Kinetics for Singlet Oxygen Formation by Riboflavin Photosensitization and the Reaction between Riboflavin and Singlet Oxygen

ABSTRACT: The formation of singlet oxygen by riboflavin and the kinetics and mechanisms of riboflavin degradation in aqueous solution under light were determined. The singlet oxygen formation rate by riboflavin was 2.31 μmole oxygen/mL headspace/h of serum bottle. The degradations of riboflavin were 66% in D₂O and 40% in H₂O, respectively, under light after 24 h. The results indicate that singlet oxygen is involved in riboflavin destruction under light. The riboflavin destructions were 94.0% and 15.7% with 0 mM or 160 mM ascorbic acid, respectively, under light after 96 h. The reaction rate between riboflavin and singlet oxygen was 1.01 × 10¹⁰/M/s, which is a diffusion-controlled reaction rate. This explains the extremely fast degradation of riboflavin in foods under light. Ascorbic acid and sodium azide reduce the degradation of riboflavin under light with different quenching mechanisms. Ascorbic acid quenched both singlet oxygen and excited triplet riboflavin. Sodium azide quenched only the singlet oxygen in riboflavin solution with a quenching rate of 1.547 × 10⁷/M/s. With the involvement of both the Type-I and Type-II mechanisms in the riboflavin degradation under light, singlet oxygen quencher alone could not protect the riboflavin from degradation completely. Addition of ascorbic acid can protect riboflavin oxidation in foods exposed to light.     

EFFECT OF SEVERAL EXPERIMENTAL PARAMETERS ON COMBINATION OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) α-AMYLASE INHIBITOR WITH PORCINE PANCREATIC α-AMYLASE

Purified red kidney bean (Phaseolus vulgaris) amylase inhibitor forms a 1:1 stoichiometric complex with porcine pancreatic α-amylase leading to complete loss of enzyme activity on starch. Rate of complex formation is pH dependent and is maximal at pH 5. The rate constants for complex formation, as measured by loss of amylase activity, were 2.85 × 10⁴ M^-1 sec^-1 at pH 6.9 (ionic strength of 0.918) and 2.55 × 10⁵ M^-1 sec^-1 at pH 5 at 30°C. At pH 6.9, rate of complex formation was 4.8 times faster at 0.918 ionic strength as compared with the rate at 0.138 ionic strength. At 30°C, pH 6.9 and ionic strength of 0.168 the dissociation constant of the enzyme-inhibitor complex was determined to be 3.5 × 10^-11 M. The rate constant for dissociation of the complex was calculated to be 8.7 × 10^-8 sec^-1 under the same conditions. The rate constant for complex formation, at ionic strength of 0.168, was 1.1 × 10⁴ M^-1 sec^-1 at 37⁰ and 9.77 × 10² M^-1 sec^-1 at 25.7°C. The calculated activation energy for complex formation is 39.5 kcal/mole suggesting a rate-controlling conformational change. Oxidation of the carbohydrate moiety of the glycoprotein inhibitor caused complete loss of activity. Maltose, a competitive inhibitor of α-amylase, bound as readily to the enzyme-inhibitor complex as to free α-amylase. Trypsinized α-amylase, although still able to bind to Sephadex, did not bind inhibitor. The experiments with maltose and trypsinized amylase suggest the inhibitor may not bind at the active site of α-amylase.     

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.     

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.     

Stability and Photochemistry of Vitamin D2 in Model System

Oxidation of vitamin D₂ in a model system of 12% water and 88% acetone in the presence of 15 ppm riboflavin under light and dark was studied by measuring the depleted headspace oxygen. Riboflavin accelerated the oxidation of vitamin D₂ by singlet oxygen under light, but did not affect the vitamin D₂ oxidation under dark. The effect of 0 and 15 ppm riboflavin on the stability of vitamin D₂ during storage at 25 and 60°C was studied by measuring the contents of vitamin D₂ during 48h. Results indicated that photosensitized singlet oxygen oxidation of vitamin D₂ under light was temperature-independent, and triplet oxygen oxidation of vitamin D₂ both under light and in the dark was temperature-dependent.     

SHELF-LIFE OF FRESH FILLED PASTA. HAZARD ANALYSIS AND CRITICAL CONTROL POINTS OF THE MANUFACTURING PROCESS AND HOUSEHOLD PRACTICES

Hazard Analysis and Critical Control Point (HACCP) approach was used to assess the safety of a fresh filling pasta (ricotta-filled ravioli) manufacturing plant in La Plata region, Argentina. Household practices like cooking and holding meals before serving were also evaluated. Samples of ricotta, main raw material of the filling, showed colony counts of Enterobacteriaceae total microorganisms, molds and yeasts of (5 × 10³-1 × 10⁵), (1x10⁵−1x10⁸) and 1 × 10⁵ CFU/g, respectively. E. coli was also detected in one out of five samples, suggesting a hazardous condition of this raw material. Salmonella spp. was not isolated from any of the dough samples tested. Ricotta filling showed high colony counts of mesophilic microorganisms (6 × 10⁶ CFU/g), Enterobacteriaceae (1 × 10⁵ - 1 × 10⁶ CFU/g), while E. Coli was detected in 20% of the samples. Counts of B. cereus, S. aureus were less than 1 × 10² CFU/g in the analyzed materials. In the finished product (ricotta-filled ravioli), the colony counts of mesophilic microorganisms and Enterobacteriaceae were 3 × 10⁸ and 8 × 10⁵ CFU/g, respectively. Critical Control Points found were cooking and holding time before serving. The implementation of suggested corrections allowed the microbial quality of the final product (ricotta-filled ravioli) to be improved considerably. The growth of total microbial counts during refrigerated storage (0, 4, 8 and 10C) were measured and the shelf-life of ricotta and ricotta-filled ravioli was calculated.     

Rapid dye reduction tests for the determination of microbiological quality of meat

Rapid dye reduction tests have been developed to determine the quality of meat. Three chemical indicators, resazurin and two tetrazolium compounds, were used to correlate the microbial numbers and reduction times in meat samples. Twenty-five surface samples from sheep carcasses were subjected to each reduction test. Total viable counts given were obtained at 37°C. Resazurin reduction time was 90–120 min when the bacterial counts ranged from 1.5×10⁶ to 7.7×10⁶/cm². Samples showing bacterial counts between 1.5×10⁶ and 6.0×10⁶/cm² reduced tetrazolium (NBT) in 360–390 min whereas samples containing bacterial counts of 2.1×10⁶/cm² took 420–450 min to reduce iodophenyl nitrophenyl tetrazolium (INT) dye. Regression equations relating the number of organisms per cm² and reduction time were applied to predict the microbiological quality of meat samples from reduction time data. Among the three dyes, resazurin gave the lowest reduction time.     

EVALUATION OF BACTERIOLOGICAL QUALITY IN SELECTED COMMERCIAL INFANT FORMULAS AVAILABLE IN POLAND AND EGYPT

One hundred samples of commercial infant formula bought in shops in the Poznan region (Poland) and Cairo region (Egypt) were investigated. Samples were analyzed for aerobic plate counts (APC), total Bacillus cereus (TBC), and incidences of Bacillus spp. and coliforms. The mean APC and TBC did not show any important variation with country, being practically the same in products bought in Poland and Egypt. All commercial infant formula analyzed immediately after opening were of satisfactory bacteriological quality, exhibiting APC lower than 10⁴ CFU g⁻¹ (mean 4.9 × 10²) and TBC lower than 10³ CFU g⁻¹ (mean 1.1 × 10²). However, 60% of the examined fruit juice and ready-to-feed infant formula presented TBC above the recommendation safety limit after storage at 22C for 72 h and at 35C for 48 h. In most cases the mean log APC and TBC were highest (P > 0.05) for fruit juice and ready-to-feed infant formula stored at elevated temperature (35 ± 1C).     

A STUDY ON SURVIVAL OF Staphylococcus aureus IN DARK AND MILK CHOCOLATE

Dark and milk chocolate bars were inoculated with Staphylococcus aureus to establish an initial population of approximately 10², 10⁴ and 10⁶ cells per gram. Samples were stored at room temperature and examined for the survival of staphylococci at 2-day intervals for the first 6 days and every 8 days thereafter. When samples were inoculated with 10² cells per gram, absence of staphylococci was observed after 2-days in dark and 14 days of storage in milk chocolate. Bars with 10⁴ cells per gram were free of cells after 38 days in dark and 86 days of storage in milk chocolate. Dark chocolate bars with 106 cells per gram, were staphylococci free after 86 days of storage; whereas the milk chocolate bars with same level of inoculation were shown to be staphylococci free after 110 days of storage     

QUANTITATIVE STUDIES OF PROTEOLYTIC AND LIPOLYTIC BACTERIA IN FROZEN MINCED BEEF AND HAMBURGER

Sixty frozen samples (30 hamburger patties and 30 minced meat) purchased from different retail markets in Ismailia, Egypt were examined for incidence of proteolytic and lipolytic bacteria. Mean values of proteolytic psychrophiles in the examined samples of hamburger and minced meat were 2×10⁵ and 6×10⁴, respectively. Lipolytic psychrophiles in hamburger were 8×10⁵ and 3×10⁵ in minced meat. Proteolytic mesophiles in hamburger and minced meat were 6×10⁵ and 5×10⁴, respectively. Mean values of lipolytic mesophiles were 5×10⁴ for hamburger and 5×10³ for minced meat. Proteolytic thermophiles in hamburger and minced meat were 3×10³ and 10³, respectively. Both proteolytic and lipolytic activities were exhibited by various bacteria that were identified.     

MICROBIOLOGICAL STATUS OF EGYPTIAN SALTED MEAT (BASTERMA) AND FRESH SAUSAGE

The microbiological quality of 125 samples of the most popular Egyptian meat products (75 Egyptian fresh sausage "EPS" and 50 basterma) was determined. The aerobic plate count (APC) and Lactobacillaceae count of basterma ranged from 1 × 10⁴ to 9 × 10⁶ CFU/g, respectively . Enterobacteriaceae, mold and yeast counts for basterma were similar (<1 × 10² CFU/g). Artificially contaminated slices of meat and garlic paste of basterma showed that the paste inhibited growth of Salmonella typhimurium. APC and Enterobacteriaceae counts of EFS ranged from 1.1 × 10⁴ to 1 × 10⁸ and from 1 × 10² to 1 × 10⁷ CFU/g, respectively. Nearly 26% and 29% of EFS were positive for Clostridium perfringens and coagulase-positive Staphylococcus aureus, respectively . Salmonella could not be detected in any examined samples. Bacteriologically, EFS might pose a potential health hazard, making it imperative to institute sanitary measures during its production and sale .; Accepted for Publication July 2, 1997     

RHEOLOGICAL AND CHEMICAL PROPERTIES OF MOZZARELLA CHEESE

Dynamic viscoelastic parameters and chemical properties of Mozzarella cheese produced using a "no-brine" cheese making method with 3 different cooking temperatures (38, 41, and 44C) were determined. Samples were stored for 3 weeks at 4C before dynamic mechanical analysis at 22C. G', G" and tan δ were 5.8 – 6.4 × 10⁵ dyne/cm², 1.9 – 2.1 × 10⁵ dyne/cm², and 0.33 – 0.35, respectively, at 1% strain and 10 rad/s. The percentage of intact α_s-casein and β-casein were 38–40% and 33–35% of total protein in the cheese, respectively. The range of cooking temperatures used in this experiment had little effect on dynamic viscoelastic properties or the amount of intact protein for the cheese.     

CONTROLLED ATMOSPHERE STORAGE AND EDIBLE COATING EFFECTS ON STORAGE LIFE AND QUALITY OF TOMATOES

Tomatoes at pink stage maturities were coated with Semperfresh^TM edible fruit coating which is composed of sucrose esters of fatty acids, sodium carboxymethyl cellulose and mono-diglycerides of fatty acids. One group of coated and uncoated tomatoes were stored at 23 × 2C under air atmosphere, another group was stored at 12C under air atmosphere and third group was stored at 12C, 93–95% RH in a controlled atmosphere (CA) containing 3% CO₂, 3% O₂ and 94% N₂. Samples of tomatoes were analyzed for firmness, weight loss, titratable acidity, pH, soluble solids, sugars, ascorbic acid and lycopene to examine changes in quality during storage. Semperfresh^TM edible fruit coating was found to be significantly effective at both storage temperatures (23 × 2C and 12C) in air to delay changes in firmness, titratable acidity, pH, soluble solids, sugars, ascorbic acid and lycopene. Semperfresh^TM fruit coating reduced fruit weight loss as compared to fruit without coating but the difference between coated and uncoated tomatoes was not significant. Shelf-life of pink tomatoes was increased 3 days at 23 × 2C and 6 days at 12C in air by coating. CA storage delayed compositional changes in tomatoes significantly as compared to air storage of coated and uncoated tomatoes. CA storage also had a significant potential for reducing weight loss and microbial spoilage. Tomatoes were stored 40 days in CA. Coated and uncoated tomatoes exhibited the same results in CA.     

EXAMINATION OF THE MICROBIAL STATUS OF SELECTED INDIGENOUS FERMENTED FOODS IN NIGERIA

Four Nigerian traditionally fermented foods (wara, nono, ogi and kununzaki) were evaluated for the presence of some microorganisms of public health concern. Among the dairy foods , Staphylococcus aureus and Klebsiella sp. were isolated from wara while Escherichia coli, Salmonella sp. and Klebsiella sp. were isolated from nono. The cereal-based fermented foods (ogi and kunu-zaki) contained Bacillus subtilis, E. coli, S. aureus, Klebsiella sp. and Enterococcus faecalis. The mesophilic aerobic counts were: 5 × 10⁵ for wara; nono, 1.53 × 10⁷; ogi, 3.6 × 10⁶ and kunu-zaki, 2.6 × 10⁶ cfu/mL. The enterobacteriaceae counts on nono, wara, ogi and kunu-zaki were 1.79 × 10⁷, 4.5 × 10⁵, 4.0 × 10⁵ and 1.2 × 10⁶ cfu/mL, respectively. No Vibrio count (detection limit: <10 cfu/mL) was recorded in all the food samples considered. The yeast and mold counts ranged from 1.0 × 10⁵– 3.31 × 10⁷ among the food products. The antimicrobial susceptibility patterns of the organisms isolated from dairy products (nono and wara) revealed that they were resistant to ampicillin (100%) and sensitive to gentamicin (100%) and nalidixic acid (100%). Most isolates from cereal based products (ogi and kunu-zaki) were 100% resistant to penicillin, ampicillin and chloramphenicol. This work highlights the need to maintain hygienic standards in the preparation of our locally fermented cereal and dairy foods.     

Antioxidant mechanisms of Trolox and ascorbic acid on the oxidation of riboflavin in milk under light

Antioxidant activities and the mechanism of water-soluble Trolox and ascorbic acid on the oxidation of riboflavin in milk were studied. Trolox or ascorbic acid at 0, 100, 250, 500, or 1000 ppm was added to milk with or without added 50 ppm riboflavin and stored under light at 27 °C. Headspace oxygen was analysed by GC and Trolox, ascorbic acid, and riboflavin were determined by HPLC. The headspace oxygen of milk with added 50 ppm riboflavin depleted faster than that of milk without added riboflavin (p < 0.05). Trolox and ascorbic acid decreased during storage under light and riboflavin was completely destroyed within 24 h. As the concentration of Trolox or ascorbic acid increased, the riboflavin loss decreased. Riboflavin, Trolox, and ascorbic acid competed to react with singlet oxygen which was formed in the presence of riboflavin under light. Trolox and ascorbic acid protected riboflavin in milk under light by reacting with singlet oxygen.     

Ionizing Radiation Effects on Starch as Shown by Staudinger Index and Differential Thermal Analysis

SUMMARY— Corn starch in the form of raw granules at commercial moisture was irradiated at two levels: 3 × 10⁵ and 6 × 10⁶ rad from a Co⁶⁰ source. The irradiated samples were completely dissolved in alkali, indicating there was no cross linking induced in the starch molecules by irradiation. Viscosity determinations of starch solution diluted with distilled water exhibited the ion charge effect generally observed in other macromolecules.
The Staudinger indices of unirradiated, irradiated at 3 × 10⁶ rad and 6 × 10⁶ rad were 42, 22 and 16 respectively, which were an indication of depolymerization of starch macromolecules with increasing irradiation. The differential thermal analysis of the three samples also showed the depolymerization of the polymer with irradiation.
It is suggested that these two simple techniques–the Staudinger index and D.T.A.–could be usefully employed to characterize small differences in starches.     

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 .     

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 .     

Chemical Reactions and Stability of Riboflavin in Foods

ABSTRACT: Riboflavin is relatively stable during thermal and nonthermal food processing and storage but is very sensitive to light. It can accept or donate a pair of hydrogen atoms. It can act as a photosensitizer (through either Type I or Type II mechanism) or a prooxidant for food components under light. Photosensitization of riboflavin causes production of reactive oxygen species such as superoxide anion, singlet oxygen, hydroxy radical, and hydrogen peroxide. Radicals and reactive oxygen species accelerate the decomposition of proteins, lipids, carbohydrates, and vitamins, and could cause significant nutrient loss in foods. Carbohydrates are less sensitive to riboflavinphotosensitized oxidation than proteins, lipids, or vitamins. Riboflavin is an excellent photosensitizer for singlet oxygen formation and a superb reactant for singlet oxygen, with the reaction rate of 1.01 ± 10¹⁰/M/s.