Neutral and acidic ascorbate oxidases from satsuma mandarin (Citrus unshiu Marc): Isolation and properties

The neutral and acidic forms of ascorbate oxidase (AAO) were isolated and purified from young fruits of satsuma mandarin by a combination of (NH₄)₂SO₄ fractionation, ion-exchange and hydrophobic chromatography, and gel filtration. The neutral and acidic AAO were both diamers of two subunits with native molecular weights of 133 and 136 kDa, respectively. Neutral AAO was stable in the range of pH 5 and 8 and exhibited optimal activity at pH 5.5 and 45°C. In contrast, acidic AAO was stable between pH 5 and 10 and exhibited optimal activity at pH 5.5 and 50°C. When L-ascorbate and D-isoascorbate were used as substrates, K_m values of neutral AAO were 2.98 × 10^?4 M and 3.36 × 10^?4 M and those of acidic AAO were 1.99 × 10^?4 M and 1.86 × 10^?3 M, respectively. The activities of the two forms of AAO were totally inhibited by metabisulphite and cyanide, substantially suppressed by higher concentrations of diethyldithiocarbamate and fluoride, and not inhibited at all by monovalent and divalent metal ions tested.     

Carbon Nanotube–Ionic Liquid (CNT–IL) Nanocamposite Modified Sol-Gel Derived Carbon-Ceramic Electrode for Simultaneous Determination of Sunset Yellow and Tartrazine in Food Samples

Carbon-ceramic electrode modified with multi-walled carbon nanotubes–ionic liquid (MWCNTs–IL) nanocomposite was constructed. This electrode was used for electrochemical determination of food dyes Sunset Yellow (SY) and tartrazine (Tz). The modified electrode based on high surface area and high ionic conductivity of nanocomposite exhibited electrocatalytic effect for oxidation of SY and Tz; also, oxidation peak potentials of SY and Tz effectively separated on modified electrode, and their simultaneous determination was possible. Operational parameters, such as the amount of MWCNTs in suspension, IL volume, solution pH, and scan rate, which affect the analytical performance of determination, were optimized. The present electrode behaved linearly to Sunset Yellow and tartrazine in the concentration range of 4?×?10^?7 to 1.1?×?10^?4?M and 3?×?10^?6 to 0.7?×?10^?4?M with a detection limit of 10^?7?M (0.045 mg?L^?1) and 1.1?×?10^?6?M (0.59 mg?L^?1), respectively. The proposed method was successfully utilized for simultaneous determination of SY and Tz in different food samples, and the obtained results were in good agreement to those obtained by HPLC method.     

Development of an Electrochemical Sensor for the Determination of Amaranth: a Synthetic Dye in Soft Drinks

The voltammetric behaviour of synthetic food colourant, amaranth, was studied using a multiwalled carbon nanotube (MWCNT) thin film-modified gold electrode in 0.1 M acetate buffer solution of pH 5.0. A well-defined oxidation peak was obtained for amaranth at 0.792 V with the modified electrode. The diffusion controlled oxidation of amaranth at the modified electrode can be attributed to the electrocatalytic nature of MWCNT, since the bare electrode has not produced an electrochemical signal under the same experimental conditions. The operational parameters such as supporting electrolyte, pH of the solution and amount of MWCNT–Nafion suspension were optimised. Under optimum conditions, the oxidation peak current varies linearly with concentration in the range from 1.0?×?10^?5 M to 1.0?×?10^?6 M with a limit of detection at 6.8?×?10^?8 M. The developed sensor has good sensitivity and selectivity towards oxidation of amaranth. The sensor was successfully employed for the determination of amaranth in three different samples of soft drinks, and the results were in good agreement with the standard spectrophotometric method.     

Determination of arginine in dietary supplements

We present a rapid and sensitive flow injection method for the determination of arginine in dietary supplements. Detection was based on the chemiluminescence reaction of arginine with alkaline hypobromite. The response is proportional to analyte concentration over the calibration range, from 2.5 × 10^?6 to 1 × 10^?4 M , and a relative standard deviation of 1% was calculated (1 × 10^?5 M , n = 12). Samples required only aqueous dilution prior to analysis, and over 100 samples could be analysed per hour, which is superior to that achieved with conventional colorimetric and enzymatic procedures. The results obtained with the flow injection methodology were concordant with those achieved using high‐performance liquid chromatography employing pre‐column derivatisation and fluorescence detection. Copyright © 2005 Society of Chemical Industry     

Spectrofluorometric Method for Determination of Al3+ with 3-Hydroxy-2-Naphthalenecarboxylic Acid and its Application to Milk Samples

On the basis of affinity of Al³⁺ ions toward functional groups of 3-hydroxy-2-naphthalenecarboxylic acid (3H2NA), a high stable complex between Al³⁺ and 3H2NA in 5 % methanol/water solution was established. The mole ratio method showed that a stable complex by taking part of acetate and 3H2NA with the stoichiometric ratio of 1:1:1 and stability constant of 8.6?×?10⁶ is formed. Under the optimum conditions for the complex formation, a rapid spectrofluorometric determination of trace amounts of Al³⁺ (λ _ex?=?366 nm, λ _em?=?460 nm) was proposed. The optimum conditions involve 3H2NA concentration of 1.0?×?10^?5 M, buffering pH of 4.5 (acetic/acetate system), and ionic strength of 0.01 M. The fluorescence intensity exhibited a good linearity against the Al³⁺ concentration in the range 5.0?×?10^?8–5.0?×?10^?6 M with the detection limit of 1.3?×?10^?8 M. No considerable interference was observed due to the presence of coexisting anions and cations. The method was applied successfully to the determination of trace amounts of Al³⁺ in milk. Validation of the measurements was confirmed using graphite furnace atomic absorption spectrophotometry.     

Synergism between lipoxygenase‐active soybean flour and ascorbic acid on rheological and sensory properties of wheat bread

BACKGROUND: The interaction between lipoxygenase‐active soybean flour (LOX) and ascorbic acid (AA), on colour, rheological and sensory properties of wheat bread was studied with the aim of reducing the applied quantity of additives in bread formulations. RESULTS: The ascorbic acid (0–500 ppm) and active soybean flour (0–1%) mixture improved bread‐crumb colour by lowering the yellow hue in a higher proportion than those expressed by the components alone, characterising a synergistic mechanism (?_b = 15.1? (1.7 × LOX) ? (0.5 × AA) ? (5.8 × LOX × AA), where ?_b represent the estimated value for the yellow hue parameter). No differences in flavour and porosity were seen between the samples. As supported by the instrumental methods, breads made with active soybean flour and ascorbic acid (LOX + AA) had whiter crumbs and were softer and springier than controls as assessed by a trained sensory panel. In summary, the combination of both active soybean flour and ascorbic acid showed synergism, promoting a greater bleaching effect than when used alone. CONCLUSION: These results suggest the potential use of active soybean flour as a synergistic ingredient in the substitution of artificial additives in bread making. Since the interaction on the bleaching response was not linear and active soybean flour showed a higher iron concentration (66.40 ± 4.23 µg g^?1) than non‐active soybean flour (52.30 ± 0.40 µg g^?1), more studies are warranted to establish the biochemical mechanisms involved in this interaction. Copyright © 2007 Society of Chemical Industry     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

α-, γ- and δ-Tocopherol Effects on Chlorophyll Photosensitized Oxidation of Soybean Oil

The effects of 0, 1.0, 2.0 and 4.0 (x 10^?3 M) α-, γ- or δ-tocopherol on chlorophyll b photosensitized oxidation of soybean oil in methylene chloride were studied by peroxide values and headspace oxygen. As concentrations of tocopherols increased, peroxide values decreased and headspace oxygen increased (P < 0.05). At 1.0 × 10^?3 M, α-tocopherol showed highest antioxidant effect, γ-tocopherol second and then δ-tocopherol. α- and -γ-Tocopherols had similar effects and δ-tocopherol had lower effect at 2.0 × 10^?3 M (P < 0.05). However, the three tocopherols were not different (P > 0.05) at 4.0 × 10^?3 M. α-Tocopherol quenched singlet oxygen to reduce the photosensitized oxidation of oil. The quenching rate constants of α-tocopherol were 2.7 × 10⁷M^?1sec^?1 by peroxide value and 2.6 × 10⁷ M^?1sec^?1 by headspace oxygen.     

Isophtalic acid terminated graphene oxide modified glassy carbon nanosensor electrode: Cd2+ and Bi3+ analysis in tap water and milk samples

In this study, graphene oxide was derivative with 5-aminoisophtalic acid by amidization reaction. The nanomaterial in suspension was denoted as graphene oxide-isophtalic acid. The graphene oxide-isophtalic acid suspension was covered on the glassy carbon electrode surface under the infrared lamb. The graphene oxide was characterized with transmission electron microscopy and x-ray diffraction. Surface characterization of the glassy carbon/graphene oxide-isophtalic acid was performed with x-ray photoelectron spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The ultrasensitive nanoplatform for the simultaneous electrochemical square-wave anodic stripping voltammetry assay of Bi³⁺ and Cd²⁺ in aqueous solution has been developed on the glassy carbon/graphene oxide-isophtalic acid. The linearity range of Bi³⁺ and Cd²⁺ were 1.0×10^?8 – 1.0×10^?12 M (S/N = 3). The responses of species were practically unaltered with the increase of various species concentration. The detection limits of Cd²⁺ and Bi³⁺ were determined as 8.1×10^?13 M and 1.06×10^?13 M, respectively. The electrode performance was checked with tap water and commercially milk samples.     

KINETICS OF THE INTERACTION OF PANCREATIC α-AMYLASE WITH A KIDNEY BEAN (PHASEOLUS VULGARIS) - AMYLASE INHIBITOR

An amylase inhibitor isolated from black beans (Phaseolus vulgaris) can completely inhibit porcine pancreatic α-amylase forming a 1:1 stoichiometric complex. The kinetic pattern of complex formation is pH dependent. At pH 5.5 it follows a first order reaction with rate constant of 0.029 min^?1 and 0.017 min^?1 at 37°C and equimolar inhibitor and enzyme concentration, respectively, of 10^?8 M and 10^?9 M. At pH 6.9 it is a second order reaction, with a rate constant of 0.25 × 10⁶ M^?1 min^?1 at 37°C, with 4 × 10^?8 M concentrations of enzyme and inhibitor. The dissociation constants of the enzymeinhibitor complex are 1.7 × 10^?10 M at pH 5.5 and 4.4 × 10^?9 M at pH 6.9, at 37°C. The kinetic data obtained at pH 5.5 suggested the formation of an initial reversible complex followed by a conformational change step. The complex can be dissociated either in acid pH (4.3) or at pH values higher than 6, 5 with partial recovery of the amylase activity.     

Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR

A quantitative assay for Plesiomonas shigelloides in pure culture and oysters based on the real-time polymerase chain reaction (Rti-PCR) was developed. The methodology involved the treatment of oyster tissue homogenates with formaldehyde, differential centrifugation, and treatment of samples with activated charcoal coated with Pseudomonas fluorescens. With seeded oyster tissue homogenates, without formaldehyde or coated charcoal treatments, the lowest level of detection for P. shigelloides was 1?×?10⁷ genomic targets per gram of tissue, equivalent to 2.5?×?10⁵ genomic targets per Rti-PCR. The addition of 4% formaldehyde to tissue homogenates reduced the minimum level of detection of P. shigelloides to 1?×?10⁵ genomic targets per gram of tissue, equivalent to 2.5?×?10³ genomic targets per real-time PCR. The treatment of tissue homogenates with only activated charcoal coated with P. fluorescens reduced the minimum level of detection of P. shigelloides to 1?×?10⁴ genomic targets per gram, equivalent to 2.5?×?10² genomic targets per real-time PCR, without DNA purification or enrichment. The combination of adding 4.0% formaldehyde to oyster tissue homogenates and treating with coated charcoal reduced the level of detection of P. shigelloides to 1?×?10³ genomic targets per gram, equivalent to 25 genomic targets per real-time PCR. The linear range of detection was from 1?×?10³ to 1?×?10⁶ genomic targets per gram without enrichment.     

Determination of lactose in sugar-free milk powder by capillary electrophoresis with electrochemical detection

Lactose in three sugar-free milk powder samples were satisfactorily determined by capillary electrophoresis with electrochemical detection using a 300 μm copper disk electrode as the working electrode in 0.1 mol/l NaOH medium. There was acceptable linearity (0.9969) between the peak current and concentration of lactose in the range from 5.0×10⁻⁶ to 5.0×10⁻³ g/ml with the detection limit (S/N=3) of 1×10⁻⁷g/ml. The proposed method was successfully applied to analyze the actual milk powder samples.     

Monitoring of Zn(II), Cd(II), Pb(II) and Cu(II) During Refining of Some Vegetable Oils Using Differential Pulse Anodic Stripping Voltammetry

In this study, a novel approach has been developed using differential pulse anodic stripping voltammetry (DPASV) for the simultaneous determination of Zn(II), Cd(II), Pb(II) and Cu(II) in edible oils at hanging mercury drop electrode. The microwave digestion of oil samples was carried out with concentrated HNO₃ and H₂O₂. KNO₃ was used as a supporting electrolyte. The experimental conditions optimized such as deposition time, stirring rate and size of mercury drop were 300 s, 600 rpm and 10 mm², respectively. The method was applied to quantify Zn(II), Cd(II), Pb(II) and Cu(II) in crude and refined hazelnut, corn, sunflower and olive oils. During refining of different vegetable oils, the removal efficiencies of Zn(II), Cd(II), Pb(II) and Cu(II) were calculated as 98.20–99.91, 98.50–99.90, 95.26–99.76 and 95.93–99.92 %, respectively. The limits of detection for Zn(II), Cd(II), Pb(II) and Cu(II) were 2.1?×?10^?8, 8.7?×?10^?10, 7.1?×?10^?9 and 3.4?×?10^?9 and the limits of quantification were 6.8?×?10^?8, 2.9?×?10^?9, 2.3?×?10^?8 and 1.1?×?10^?8 M with linear regression coefficients (R ²) of 0.9930, 0.9928, 0.9893 and 0.9931, respectively. It was observed that the above metals in crude and refined vegetable oils could be determined simultaneously by the DPASV method.     

Comparison of the modeled potential yield versus the actual yield of maize in Northeast China and the implications for national food security

A precise spatial knowledge of potential yield and actual yield is crucial to assessing an increase in grain yield and is relevant to national food security. In this paper, the potential maize yields at the county level in 2013 in Northeast China were estimated using a Miami model in combination with an integrated fertility index and the effect of chemical fertilizers on yield increase. Then, the spatial characteristics of the climate, farmland and grain production potential were presented, and the potential yield increase and food security implications were analyzed. The estimated production potentials of the climate, farmland and grain in 2013 were approximately 4.65?×?10³–13.06?×?10³ kg/ha, 2.77?×?10³–9.38?×?10³ kg/ha, and 2.97?×?10³–12.1?×?10³ kg/ha, respectively, whereas the actual maize yield in 2013 was 1.50?×?10³–8.60?×?10³ kg/ha, accounting for 41.86–95.84 % of the grain production potential. The total average potential maize increase in Northeast China was 3.32?×?10³ kg/ha, measured from the difference between the climate production potential and the actual yield. Furthermore, the main regions with lower surplus production but a higher potential for increase were located in the eastern Liaoning, Jilin and Heilongjiang Provinces. In addition, the surplus production, which was 136.56 million tons, could feed 341.4 million people in other areas of China. In conclusion, we suggest that improving access to agronomic practices (such as fertilizer and high-yielding seed) and developing agricultural policies and strategies could increase the maize yield and further narrow the yield gap.     

Efficiency of modified skimmed milk base media to achieve high exopolysaccharide/cell ratios by Lactobacillus delbrueckii subsp. bulgaricus SZ2 in optimised conditions defined by the response surface methodology

Exopolysaccharide (EPS) production by Lactobacillus delbrueckii subsp. bulgaricus SZ2 was optimised in modified MRS (M‐MRS) using the response surface methodology (RSM). Maximum EPS production was 74.3 ± 2 mg/L, and the optimised values of the three variables predicted for maximum EPS production included a temperature of 38.7 °C, Bacto‐casitone and glucose concentrations of 24.5 and 29.6 g/L, respectively. To compare EPS production in MRS and skimmed milk (SM), the kinetics of EPS formation and growth were monitored in M‐MRS, SM, skimmed milk plus 2% additional sucrose (Suc‐SM) and skimmed milk containing Bacto‐casitone (20 g/L) and yeast nitrogen base (5 g/L) (BY‐SM). EPS production in all the media tested seemed to be growth‐related. The EPS/cell ratios were determined to be 3.12 × 10^?10, 1.43 × 10^?10, 4.42 × 10^?11 and 3.16 × 10^?11 mg/cell, in Suc‐SM, SM, M‐MRS and BY‐SM, respectively, clearly indicating the greater effect of C/N ratio when cell behaviour in EPS production is considered.     

STUDIES ON BLACK GRAM (Phaseolus mungo L.) TRYPSIN INHIBITOR

ABSTRACT A trypsin inhibitor isolated from black gram (Phaseolus mungo L.) had 75 amino acid residues with an estimated molecular weight of 7892. The kinetic constants K_m and V_max as evaluated by the Dixon and Cornish-Bowden plots were 2.7 × 10^?5and 6 × 10^?3M/min, respectively. The dissociation constants of the enzyme-inhibitor complex (Ki) and the enzyme-inhibitor-substrate complex (Ki') were respectively 4 × 10^?7M and 1.9 × 10^?6M. Trypsin inhibition by black gram trypsin inhibitor was of a linear-mixed type. Chemical modification studies suggested the possible involvement of lysine and arginine at the active site of the inhibitor.     

Effect of Temperature and Moisture Content on the Kinetics of Trypsin Inhibitor Activity, Protein in vitro Digestibility and Nitrogen Solubility of Cowpea Flour

Samples of finely ground cowpea flour containing 7.5%, 19.4% and 25.5% moisture were heated in sealed tubes at 100° 125° and 150°C for periods of 0.5 to 120 min. First order rate constants for losses of trypsin inhibitor activity and nitrogen solubility ranged from 1 × 10^?2 to 18 min^?1 and from 4 × 10^?2 to 8 min^?1 respectively. In vitro protein digestibility (IVD) increased, then decreased with heating as described by sequential first order kinetics. Rate constants for increase of IVD varied from 0.13 to 12 min^?1, while for decrease in IVD the range was 5 × 10^?5 to 3 × 10^?2 min^?1. Activation energies and relationships between In k and water activity were computed.     

Thermal Properties of Sweet Potato with its Moisture Content and Temperature

Thermal properties of sweet potato were experimentally determined and modeled as a function of temperature and moisture content. The purpose is to develop empirical correlations that could predict thermal properties during sweet potato processing. Thermal conductivity from the study was 0.49 ± 0.038 Wm^?1K^?1 (mean ± s.d.), thermal diffusivity was 1.2?×?10^?7 ± 9.05?×?10^?9 m²s^?1, specific heat was 3660 ± 477.4 Jkg^?1K^?1, and density was 1212 ± 73.5 kgm^?3. All properties were determined within temperature range of 20 to 60°C and moisture content range of 0.45 to 0.75 w.b. Prediction models for the thermal properties of sweet potato were developed as a function of product temperature and moisture content with experimental data from this study. Mechanistic models were also developed for thermophysical properties of sweet potato using major food components of the product. Developed models were all presented and compared.     

Mass transfer kinetics and mathematical modelling of the osmotic dehydration of orange‐fleshed honeydew melon in corn syrup and sucrose solutions

A simple mathematical model to predict dehydration and impregnation process during osmotic dehydration of orange‐fleshed honeydew in sucrose and corn syrup solutions was proposed. Results showed low dispersion and a good fitting capability for WL and SG kinetics. Diffusivity values for WL ranged from 0.96 × 10^?10 to 2.22 × 10^?10 and 1.04 × 10^?10 to 3.10 × 10^?10 m² s^?1 in corn syrup and sucrose solutions, respectively. For SG, the obtained range was 0.72 × 10^?10 to 2.35 × 10^?10 and 0.71 × 10^?10 to 2.46 × 10^?10 m² s^?1 in corn syrup and sucrose solutions, respectively. The half‐life of dehydration rates (t_1/2) was from 30.9 to 71.2 min and from 19.4 to 57.5 min in corn syrup and sucrose solutions, respectively. Diffusivities values obtained according to the proposed model were close to the ones observed from diffusive model; t_1/2 was a promising criterion for the process time definition.     

Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.