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.     

Sorbic Acid and Potassium Sorbate Permeability of an Edible Methylcellulose-Palmitic Acid Film: Water Activity and pH Effects

The apparent permeability constants for potassium sorbate and sorbic acid through an edible film composed of methylcellulose and palmitic acid (weight ratio 3:1) were evaluated as a function of water activity (a_w) and pH. For films with thickness 55–66 μm, potassium sorbate permeability increased from 2.3 × 10^?10 to 2.0 × 10^?8 (mg/sec cm²)(cm)/(mg/mL) as a_w increased from 0.65 to 0.80. Films were not stable at a_w levels above 0.80. Permeability of the film to sorbic acid at a_w 0.8 decreased from 3.3 × 10^?8 to 9.1 × 10^?10 (mg/sec cm²)(cm)/ (mg/mL) as pH increased from 3 to 7. At pH 3 the undissociated acid was 97.5% and at pH 7 it was 0.4%.     

A Fluorescence‐Based Method for Cyanate Analysis in Ethanol/Water Media: Correlation between Cyanate Presence and Ethyl Carbamate Formation in Sugar Cane Spirit

Based on the fluorescence properties of 2,4‐(1H,3H)‐quinazolinedione, a product of the reaction between cyanate and 2‐aminobenzoic acid, a simple, sensitive, selective, and reproducible method for the cyanate analysis in aqueous ethanolic media is proposed. In this method, λ_exc and λ_em are 310 and 410 nm, respectively, and the limits of detection and quantification are 2.2 × 10^?7 and 6.7 × 10^?7 mol/L, respectively. Under optimal conditions (pH = 4.5, 40% ethanol), a concentration of 5.0 × 10^?6 mol/L cyanate can be determined in a single measurement, at a 95% level of confidence, with an uncertainty of ± 0.13 × 10^?6 mol/L. Cyanide, thiocyanate, chloride, nitrate, and sulfate ions, as well as urea and urethane in concentrations 1 × 10³ higher than that of cyanate do not interfere with the measurement. The methodology was applied to cyanate analyses in the different fractions of the sugarcane distillate and the data strongly suggest a correlation between the presence of urea in wine, and the cyanate and ethyl carbamate concentrations in the spirit.     

Kinetics of ascorbic acid degradation and colour change in ground cashew apples treated at high temperatures (100–180 °C)

Ascorbic acid (AA) degradation and colour changes, measured by the lightness index (L*), were determined in cashew apples (at low dissolved O₂ concentrations) heated at high temperature (100–180 °C) in a hermetically sealed cell. A nonisothermal method was developed to estimate thermal degradation kinetics. The results showed that reaction kinetics during heat treatments were well represented by first‐order reactions. The temperature dependence of the kinetic constants was described by an Arrhenius type equation. The activation energy (E_a) for AA degradation and lightness index were 94 ± 3 and 98 ± 3 kJ mol^?1, respectively. The reaction rate constant at 140 °C for AA degradation (64 × 10^?5 ± 3 × 10^?5 s^?1) was twice that for the lightness index change (33 × 10^?5 ± 2 × 10^?5 s^?1). Results allow generating temperature profiles of heat processes that would help preserve the AA of cashew apples as well as control the colour formation during high‐temperature processes.     

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.     

Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

Reduction of sodium and increment of calcium and ω‐3 polyunsaturated fatty acids in dry fermented sausages: effects on the mineral content,lipid profile and sensory quality

BACKGROUND: A combined technological approach was applied in the development of healthier dry fermented sausages: a partial substitution of the pork back fat by pre‐emulsified linseed oil and a partial replacement of sodium chloride with calcium ascorbate at two different levels, leading to low amounts of salt (14gSalt and 10gSalt, with 14 g and 10 g NaCl per kg of mixture, respectively). RESULTS: The developed products (14gSalt and 10gSalt) showed adequate results for a_w (0.85 and 0.87) and pH (4.98 and 5.21), and low lipid oxidation values (1.4 × 10^?4 and 1.5 × 10^?5 g malondialdehyde (MDA) kg^?1). The lipid modification led to a significantly higher supply of ω‐3 (23.3 g kg^?1) compared to the control (3.2 g kg^?1). Simultaneously, reductions of 38% and 50% in sodium content and a calcium supply of 4 and 5.2 g kg^?1 were achieved in the 14gSalt and 10gSalt formulations, respectively, compared to the control products (26 g salt and 0.87 g kg^?1 Ca). Instrumental analysis of colour and texture and sensory studies demonstrated that the organoleptic quality of the new formulations was similar to that of traditional products. CONCLUSIONS: The developed dry fermented sausages showed healthier properties than traditional ones owing to their reduced sodium and higher calcium content and a significant supply of ω‐3 fatty acids. © 2012 Society of Chemical Industry     

Some Enzyme Properties of Raw Starch Digesting Amylases from Streptomyces sp. No. 4

Streptomyces sp. No. 4 produce two forms of amylase that attack raw cassava starch. Both forms, amylase‐1 and amylase‐2, were purified by starch adsorption, affinity chromatography, and ion exchange chromatography. The molecular weights of amylase‐1 and amylase‐2 as determined by SDS‐PAGE were 56 kD and 77 kD, respectively. Optimal enzyme activities occurred at pH 5.5 and at 50°C for amylase‐1 and at 45°C for amylase‐2. The activation energy of amylase‐1 and amylase‐2 were 67 and 42 kJ/mol, respectively. Hg²⁺ and pCMB inhibited both enzymes, whereas 2‐mercaptoethanol activated only amylase‐2. EDTA inhibited amylase‐1 but activated amylase‐2. The main product of hydrolysis of raw cassava starch by amylase‐1 was maltose, followed by maltotriose, maltotetraose and dextrin. Amylase‐2 cleaved raw cassava starch to produce glucose and maltose as main products. Both amylase‐1 and amylase‐2 are α‐amylases, as shown by the fast disappearance of iodine staining, the corresponding reaction products and the ability of both enzymes to hydrolyze crosslinked blue starch.     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

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.     

Antigenicity of β-lactoglobulin reduced by combining with oleic acid during dynamic high-pressure microfluidization: Multi-spectroscopy and molecule dynamics simulation analysis

Some food components can modulate the antigenicity of β-lactoglobulin (β-LG). This study investigated the role of oleic acid (OA) in reducing the antigenicity of β-LG. The results indicate the antigenicity of β-LG gradually decreased from 15 (sample with no OA) to 9.86, 7.51, and 6.01 µg/mL when interacting with OA during dynamic high-pressure microfluidization treatment at 0.1, 80, and 160 MPa. Although binding sites (n) of β-LG combined with OA at 0.1, 80, and 160 MPa decreased from 0.79 to 0.5 and 0.66, β-LG had a higher binding affinity (K_a) to OA than that of untreated β-LG. The values of K_a for β-LG/OA at 0.1, 80, and 160 MPa were 5.51 × 10⁶, 17.43 × 10⁶, and 49.75 × 10⁶M^?1, respectively. The molecule dynamic simulation showed that the OA molecules located at both β-barrel (site 1) interacted with Lys60, Glu62, and Lys69 and outer surface site 2 consisting of Tyr20, Tyr42, Ser21, Glu157, and His161. Additionally, when binding with OA during the dynamic high-pressure microfluidization treatment, the conformation of β-LG changed, reflected by the decrease of fluorescence intensity and total sulfhydryl group content, the increase of surface sulfhydryl group content, and secondary structure changes of β-LG. These results deduce that some epitopes may be masked by OA or modified by the conformational changes, resulting in the decline of antigenicity of β-LG molecules.     

Gamma irradiation as a means to eliminate Listeria monocytogenes from frozen chicken meat

Cells of Listeria monocytogenes ATCC 35152 were sensitive to gamma irradiation in phosphate buffer, pH 7.00 (D₁₀, dose required for 10% survival—0.15 kGy) at 0–5°C. The cells showed higher radiation survival when irradiated under frozen condition, with a D₁₀ of 0.3 kGy. The protection offered by shrimp/chicken/kheema homogenates (100 g litre^?1) was evidenced by even higher D₁₀ values (0.5 kGy) at both 0–5°C and cryogenic temperature. Boneless chicken meat samples were artificially inoculated with L monocytogenes ATCC 35152 cells at low (5 × 10³) colony-forming unit (cfu) g^?1 and high (5 × 10⁶ cfu g^?1) concentrations and irradiated at 1, 3, 4, 6 kGy doses under cryogenic conditions. The efficacy of the radiation process was evaluated by detecting L monocytogenes during storage at 2–4°C in the irradiated samples. These studies, when repeated with three other serotypes of L monocytogenes, clearly suggested the need for a dose of 3 kGy for elimination of 10³ cfu cells of L monocytogenes g^?1 from air-packed frozen chicken meat.     

Dephosphorylation of phytate compounds by means of acid phosphatase from Aspergillus niger

A non-purified preparation of intracellular acid phosphatase (EC 3.1.3.2) from a waste mycelium of Aspergillus niger was utilised for dephosphorylation of phytate compounds present in food and feed ingredients. The enzymic hydrolysis of p-nitrophenylphosphate was used for assaying acid phosphatase activity, expressed in standard units (u). The hydrolysis of phytate phosphorus in wheat bran, soya bean meal and fully formulated feedstuffs for broilers (Galus galus; ‘Cornish’ × White Rock') was carried out at 40°C, a pH value of 4.5 and an enzyme dosage ranging from 12 to 30 u g^?1. Complete dephosphorylation of soya bean protein isolates was performed at 60°C, a pH value of 4.5 and an enzyme dosage of 12 u g^?1. In the gastrointestinal tract of broilers the in-vivo dephosphorylation of phytates present in feed was observed when the preparation of acid phosphatase was added to the diet.     

Evaluation of drying models of apple (var. McIntosh) dried in a convective dryer

Drying behaviour of apple particles was investigated in a laboratory type dryer. The effect of drying air temperature, airflow velocity, initial height of layer, particles shape and size on the dehydration characteristics of apples was investigated. Increase in drying air temperature and increase in the airflow velocity caused a decrease in the drying time and an increase in drying rate. Increase in initial height of layer and increase in the sample thickness caused an increase in the drying time and decrease in drying rate. Drying time of the cubes was shorter and their drying rate was higher than for slices. The experimental dehydration data of apple particles obtained were fitted to the semi‐theoretical, empirical and theoretical models. The accuracies of the models were measured using the correlation coefficient (R), mean bias error (MBE), root mean square error (RMSE), reduced chi‐square (χ²), and t‐statistic method. All models described the drying characteristics of apple particles satisfactorily (R > 0.9792). The Logarythmic model can be considered as the most appropriate (R > 0.9976, MBE = ?10^?11?4.5 × 10^?6, RMSE = 0.00287–0.01746, χ² = 8.5 × 10^?6?3.1 × 10^?4, t‐stat = 7.3 × 10^?9?1.2 × 10^?3). The effect of drying air temperature, airflow velocity, characteristic dimension of the particle and initial height of layer on the drying models parameters were also determined.     

Characterization of Two α-Amylase Inhibitors from Black Bean (Phaseolus vulgaris)

Two inhibitors of α-amylase, I-1 and I-2, were purified from the black bean (Phaseolus vulgaris). The rate of complexation with porcine pancreatic α-amylase was very slow. I-l required 12 hr and I-2 6 hr for maximum inhibition at 30°C. The stoichiometry of complexation of α-amylase with I-1 was 2:1 and 1:1 with I-2. Optimum pH for complexation with I-1 ranged from 4.5 to 5.0 and 5.0 to 5.5 for I-2 and both were most stable at pH 3.0 to 4.0. An increase in ionic strength of the preincubation buffer from 0.106 to 0.906 enhanced the rate of inhibition 3 fold for both inhibitors. Maltose, a competitive inhibitor of α-amylase, added to the preincubation solution prevented complex formation for both inhibitors; however, it did not reverse the inhibition of the preformed complex. The dissociation constant for I-1 was 2.2 × 10^?9M and 3.8 × 10^?9M for I-2 at 30°C and pH 6.9. Both inhibitors functioned via a noncompetitive mechanism. I-2 was also stable at pH 2.0. I-2 was more stable to proteolytic digestion by trypsin than I-1 and was also stable to pepsin digestion.     

Diffusion of lactic acid in a buffered gel system supporting growth of Lactobacillus curvatus

Diffusion of lactic acid in a buffered gel system was investigated using fluorescent ratio imaging. The range of pH studied was from 5.5 to 3.8, which included the minimum pH of growth of Lactobacillus curvatus (4.26). Diffusion of lactic acid in this pH range was found to be Fickian and the estimated effective diffusion coefficient of lactic acid at 20 °C was 2.81 × 10^?10 m² s^?1 with a standard deviation of 0.21 × 10^?10 m² s^?1. This effective diffusion coefficient was estimated by fitting the one‐dimensional solution of the Fickian diffusion equation for infinite systems to a transformed experimental data set. The original data set as recorded by the fluorescent microscope consisted of concentration–distance curves at specific time intervals. This was transformed by realigning the distance scale of all the concentration–distance curves. © 2002 Society of Chemical Industry     

Sensitive and Selective Determination of Riboflavin in Milk and Soymilk Powder by Multi-walled Carbon Nanotubes and Ionic Liquid [BMPi]PF<Subscript>6</Subscript> Modified Electrode

A novel electrochemical method to detect riboflavin was proposed using a multi-walled carbon nanotubes and ionic liquid N-butyl-N-methyl-piperidinium hexafluorophosphate composite film modified glassy carbon electrode (MWNTs-[BMPi]PF₆/GCE). A well-defined CV behavior with a pair of sensitive and well-shaped redox peak was observed, and the response value of riboflavin at MWNTs-[BMPi]PF₆/GCE is much higher than that at MWNTs/GCE in 0.1 mol L^?1 HAc-NaAc buffer solution (pH 4.5). The electrochemical approach based on a sensitive DPV analytical signal exhibits an excellent analytical performance with a wide linear range (2.6 × 10^?8 to 1.3 × 10^?6 mol L^?1) and low detection limit (4.7 × 10^?9 mol L^?1) for the riboflavin. Moreover, the proposed method possesses a potential practical application value and can be employed for the quantitative analysis of trace riboflavin with a recovery of 95.8–102.4 % in food samples such as milk and soymilk powder.     

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.     

Development and Characterization of Biodegradable/Edible Wheat Protein Films

Biodegradable and edible films were prepared from three types of wheat flours: commercial bread, hard red winter, and soft white. Films were produced at two pH values (4 and 11) and tested for oxygen permeability as related to temperature. Films were also produced with a cross-linked agent and tested for tensile strengths. Oxygen permeability was 5.9 × 10^?20 to 18.5 × 10^?20 m³O₂ m m^?2 s^?1 Pa^?1 similar to values for commercial nylon. The oxygen permeability activation energy varied from 9.1 to 14.5 kcal mol^?1, depending on type of flour and pH did not affect oxygen permeability. Presence of the cross-linking agent increased the strength of films and elongation at break ranged from 490% to 640%, while tensile stress at break ranged from 25.8 × 10^?3 kg m^?2 to 44.1 × 10^?3 kg m^?2, lower than commercial nylon.