometric biosensor based on lactate oxidase for determination of lactate has been developed, and two methods of immobilization of lactate oxidase on the surface of industrial screen-printed platinum electrodes SensLab were compared. A sensor with immobilized in the Resydrol polymer lactate oxidase by the method of physical adsorption is characterized of narrow dynamic range and greater response value in comparison with a biosensor based on immobilised in poly(3,4-ethylenedioxythiophene) lactate oxidase by the method of electrochemical polymerization. Operational stability of the biosensor developed was studied and it was shown, that the immobilization method does not influence their stability. The analysis of the lactate in wine and during wine fermentation has been conducted. High correlation of the data obtained by means of amperometric lactate biosensor and a standard method of an ionic chromatography has been shown. The developed biosensor could be applied in the food industry for the control and optimization of the wine fermentation process, and quality control of wine.     

Amperometric tyrosinase biosensor based on polyacrylamide microgels

An amperometric enzyme sensor using tyrosinase (PPO) entrapped in polyacrylamide microgels has been developed for determination of phenolic compounds. Polyacrylamide microgels were obtained by the concentrated emulsion polymerization method. The crosslinking of the polymer matrix optimum to retain the enzyme and to allow the diffusion of the compounds involved in the enzyme reaction has been studied (4.0%) as well as the influence on the response of analytical parameters such as pH, temperature, enzyme load and working potential. The useful lifetime of the biosensor was 27 days and it was useful to determine monophenolics compounds (e.g. cresol, chlorophenol) and diphenolics compounds (e.g. catechol and dopamine) by amperometric measurements at -100mV (versus SCE) in a batch system. The results showed that the substrate structures have a great influence on the sensor response.     

Lack of involvement of polyphenol oxidase in ortho-hydroxylation of phenolic compounds in mung bean seedlings

Complete elimination of polyphenol oxidase activity in hypocotyls and leaves of developing mung bean ( Vigna radiata L. Wilczek cv. Berkin) seedlings by tentoxin had no effect on the content of the ortho-hydroxylated flavonoids delphinidin and rutin. Tentoxin completely eliminated polyphenol oxidase-mediated ortho-hydroxylation of p -coumaric acid to caffeic acid. Despite this, tentoxin had no effect on caffeic acid derivative contents in the seedlings. High performance liquid chromatography profiles indicated that elimination of polyphenol oxidase had no effect on either the quality or the quantity of soluble phenolic compounds, These data strongly indicate that polyphenol oxidase is not involved in metabolism of phenolic compounds in developing plant tissues.     

An amperometric biosensor for determining amino acids using a bienzymatic system containing amino acid oxidase and protease

An amperometric biosensor for rapid determination of the concentration of l-amino acids has been developed using l-amino acid oxidase (l-AAO) immobilized by gel entrapment with poly(carbamoyl) sulfonate hydrogel. The broad substrate range of l-AAO allows this biosensor to be flexible in application. The artificial sweetener, aspartame, was determined by coupling l-AAO with pronase.     

Biosensor for the enantioselective analysis of S-perindopril

Because S-perindopril enantiomer is the eutomer which is responsible for the angiotensin-converting enzyme inhibition activity, it is necessary to develop a reliable method for its assay from its distomer, the R-enantiomer. For this purpose, an amperometric biosensor was developed based on L-amino acid oxidase. The working range of the described biosensor was 20pmol/L-10 micromol/L on the 7.0-7.4 pH range, with a detection limit of 2pmol/L. The low enantioselectivity for R-perindopril, as compared with S-enantiomer, was demonstrated by both mixed solutions and separate solutions methods (amperometric selectivity coefficient is 1.0 x 10(-4)). The biosensor was also selective towards D-proline and polyvinylpyrrolidone. The amperometric biosensor can be used for enantioselective analysis of S-perindopril in raw material, with an RSD < 1%. The life time (t95%) of the biosensor is three weeks.     

Barley (Hordeum vulgare) seed dormancy as related to glumella characteristics

Dormancy of freshly harvested barley ( Hordeum vulgare L. cv. Sonja) caryopses results mainly from glumellae which fix oxygen by polyphenol oxidase (EC 1.14.18.1)-mediated oxidation of phenolic compounds present in high amounts. The breaking of dormancy during dry storage is not due to qualitative or quantitative modifications of the phenols or polyphenol oxidases. Glumellae of dormant caryopses start to take up oxygen at the beginning of inbibition, whereas those of non-dormant caryopses start to take up oxygen only after about 10 h. That delay should allow germination.     

Synthesis and characterization of a pyrrole-alginate conjugate and its application in a biosensor construction

N-(3-Aminopropyl)pyrrole was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a pyrrole-alginate conjugate for subsequent use in biosensor applications. The pyrrole-alginate conjugate was quantified by UV spectroscopy at 230 nm, by an HPSEC-MALLS analytical method, as well as by FTIR and 13C NMR spectroscopies. The new pyrrole-alginate conjugate was used for the immobilization of polyphenol oxidase (PPO) onto an electrode surface by physical entrapment resulting from the gellification process and electrochemical polymerization of the pyrrole groups. The efficiency of this cross-linking approach (chemical and electrochemical) was investigated by comparing the amount of enzyme released from polypyrrole-alginate and regular alginate. In addition, biosensors were prepared by entrapment of the PPO in polypyrrole-alginate and regular alginate matrixes and their performance for the amperometric determination of catechol chosen as a model analyte was examined, yielding a sensitivity of 350 and 80 microA M(-1) cm(-2), respectively, for polypyrrole-alginate and alginate biosensors.     

A novel glucose sensor based on monodispersed Ni/Al layered double hydroxide and chitosan

A novel cheap and simple amperometric glucose biosensor, based on the electrode modified with the Ni/Al layered double hydroxide (LDH) nanoflakes and chitosan (CHT), without glucose oxidase, is presented. The glucose biosensor based on monodispersed high active Ni/Al-LDH nanoflakes and CHT exhibits an appropriate linear range of 0.01-10mM and good operational stability. The amperometric sensor shows a rapid response at the potential value 0.48V. In addition, optimization of the biosensor construction, the effects of the applied potential, the scan rate as well as common interfering compounds on the amperometric response and human serum samples analysis of the sensor were investigated and discussed.