Riboflavin Photosensitized Singlet Oxygen Oxidation of Vitamin D

Samples containing various levels of vitamin D and riboflavin, with and without ascorbic acid or a-tocopherol were prepared in a model system. Samples were stored in the light or in the dark at 45®C for up to 8 h. Headspace oxygen was determined by gas chromatography with thermal conductivity detection. Oxidation of vitamin D was not observed in samples without riboflavin stored in the light nor in samples with riboflavin stored in the dark. Vitamin D with riboflavin was oxidized under light. α-Tocopherol quenched singlet oxygen at a rate of 2.52 × 10⁸ M^-1 sec^-1, whereas ascorbic acid quenched singlet oxygen at a rate of 2.23 × 10⁷ M^-1 sec^-1.     

STRUCTURAL FEATURES OF RED KIDNEY BEAN á-AMYLASE INHIBITOR IMPORTANT IN BINDING WITH á-AMYLASE

The structural features of the glycoprotein α-amylase inhibitor from red kidney bean (Phaseolus vulgaris) important for binding to α-amylase were examined. The inhibitor contained 13.0% carbohydrate. The carbohydrate portion of the inhibitor is composed of 25 mannose, 2 xylose, 1 fucose and 17 N-cetylglucosamine residues per mol. Seventy (70) percent of the neutral carbohydrates were removed from the inhibitor by treatment with endo-β- N -acetylglucosaminidase H. The carbohydrate remaining attached to the protein consisted of 7 mannose, 2 xylose, and 1 fucose residues (N-acetylogucosamine was not determined). The intact glyco chains obtained from the pronase digest of the inhibitor failed to inhibit β-amylase (3.3 times 10⁵ fold molar excess of glyco chains to enzyme) when preincubated with the enzyme at 30°C for 30 min at pH 6.9 before adding starch as substrate.
Oxidation of one tryptophan residue of the α-amylase inhibitor with N-bromosuccinimide at pH 6.0 led to a 50% loss in inhibitory activity. Periodate oxidation of α-amylase inhibitor led to less of two tyrosine and one methionine residues per mole of inhibitor within 15 min. There was not a direct correlation between modification of these residues and loss of inhibitory activity. Modification of three of the five histidine residues with diethylpyrocarbonate resulted in about 50% loss in inhibitory activity.     

PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE- and CM-cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α-amylase was demonstrated by gel filtration on Sephadex G-100. The inhibitor has activity against porcine pancreatic α-amylase, human salivary α-amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α-amylase but is inactive against Bacillus subtilis α-amylase, Aspergillus oryzae α-amylase, barley α-amylase and red kidney bean α-amylase.     

Quenching Mechanisms and Kinetics of α-Tocopherol and β-Carotene on the Photosensitizing Effect of Synthetic Food Colorant FD&C Red No. 3

ABSTRACT: Effects of FD&C Red No. 40, Red No. 3, Yellow No. 5, Yellow No. 6, Green No. 3, Blue No. 1 and Blue No. 2 on 0.03M soybean oil oxidation in acetone at 25 °C under light were studied by measuring headspace oxygen depletion. As Red No. 3 increased from 0 to 5, 20, 100 and 200 ppm, the headspace oxygen was reduced by 2 to 70, 73, 77 and 77%, respectively, for 4 h. Only Red No. 3 acted as a photosensitizer to produce singlet oxygen in the oil. The quenching rates of α-tocopherol and β-carotene for the singlet oxygen by Red No.3 were 4.1 × 10⁷ M⁻¹s⁻¹ and 7.3 × 10⁹ M⁻¹s⁻¹, respectively. When β-carotene was below 1.86 × 10⁻⁶ M, β-carotene quenched singlet oxygen, but it quenched both singlet oxygen and Red No. 3 at or above 3.72 × 10⁻⁶ M. However, α-tocopherol quenched singlet oxygen only.     

Singlet Oxygen Oxidation Rates of ∝-, γ-, and δ-Tocopherols

ABSTRACT:  The reaction rate constants of 5 × 10⁻⁴ M, 10 × 10⁻⁴ M, and 20 × 10⁻⁴ M α-, γ-, and δ-tocopherols with singlet oxygen in methylene chloride containing 1 × 10⁻⁵ M chlorophyll under light at 25 °C for 60 min were studied. The oxidation of tocopherols determined by a spectrophotometric method showed that the losses of 20 × 10⁻⁴ M α-, γ-, and δ-tocopherols after 60 min under light were 21%, 16%, and 9%, respectively. The degradation of α-, γ-, and δ-tocopherols was undetectable in the absence of chlorophyll under light or in the presence of chlorophyll in dark. The losses of tocopherols under light were mainly due to singlet oxygen oxidation. The degradation rates of 20 × 10⁻⁴ M α-, γ-, and δ-tocopherols were 6.6 ×10⁻⁶ M/min, 5.0 × 10⁻⁶ M/min, and 2.9 × 10⁻⁶ M/min, respectively. The reaction rates between α-, γ-, or δ-tocopherol and singlet oxygen were 4.1 ×10⁶/M s, 3.3 × 10⁶/M s, and 1.4 × 10⁶/M s, respectively. The singlet oxygen oxidation rate of δ-tocopherol was significantly lower than α- or γ-tocopherol at α= 0.05. As the electron density in the chromanol ring of tocopherol increased, the singlet oxygen oxidation was increased.     

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.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

PREPARATION AND CHARACTERIZATION OF α-AMYLASE IMMOBILIZED ON COLLAGEN MEMBRANES

Crystalline pancreatic α-amylase was codispersed with hide collagen at pH 4.0 and tanned to form a membrane which degraded starch. The optimum pH for the codispersed membrane preparation was at pH 7.0 in contrast to the soluble enzyme which was as active at pH 8.0 as at pH 7.0. The immobilized enzyme responded maximally to 0.22M chloride whereas 0.02M chloride gave optimum rates for the soluble enzyme. The immobilized enzyme resisted thermal inactivation better than the soluble α-amylase. Raising the temperatures from 30 ° to 50 °C produced a 500% increase in rate for the bound enzyme. It was also demonstrated that membranes retained greater activity when stored in starch solution than in water. The effect of glutaraldehyde concentration on membrane activity was also studied.     

Inactivation of Escherichia coli O157:H7 and Salmonella enteritidis in Liquid Egg White Using Pulsed Electric Field

ABSTRACT: The effects of temperature and pulsed electric field (PEF) intensity on inactivation of pathogens such as Escherichia coli O157:H7 and Salmonella enteritidis in egg white was investigated. Liquid egg white inoculated with 10⁸ colony-forming units (CFU)/mL of each pathogen was treated with up to 60 pulses (each of 2 JAS width) at electric field intensities of 20 and 30 kV/cm. The processing temperatures were 10°C, 20°C, and 30°C. After treatment, uninjured and total viable cells were enumerated in selective and nonselective agars, respectively. Maximum inactivations of 3.7 and 2.9 log units were obtained for S. enteritidis and E. coli O157:H7, respectively, while injured cells accounted for 0.5 and 0.9 logs for E. coli O157:H7 and S. enteritidis , respectively. For both bacteria, increasing treatment temperature tended to increase the inactivation rate. There was synergy between electric field intensity and processing temperature. The inactivation rate constant k _T values for E. coli O157:H7 on both selective and nonselective agars were 8.2 × 10^-3 and 6.6 × 10^-3/μS, whereas the values for S. enteritidis were 16.2 × 10^-3 and 12.6 × 10^-3/μS, respectively. The results suggest that E. coli O157:H7 was more resistant to heat-PEF treatment compared with S. enteritidis.     

HYGIENIZATION OF INDIAN CHICKEN MEAT BY IONIZING RADIATION

Both fresh and frozen chicken meat were evaluated for microbiological status by screening for total bacterial counts and for the presence of pathogens like Enterobacteria , Bacillus cereus, coagulase positive Staphylococci and Salmonella spp. Most of the samples exhibited heavy bacterial contamination (1.2 × 10⁵ - 2.6 × 10⁶/g), mainly with Staphylococcus spp. (1.5 × 10⁴ - 2.8 × 10⁵/g). All the chicken samples also showed the presence of Salmonellae (3 × 10¹ - 2.1 × 10²/g). Among the different serotypes observed in chickens . S. typhimurium was common in fresh as well as frozen chicken. Radicidation at 2 kGy at cryogenic conditions (−40°C) was efficient in eliminating the natural pathogenic contamination of the poultry . Salmonella spp. viz. S. seftenberg and S. typhimurium differed in radiation sensitivity, the D₁₀ values in phosphate buffer (pH 7.2) being 0.25 kGy and 0.12 kGy, respectively. Chicken homogenate (10%) offered approximately 2-fold protection to these cells. Chicken samples artificially inoculated with a heavy inoculum (10⁸ cells/g) of these 2 serotypes required higher gamma radiation doses of 4–5 kGy. The findings suggested that a dose of 2 kGy is adequate for normally contaminated chicken samples, but for the heavily contaminated chicken a dose of 4–5 kGy, depending upon the predominating Salmonella serotype present, is required .     

THE MOLECULAR WEIGHT OF α-AMYLASE INHIBITOR FROM WHITE BEAN cv 858B (PHASEOLUS VULGARIS L.) IS 56 kDa, NOT 20 kDa

The molecular weights (M_rs) of α-amylase inhibitors (αAIs)fiom 18 (Ah) bean cultivars estimated by Superose 12 gelfiltration chromatography were 22–62% smaller than those determined by Sephadex G-100 gel filtration and by polyacrylamide gel electrophoresis (PAGE) methods. αAI-4 from WKB cultivar 858B was purified and the Mr was shown to be 51.0 kDa based on Sephadex G-100 gel filtration chromatography and by PAGE. A M_r for aAI-4 of 56.714 kDa was determined by laser-assisted time-of-flight mass spectrometry and appears to be the true M_r of the mature glycosylated active aAI-4. The results show that Superose 12 gelfiltration chromatography is not usefulfor M_r determination of some proteins. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that the 56.7 kDa aAI-4 molecule dissociated into 45.0, 33.6,15.2 and 12.4 kDa submolecules, with only the two small subunits, a and β, present at high SDS concentration. This provides evidence that the aAI-4 molecules composition is α₂β².     

RELEASE OF PROPYL PARABEN FROM A POLYMER COATING INTO WATER AND FOOD SIMULATING SOLVENTS FOR ANTIMICROBIAL PACKAGING APPLICATIONS

The release phenomena of propyl paraben from a polymer coating to water and three food simulating solvents (10% aqueous ethanol, 50% aqueous ethanol, n-heptane) were studied for antimicrobial packaging applications. The effects of food simulating solvent, initial concentration in the coating and temperature on the propyl paraben release were examined. The initial concentration of propyl paraben in the coating ranged from 1.26 × 10⁴ to 10.52 × 10⁴ g/m³ and the temperature from 5.5 to 30C. For water, the release was controlled by Fickian diffusion with constant diffusion coefficient (7±11 × 10^-11 cm²/s at 30C), and independent of the initial concentration. For 10% ethanol, the release followed again the Fickian model with constant diffusion coefficient (30±40 × 10^-11 cm²/s at 30C). For 50% ethanol and n-heptane, the release was instantaneous and not controlled by Fickian diffusion. For the release into water, the activation energy for diffusion from the Arrhenius relationship was around 88 kJ/mole.     

CHARACTERIZATION OF PHYTASE OF BEANS (PHASEOLUS VULGARIS)

Phytase from California Small White beans was extracted, concentrated and enriched by heat treatment and ammonium sulfate fractionation. Crude enzyme fractions tenaciously retained endogenous phytate which could be removed by autolysis at 45°C and pH 5.2. Characterization of the phytase preparation showed: (1) increase in activity with increasing temperature from 37 to 60°C, (2) an activation energy of approximately 9,200 cal/mole for the hydrolysis of inositol hexaphosphate; (3) pH optimum of 5.2; (4) a K_m value of 2.22 × 10^-4M; (5) apparently partial inhibition at high substrate concentrations; (6) fully competitive inhibition by one of the reaction products, inorganic phosphate, with K_i=3.1 × 10⁴M. These properties and kinetic constants are comparable to those reported for phytase preparations from similar sources and they adequately account for in situ autolytic loss of phytic acid from these beans.     

Quenching Mechanism and Kinetics of Ascorbic Acid on the Photosensitizing Effects of Synthetic Food Colorant FD&C Red Nr 3

ABSTRACT:  The pH effect on the oxidative stability of ascorbic acid in the presence of food colorant FD&C Red Nr 3 during storage with or without light was investigated. The quenching mechanism and kinetics of ascorbic acid on the FD&C Red Nr 3 photosensitized oxidation in an aqueous system at 25 °C were also studied by measuring the degradation of ascorbic acid or depletion of headspace oxygen. Red Nr 3 had no influence on the oxidation of ascorbic acid under dark storage, but accelerated its oxidation rate under light storage. The oxidative stability of ascorbic acid decreased as the pH increased from 4 to 7 under light without FD&C Red Nr 3. The quenching rates of ascorbic acid on the singlet oxygen by measuring the degradation of ascorbic acid in the presence of Red Nr 3 under light storage were 1.53 ± 0.15 × 10⁸, 1.86 ± 0.25 × 10⁸, and 1.19 ± 0.12 × 10⁸ M⁻¹S⁻¹ at pH 4, 5.6, and 7, respectively.     

Production of Enterotoxin-B in Cured Meats

SUMMARY— A variety of laboratory cured hams were inoculated with 10³−10⁶ cells of S. aureus strain S-6 and incubated at 10, 22 and 30°C anaerobically for up to 16 weeks. Enterotoxin-B was detected by gel-diffusion in hams with original pH over 5.30, up to 9.2% NaCl (brine) and 0.54 ppm undissociated nitrous acid. There was better toxin production at 30° than at 22° or 10°C. Toxin was detected at 10°C after at least 2 weeks incubation and in most samples after 8 weeks when pH was greater than 5.6. Toxic hams had more than 4 × 10⁶ cells/g. Contaminants were always less than 10⁵/g. Tween 80 inhibited toxin production at 30° but not at 10°C. Toxic hams looked normal even after 2 months incubation at 10°C.     

Apparent diffusivities of reducing sugars in potato strips blanched in water

The mechanism of reducing sugar losses was investigated in potatoes cut into 9.5 × 9.5 mm and 13 × 13 mm strips and blanched for 300–2400s at different temperatures between 70°C and 100°C. Experiments were carried out using large amounts of well-agitated water. Assuming no chemical reaction and using appropriate solutions of the unsteady state diffusion equation for square parallelpiped geometry, the apparent diffusivities, D_a, of reducing sugars in the potato matrix were determined at different temperatures. the incorporation of the dimensions of the strips in the solution of the diffusion equation was sufficient to explain the effect of size on losses. Values of D_a were found to be in the range 1.2 × 10^-11 1.7 × 10^-11 m²s^-1 and could be correlated with temperature according to the Arrhenius law.     

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.     

MICROBIOLOGICAL QUALITY OF TRADITIONAL MARKET CURED FISH IN TANZANIA

The microbiological quality of six varieties of retail market traditionally cured fish in Morogoro, Tanzania was investigated over a five-month period. The fish were contaminated with bacteria and molds at levels of: total aerobes, 10⁶ - 1.7 × 10⁷ c.f.u/g; faecal coliforms, 1.1 × 10¹ - 2.5 × 10³ MPN/g; faecal streptococci, 1.4 × 10¹ - 1.3 × 10³ MPN/g; Staphylococcus aureus, 1.3 × 10³ - 8.6 × 10³ c.f.u/g; Aspergillus flavus group, 2.1 × 10¹ - 2 × 10² c.f.u/g of fish. Of faecal coliform, 45% of the isolates were Escherichia coli. Twenty-five percent of the S. aureus isolates were coagulase positive. Sixteen percent of A. flavus isolates were aflatoxigenic. Aflatoxin contamination ranged from O to 18.5 μg/kg of fish. Insect infestation by Dermestes spp. and mites was observed. The results of this preliminary study emphasize the importance of proper processing and handling offish in the tropics in order to safeguard public health .     

Effect of technological parameters on the characteristics of kasseri cheese made from raw or pasteurized ewes' milk

Two groups of kasseri cheese (pasta filata type) were manufactured from raw or pasteurized ewes' milk, without starter cultures. Cheeses of each group were divided into two subgroups: the first was ripened and stored at 4°C and packaged in plastic film; the second ripened and stored at 15°C and coated with paraffin wax. Milk pasteurization and technological parameters had a significant effect on the pH ( P  < 0.05), while only technological parameters had an effect on the total solids content. At day 120, the range of mean cfu/g counts for the mesophilic aerobic flora was 9.5 × 10⁷−1.4 × 10⁸; for the thermophilic streptococci, the range was 2.6 × 10⁷−7.6 × 10⁷; and for the thermophilic bacilli, 9.8 × 10⁶−1.7 × 10⁷. Changes in the N fractions became significant after 30 days of ripening. For mature 120-day-old cheeses, the percentage of total N soluble at pH 4.6 was 22.7%–22.9% in raw milk cheeses and 19.0%–21.7% in pasteurized milk cheeses. The percentage of total N soluble at 12% TCA was 10.1%–12.2% in raw milk cheeses and 7.3%–11.5% in pasteurized milk cheeses; the percentages of total N soluble at 5% PTA were 3.1%–4.0% and 2.6%–3.6%, respectively. The residual α_s-casein percentages at day 120 ranged between 63% and 78% of the respective area at day 1; the residual β-casein ranged between 67% and 75%. There were some characteristic differences in the reverse phase-HPLC peptide profiles of the four cheeses. In general, the effect of the different ripening conditions was more pronounced in cheeses made from pasteurized milk.     

Microbiological quality of ice creams commercialized in some cities in the state of Rio de Janeiro, Brazil

The microbiological quality of 60 ice cream samples of three commercial brands (A, B and C) of various flavours, commercialized in some towns in the State of Rio de Janeiro, Brazil, was evaluated. Total bacteria count (TCB), coliforms at 35°C (CT), coliforms at 44°C (CF) and presence of Staphylococcus aureus were performed on all samples. TCB ranged from 2.0 × 10 ² to 6.9 × 10 ⁵ cfu/mL, CT from < 3–≥ 2400 MPN/mL, CF from < 3–1100 MPN/mL and S. aureus from < 10–1.4 × 10 ⁶ cfu/mL. The level of bacterial contamination found in this study reflects the unhygienic conditions prevalent in manufacturing and storage of ice creams. Actions are thus necessary by the Brazilian regulatory agencies to require the ice cream processing plants to adopt quality guarantee systems, such as good manufacturing practices and hazard analysis and critical control points system.