A new amperometric biosensor for hydrogen peroxide determination based on HRP-nanogold-PTH-nanogold-modified carbon paste electrodes

A new strategy for immobilization of horseradish peroxidase (HRP) has been developed by self-assembling gold nanoparticles to multiporous polythionine (PTH) film modified carbon paste interface. A thionine film was initially electropolymerized onto carbon paste interface in a mildly acidic thionine solution at a bias voltage of −1.0 to 1.5 V. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the PTH film with multiporous structure. The multiporous PTH film provided a biocompatible microenvironment for gold nanoparticles and enzyme molecules, and greatly amplified the coverage of HRP molecules on the electrode surface. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the derived biosensor. The performance and factors influencing the performance of the biosensor were also proposed. The immobilized HRP displayed a catalytic property to the reduction of H₂O₂. The H₂O₂ biosensor achieved 95% of the steady-state current within 2 s, and exhibited a linear range of 9.6×10⁻⁶ to 1.2×10⁻³ M H₂O₂ with a detection limit of 7.5×10⁻⁷ M (S/N = 3). Furthermore, the biosensor remained at about 90% of its original sensitivity after 2 weeks’ storage.     

Enzymatic Catalytic Spectrophotometric Determination of Thiamine in Food

A highly sensitive and simple spectrophotometric method for the determination of thiamine based on inhibitory effect of thiamine on the hemoglobin-catalyzed reaction of H₂O₂ with acid chrome blue K was developed. The concentration of thiamine is linear with the percentage inhibition of system under the optimal experimental conditions. The calibration curve is linear in the range 3 × 10⁻⁷ to 3.00 × 10⁻⁵ M with the detection limit of 1.2 × 10⁻⁸ M. This method can be used for the determination of thiamine in food with satisfactory results.     

Supercritical CO<Subscript>2</Subscript> extraction of <Emphasis Type="Italic">Alkanna</Emphasis> species and investigating functional characteristics of alkannin-enriched yoghurt during storage

Alkannin is a potent pharmaceutical substance with a wide spectrum of biological activities. In the scope of this study, supercritical CO₂ extraction and sonication with hexane were applied to various Alkanna species, which were then subjected to hydrolysis. Total alkannins were quantified by HPLC/DAD and incorporated into yoghurt. Viscosities, pH values and microbial analyses were reported at 7 days of intervals for 21 days of storage. A. tinctoria possessed the highest amounts of alkannins and total phenols (686.3 mg GAE/g extract). The results revealed no significant changes in pH values (4.1–4.0), viable counts of Streptococcus salivarius ssp. thermophilus (80–150 × 10⁶ cfu g⁻¹) and slightly lower viscosities of enriched yoghurts (8,250–6,750 cPs) compared with the control (4.15–4.0; 110–105 × 10⁶ cfu g⁻¹; 12,600–11,310 cPs) during storage. However, viable counts of Lactobacillus delbrueckii ssp. bulgaricus of enriched yoghurts (87 × 10³ cfu g⁻¹) were much better than the control (191 × 10³ cfu g⁻¹), indicating a significant decrease in post acidification and generation of bitter peptides. Among the species investigated, A. tinctoria is the most promising source, obtained at higher yields via supercritical fluid extraction technology as a green alternative to solvent extraction and thus can be utilized at industrial scale in order to develop yoghurt products with improved health benefits.     

Quantitative determination of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. in milk using a series piezoelectric quartz crystal sensor

A series piezoelectric quartz crystal (SPQC) sensor was developed for quantitative determination of Lactobacillus spp. populations in milk. When the electrodes were immersed in a reaction cell with bacterial inoculum, the change of frequency was caused by the impedance change of the microbial metabolism. A significant frequency decrease was found due to the coagulation of milk when the Lactobacillus spp. was cultivated in the media. The SPQC sensor system established in this study demonstrated the specificity and selectivity for detection of Lactobacillus spp. in milk sample. The calibration curve of detection time against density of Lactobacillus spp. shows a linear correlation coefficient (R ² = 0.8453) over the range of 10²–2.4 × 10⁵ CFU ml⁻¹. The detection time was influenced by the addition of peptone and glucose. The sensor exhibited rapid (within 4 h) and enabled real time monitoring of Lactobacillus spp. growth. This system is potentially applicable to detect Lactobacillus spp. concentration at local farm when a suitable temperature control device is adapted.     

Amperometric Determination of Quercetin in Some Foods by Magnetic Core/Shell Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>@ZnO Nanoparticles Modified Glassy Carbon Electrode

In this paper, Fe₃O₄@ZnO core/shell magnetic nanoparticles (MNPs) have been synthesized by a simple method, to modify carbon paste/glassy carbon electrode and improve its efficiency for determination of quercetin. The synthesized MNPs were characterized by X-ray powder diffraction (XRD), transmission electronic microscope (TEM), and scanning electronic microscope (SEM). SEM and TEM results show that the prepared Fe₃O₄@ZnO MNPs are made of the spherical shape particles with an average size of about 15 nm. The electrochemical behavior of quercetin at the surface of modified electrode was investigated. Under the optimal conditions, a linearity range of quercetin was 7.9?×?10^?7 to 6.1?×?10^?5 mol/L (0.24–18.44 mg/L) with detection limit (S/N?=?3) and sensitivity of 0.16 μmol/L (0.048 mg/L) and 0.04 μA/μM, respectively. The validated method was applied successfully for determination of quercetin in some foods and human breast milk.     

Photodegradation of Aflatoxin B<Subscript>1</Subscript> in peanut oil

A photodegradation study of aflatoxin B₁ (AFB₁) in peanut oil was performed under UV irradiation at different AFB₁ initial concentrations and UV irradiation intensities. The UV intensity and the irradiation duration on the AFB₁ photodegradation ratio is more effective, when compared with AFB₁ initial concentration, and AFB₁ with initial concentration of 2 mg/kg can be degraded thoroughly within 30 min under the intensity of 800 μw/cm². The photodegradation of AFB₁ between the selected ranges of concentrations was proved to follow first-order reaction kinetics well (R ² ≥ 0.99). The Ames test, employing Salmonella typhimurium tester strains TA98 and TA100, was employed to evaluate the residual toxicity of the AFB₁ subproducts in peanut oil, and the results indicated that the mutagenic activity of UV-treated samples (800 μw/cm² × 30 min) was completely lost compared with that of untreated samples, providing clues to the assessment of safety issues of UV method applied in AFB₁ decontamination.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

Cytoprotective effect of fucoxanthin isolated from brown algae <Emphasis Type="Italic">Sargassum siliquastrum</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell damage

In this study, the cytoprotective effect of fucoxanthin, which was isolated from Sargassum siliquastrum, against oxidative stress induced DNA damage was investigated. Fucoxanthin, a kind of carotenoid, was pretreated to the medium and the protective effect was evaluated via 2′,7′-dichlorodihydrofluorescein diacetate, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, and comet assays. Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H₂O₂), but the production was significantly reduced by the treatment with fucoxanthin. The fucoxanthin strongly enhanced cell viability against H₂O₂ induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner. Furthermore, a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye. These results clearly indicate that fucoxanthin isolated from S. siliquastrum possesses prominent antioxidant activity against H₂O₂-mediated cell damage and which might be a potential therapeutic agent for treating or preventing several diseases implicated with oxidative stress.     

Cytoprotective effects of polyphenolics on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell death in SH-SY5Y cells in relation to their antioxidant activities

The cytoprotective effects of five flavonoids (quercetin, rutin, catechin, kaempferol and morin) and four hydroxycinnamic acids (caffeic, ferulic, sinapic and chlorogenic acids) were evaluated by the degree of protection they provided against H₂O₂-induced damage to human neuroblastoma SH-SY5Y cells. All compounds exhibited protection against H₂O₂-mediated cytotoxicity in a dose dependent manner. The concentration required to give a 50% reduction in cell death (EC₅₀ value) were derived from their dose–response relationships. The cytoprotective activities of these phenolic compounds in the order of quercetin > caffeic acid > rutin > chlorogenic acid > catechin > ferulic acid > sinapic acid > morin > kaempferol. The EC₅₀ values of the phenolic compounds were strongly related to their chemical structures. The EC₅₀ values were compared with the antioxidant activities as determined by five different chemically based antioxidant assays [2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP) and lipid peroxidation inhibition capacity (LPIC)]. The ability of these phenolic compounds to protect from H₂O₂-induced SH-SY5Y cell death correlated (r ² = 0.85) with their determined LPIC values and weakly (r ² = 0.44) with their ABTS activities. There was no correlation between EC₅₀ values and ORAC, FRAP or DPPH activities. The cytoprotection assay is a more biologically relevant measurement than the chemically defined antioxidant activity assays because it uses human cells as a substrate and therefore accounts for some aspects of uptake, metabolism and location of antioxidant compounds within cells.     

Polysaccharide/Protein Nanomultilayer Coatings: Construction,Characterization and Evaluation of Their Effect on ‘Rocha’ Pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) Shelf-Life

Nanolayered coatings of κ-carrageenan, a polysaccharide with good gas barrier properties, and lysozyme, a protein with antimicrobial action, were in a first stage assembled on aminolysed/charged polyethylene terephthalate (PET) pieces, which acted as a support, by alternate five-layer deposition. This was performed to allow the characterization of the nanomultilayer system. PET aminolysis was confirmed by Fourier transform infrared spectroscopy and contact angle, and the subsequent layer adsorption on aminolysed PET surface was confirmed by absorbance, contact angle and SEM images. The water vapour permeability and the oxygen permeability (O₂P) of the five layers were found to be 0.013 ± 0.003 × 10⁻¹¹ and 0.1 ± 0.01 × 10⁻¹⁴ g m⁻¹ s⁻¹ Pa⁻¹, respectively. The nanomultilayer system was subsequently applied (without PET support) directly on ‘Rocha’ (Pyrus communis L.) fresh-cut pears and whole pears. Uncoated fresh-cut pears and whole pears presented higher mass loss, higher total soluble solids (TSS) and lower titratable acidity when compared with coated fresh-cut pears and whole pears. Uncoated fresh-cut pears also presented a darker colour. These results showed that the nanolayered coating assembled on the fruits’ surface has a positive effect on fruit quality and contributed to extend the shelf-life.     

Determination of Rosmarinic Acid by High-Performance Liquid Chromatography and Its Application to Certain Salvia Species and Rosemary

This study describes a method development for the determination of rosmarinic acid (RA) by using a gradient high-performance liquid chromatography (HPLC) and its application to certain plant materials. The analysis was performed by utilizing a two solvents system [A: methanol/water/formic acid (10:88:2; v:v:v); B: methanol/water/formic acid (90:8:2; v:v:v)] on a reverse-phase column. The flow rate and injection volume were 1 ml min⁻¹ and 10 μl, respectively. Signals were detected at 280 nm. In addition, an internal standard (IS) technique was applied for the analysis of RA to increase precision, and propylparaben was employed for this purpose. The repeatability results as RSD% were 1.66, 1.17 and 1.26 for intra-day and 1.38 was for inter-day with the employment of (3.67 × 10⁻⁵ M) RA. A limit of linearity (LOL) was observed in a wide (1.13 × 10⁻⁵–5.65 × 10⁻⁴ M) concentration range. Linearity parameters were also examined in the range of 5.95 × 10⁻⁶–7.14 × 10⁻⁵ M RA, and very good correlation was observed. The limit of detection (LOD) and limit of quantification (LOQ) (for inter-day) were 1.60 × 10⁻⁶ M (signal/noise [S/N] = 3.3) and 4.80 × 10⁻⁶ M (S/N = 10), respectively. The method was applied to the extracts of certain Lamiaceae plants (Salvia candidissima Vahl. subsp. candidissima, S. sclarea L., S. verticillata L. subsp. verticillata and R. officinalis L.), and reasonable results were obtained.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

<Emphasis Type="Italic">In silico</Emphasis> phylogenetic analysis of lactic acid bacteria and new primer set for identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in food samples

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 10³ and 7 × 10² CFU mL⁻¹, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.     

Study of the diffusion coefficients of diphenylbutadiene and triclosan into and within meat

The migration of two chemicals, diphenylbutadiene and triclosan, was the target of this paper. Pork meat with different fat contents was prepared to study the influence of this parameter in the migration levels and to study the rate of diffusion of these migrants into the whole plastic/foodstuffs system and within the foodstuff. The whole system plastic/foodstuff diffusion coefficient (effective D) was calculated according to an equation based on the Fick’s Second Law, and D within the foodstuff (D _F) was calculated according to the Moisan equation. At 5 °C, D _F was 1.6 × 10⁻⁷ and 1.7 × 10⁻⁷ cm²/s for DPBD and triclosan, respectively. At 25 °C, D _F was 3.7 × 10⁻⁷ and 3.9 × 10⁻⁷ cm²/s for DPBD and triclosan, respectively. As expected, D within the pork meat is faster than the whole system D, which means that the interface plastic/foodstuff may be the limiting step for the mass transport of chemicals from the packaging to the foodstuff.     

Lipase-catalyzed acylation of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine with conjugated linoleic acid in [Bmim]PF<Subscript>6</Subscript> ionic liquid

Improving significantly the acylation reaction of l-carnitine with conjugated linoleic acid catalyzed by lipase needs to select an efficient and suitable reaction medium that is not only polar but also hydrophobic. [Bmim]PF₆ which is satisfied with the above two requirements was applied as the reaction medium. The optimal reaction conditions were: Novozyme 435 as the catalyst, 5:1 of molar ratio of fatty acid to l-carnitine, 40 g L⁻¹ lipase, 0.22 a_W of initial water activity, 125 g L⁻¹ molecular sieves (they were added within 0–3 h of reaction time), 60 °C of reaction temperature, 200 rpm of rotation speed, 32 h of reaction time. The overall conversion could reach 98.27%. The conversion in [Bmim]PF₆ was 2.13 and 1.56 times higher than that in acetonitrile and in solvent-free system, respectively. The lipase in the present work could be used eight times. [Bmim]PF₆ as the reaction medium was better than acetonitrile and solvent-free system.     

Spectrophotometric Determination of Trace Nitrite with Brilliant Cresyl Blue Using β-Cyclodextrin as a Sensitizer

A simple and sensitive spectrophotometric method for determination of nitrite has been described. The method is based on the oxidation of brilliant cresyl blue (BCB) by nitrite in acidic medium, which results in the decrease in absorbance at 636 nm. The decrease in absorbance is directly proportional to nitrite concentration obeying Beer’s law. The sensitivity is largely enhanced in the presence of β-cyclodextrin (β-CD) because of inclusion complexation. It was calculated that β-CD and BCB could form 1:1 inclusion complexation with a formation constant of 546.6 L/mol. Linear calibration graphs were obtained for 0.02 × 10⁻³–0.8 × 10⁻³ g/L sodium nitrite at 636 nm. The detection limit for the analytical procedure was 4.0 × 10⁻⁶ g/L sodium nitrite. The relative standard deviation for determination of 0.1 × 10⁻³ g/L and 0.5 × 10⁻³ g/L sodium nitrite were 0.69% and 0.38%, for ten determinations, respectively. Twenty-two coexistent ions or species were examined, and no serious interference for most of ions was observed. The method has been applied to determine nitrite in water and vegetable samples with satisfactory results.     

Application of Catalytic Hydrogen Evolution in the Presence of Neonicotinoid Insecticide Clothianidin

Clothianidin, a new generation of pesticide, was determined in spiked tap water, apple juice, and soil by square-wave adsorptive stripping voltammetry. The method of determination is based on the hydrogen evolution reaction catalyzed by clothianidin at the hanging mercury drop electrode. The optimal signal was detected at −1.4 V versus Ag/AgCl in citrate buffer at pH 2.2. Various parameters such as pH, buffer concentration, frequency, amplitude, step potential, accumulation time, and potential were investigated to enhance the sensitivity of the determination. The optimal results were recorded at an accumulation potential of −0.35 V, accumulation time of 7 s, amplitude of 100 mV, frequency of 200 Hz, and step potential of 7 mV. The mechanism of catalytic hydrogen evolution was considered under experimental and theoretical conditions. This electroanalytical procedure enabled to determine clothianidin in the concentration range 9 × 10⁻⁹–4 × 10⁻⁶ mol L⁻¹ in supporting electrolyte and tap water, 1 × 10⁻⁷–4 × 10⁻⁶ mol L⁻¹ in diluted apple juice, and 2 × 10⁻⁷–1 × 10⁻⁶ mol L⁻¹ in soil. The detection and quantification limits in supporting electrolyte and diluted apple juice were found to be 2.6 × 10⁻⁹, 8.6 × 10⁻⁹ and 3 × 10⁻⁸, and 1 × 10⁻⁷ mol L⁻¹, respectively. A standard addition method was successfully used to determine clothianidin in spiked tap water, spiked apple juice, and spiked soil.     

Anthocyanin effectively scavenges free radicals and protects retinal cells from H<Subscript>2</Subscript>O<Subscript>2</Subscript>-triggered G2/M arrest

It has been previously shown that anthocyanins effectively neutralize free radicals and can act as an antioxidative and anti-aging agent and prevent dementia. In addition, anthocyanins promote expression of rhodopsin, which facilitates night vision impairment, blurred vision, eye fatigue due to physical and mental fatigue, and a loss in rhodopsin has been shown to result from various eye diseases. In this study, the free radical scavenging properties of anthocyanins were evaluated for the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, alkyl radical, and hydroxyl radical using electron spin resonance spectroscopy. The DPPH radical scavenging activity of anthocyanins increased in a dose-dependent manner, with a 50% inhibitory concentration (IC₅₀) value of 2.9 μg/mL. The alkyl radical scavenging activity of anthocyanin was also high, with a IC₅₀ value of 52.2 μg/mL. In addition, the hydroxyl radical scavenging activity of anthocyanins was concentration-dependent. The inhibitory effect of anthocyanins on lipid peroxidation was examined using the ferric thiocyanate and thiobarbituric acid assays. The inhibitory activity of anthocyanins was found to be comparable to that of Vitamin E. In addition, the ability of anthocyanins to reduce oxidative DNA damage was assessed in vitro by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular form. Also, we have found that anthocyanins’ inhibitory activity of the H₂O₂-induced G2/M phase arrest in ARPE-19 cells. Anthocyanins enhanced the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase in ARPE-19 cells. Taken together, the present results demonstrate that anthocyanins possess potent antioxidative activity.