Effects of Trolox and ascorbic acid on the riboflavin photosensitised oxidation of aromatic amino acids

The effects of 0, 100, 250, 500, 750 or 1000 ppm Trolox and ascorbic acid on the singlet oxygen oxidation of aromatic amino acids viz. phenylalanine, tryptophan, tyrosine in the presence of 25 ppm riboflavin were determined by measuring depleted headspace oxygen by gas chromatography and aromatic amino acid content by high performance liquid chromatography. The coefficients of variations (CVs) for headspace oxygen analysis and HPLC analysis of aromatic amino acids were 1.4% and 4.7%, respectively. Samples were stored under light (1000 lux) at 30 °C for 10 h. Both Trolox and ascorbic acid acted as antioxidants. As the concentration of Trolox and ascorbic increased from 0 to 1000 ppm, the head space oxygen depletion increased. This was due to the oxidation of Trolox and ascorbic acid along with amino acids in the presence of riboflavin. High performance liquid chromatography analysis of the samples clearly indicated that both Trolox and ascorbic acid minimised the degradation of phenylalanine, tryptophan, tyrosine significantly (p < 0.05), but did not prevent their oxidation completely. Trolox acted as a better antioxidant than ascorbic acid in protecting phenylalanine, tryptophan and tyrosine. Type-I mechanism was mainly responsible for riboflavin photosensitised degradation of aromatic amino acids.     

Changes of Headspace Volatiles in Milk with Riboflavin Photosensitization

ABSTRACT:  Effects of fluorescent light, riboflavin, ascorbic acid, sodium azide, and butylated hydroxyanisole (BHA) on the volatiles in milk at 4 °C were determined using a combination of headspace-solid phase microextraction (HS-SPME), gas chromatography (GC), and mass spectrometry (MS). Pentanal, hexanal, heptanal, and dimethyl disulfide were formed only in the milk stored under light and increased significantly as the duration of light exposure increased from 0 to 8 h and the concentration of added riboflavin increased from 5 to 50 ppm ( P  < 0.05). As fat content in milk increased, peak areas of pentanal, hexanal, and heptanal increased significantly ( P  < 0.05) while those of dimethyl disulfide did not change significantly ( P  > 0.05). Sodium azide prevented the formation of dimethyl disulfide in milk, implying that dimethyl disulfide can be formed through singlet oxygen oxidation (type II pathway). Addition of ascorbic acid and BHA reduced the formation of hexanal, heptanal, and dimethyl disulfide significantly ( P  < 0.05). Generation mechanisms of pentanal seem to be different from those of hexanal and heptanal in milk. Both singlet oxygen oxidation (type II pathway) and free radicals (type I pathway) play important roles in the formation of light-induced volatiles in milk.     

Antioxidant capacities of α-tocopherol,trolox, ascorbic acid,and ascorbyl palmitate in riboflavin photosensitized oil-in-water emulsions

Antioxidant capacities of α-tocopherol, trolox, ascorbic acid, and ascorbyl palmitate at 0.01, 0.1, and 1.0 mM in riboflavin photosensitized oil-in-water (O/W) emulsions were determined using headspace oxygen depletion, lipid hydroperoxide, and headspace volatile analyses. After 32 h visible light irradiation, headspace oxygen in O/W emulsions without adding antioxidants, with 1.0 mM α-tocopherol, trolox, ascorbic acid, and ascorbyl palmitate decreased to 18.50%, 18.85%, 16.01%, 17.92%, and 19.88%, respectively, whereas those samples in the dark were 20.74%. Trolox and ascorbic acid acted as prooxidants while their lipophilic counterparts, α-tocopherol and ascorbyl palmitate, respectively showed antioxidant properties. Similar antioxidative or prooxidative properties of the tested compounds can be observed in the results of lipid hydroperoxides and headspace volatiles. However, the prooxidant and antioxidant properties of the tested compounds were not clearly shown at 0.01 and 0.1 mM concentration. Both the type and concentration of antioxidants influenced the antioxidant capacities in riboflavin photosensitized O/W emulsions.     

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.     

ESR Study of the Singlet Oxygen Quenching and Protective Activity of Trolox on the Photodecomposition of Riboflavin and Lumiflavin in Aqueous Buffer Solutions

ABSTRACT:  Singlet oxygen quenching activity of Trolox, a water-soluble derivative of tocopherol, was studied by electron spin resonance (ESR) spectroscopy in a buffer solution (pH 7.4) containing methylene blue (MB), 2,2,6,6-tetramethyl-4-piperidone (TMPD) after light illumination for 30 min. Trolox at the concentration of 125 μM quenched 89.1% singlet oxygen in the system. Trolox showed significantly higher singlet oxygen quenching activity than ascorbic acid in the buffer solution ( P  < 0.05). Riboflavin in phosphate buffer solutions was degraded very fast under fluorescent light illumination. The photodegradation rate of riboflavin at pH 8.5 was significantly higher than pHs 4.5 and 6.5 ( P  < 0.05). Lumiflavin was also degraded under the fluorescent light illumination, but its degradation rate was much lower than that of riboflavin under the same light intensity. Unlike riboflavin, the rate of lumiflavin photodegradation was the greatest at pH 4.5 and followed by pHs 6.5 and 8.5, in a decreasing order. Trolox greatly protected the photodegradation of riboflavin and lumiflavin. The protective activities of Trolox against the photodegradation of riboflavin and lumiflavin were also pH dependent. The treatments of 5 mM Trolox in the buffer solutions of pHs 8.5 and 6.5 exhibited 56.1% and 31.7% protection of riboflavin against degradation during 120 min light illumination, respectively. The treatments of Trolox at the concentrations of 1, 3, and 5 mM in the buffer of 6.5 exhibited 14.8%, 58.4%, and 81.4% protection of lumiflavin against degradation during 24 h light illumination, respectively.     

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.     

Effects of lipophilic and hydrophilic antioxidants in oil-in-water emulsions under chlorophyll photosensitization

Antioxidative or prooxidative properties of α-tocopherol, Trolox, ascorbic acid, and ascorbyl-palmitate at the concentration of 0.1 and 1.0 mM were determined in oil-in-water (O/W) emulsions under chlorophyll photosensitization. Headspace oxygen depletion, lipid hydroperoxides, and headspace volatile analyses were conducted to determine the oxidative stability of O/W emulsions. For 32 h visible light irradiation, depleted headspace oxygen content in O/W emulsions were in the order of samples containing Trolox, ascorbic acid, ascorbyl palmitate, α-tocopherol, without antioxidants under light, and samples in the dark, which implies that all the added compounds acted prooxidant. These prooxidative properties of added compounds can be observed in the results of lipid hydroperoxides and headspace volatiles. Samples containing ascorbic acid and ascorbyl palmitate retained higher chlorophyll content than those containing Trolox up to 16 h. Increases of concentration of Trolox, ascorbic acid, and ascorbyl palmitate from 0.1 to 1.0 mM increased the lipid oxidation products whereas α-tocopherol decreased the degree of lipid oxidation implying α-tocopherol may not share the same prooxidant mechanisms compared to other compounds in chlorophyll sensitized O/W emulsions.     

Effects of Riboflavin Photosensitized Oxidation on the Volatile Compounds of Soymilk

ABSTRACT: Soymilks with or without added riboflavin in serum bottles were stored under light or in dark at 20 °C. The headspace oxygen and volatile compounds were determined by gas chromatography. Riboflavin had significant effects on the headspace oxygen depletion and volatile compounds formation in soymilk under light ( P < 0.05). Riboflavin did not have significant effects on the formation of volatile compounds and the depletion of headspace oxygen in dark ( P > 0.05). The volatile compounds increased under light, but not in dark as the added riboflavin increased. Storage temperature at 4 °C or 20 °C did not have significant difference in the effect of riboflavin on the headspace oxygen depletion in soymilk under light. Hexanal, an important beany flavor compound, was identified as the major volatile compound in the riboflavin photosensitized soymilk. Singlet oxygen oxidation was involved in the formation of volatile compounds in soymilk under light. Hexanal could be formed by singlet oxygen oxidation. Ascorbic acid, a quencher for singlet oxygen and the excited triplet sensitizer, significantly inhibited the formation of hexanal and total volatiles in soymilk under light.     

Effects of green tea catechin on the lipid oxidation,volatile compound formation,and losses of retinol and α-tocopherol in whole milk during light illumination as compared with ascorbic acid

Effects of green tea catechin (GTC, catechin content 61.4%) on the formations of hydroperoxide, malonaldehyde, and volatile compounds, and losses of retinol and tocopherol in milk during light illumination were studied. GTC showed surprisingly high protective activity of milk photooxidation, in a dose dependent manner. GTC at 25 ppm exerted 82.1 and 75.0% inhibitions of hydroperoxide and malonaldehyde formation during 30 h light illumination, respectively. GTC at 25 ppm exerted 88.7 and 94.3% inhibition of hexanal and heptanal formation in milk during 30 h light illumination, respectively. The 25 ppm GTC greatly suppressed the losses of retinol and α-tocopherol, showing 53.6 and 58.7% protections of their losses in milk after 12 h light illumination, respectively. The protective activity of GTC was at least 4 times higher than ascorbic acid. The inhibitory activities of GTC and ascorbic acid against the photooxidation of milk were in consistent with their singlet oxygen quenching abilities.     

Singlet Oxygen and Ascorbic Acid Effects on Dimethyl Disulfide and Off-Flavor in Skim Milk Exposed to Light

The light-induced off-flavor and dimethyl disulfide increased as fluorescent light exposure increased from 0,2,4,6, to 8h (p<0.05). Sensory evaluation and identified compounds suggest that dimethyl disulfide was mainly responsible for the light-induced off-flavor in skim milk. Dimethyl disulfide was formed by the singlet oxygen oxidation of methionine in milk. Gas chromatographic analysis and sensory evaluation showed that 200,500, and 1000 ppm ascorbic acid, which is a singlet oxygen quencher, lowered the formation of dimethyl disulfide and off-flavor in skim milk (p<0.05).     

Effects of soybean oil and oxidized soybean oil on the stability of β-carotene

The effects of 1.0%, 2.5%, and 5.0% purified soybean oil and thermally oxidized soybean oil on the stability of 100 ppm β-carotene as a fat-soluble vitamin A and singlet oxygen quencher in isooctane have been studied. The samples were stored under 1000, 2000, or 4000 lx at 20 °C for 2 days and at 50 °C for 16 days in the dark. The β-carotene was determined by high-performance liquid chromatography. The centrifugation and filtration of vegetable mixture, during sample preparation for β-carotene analysis by HPLC, decreased the coefficient of variation from 4.13% to 1.02%. The purified soybean oil and thermally oxidized soybean oil stabilized β-carotene in isooctane under light and in the dark at α = 0.05. The losses of β-carotene, with 1.0% purified oil, 1.0% thermally oxidized oil and without any oil during 48 h under light, were 11.2%, 80%, and 100%, respectively. 100 ppm TBHQ had a protective effect on the stability of β-carotene in isooctane at α = 0.05. The β-carotene stability decreased as the light intensity increased from 1000 to 2000 or 4000 lx at α = 0.05. The stability of vitamins in fruit and vegetable drinks enriched with fat-soluble vitamins and antioxidants during storage can be greatly improved by adding approximately 1.0% high quality non-oxidized soybean oil.     

Retinyl Palmitate Isomers in Skim Milk During Light Storage as Affected by Ascorbic Acid

Effects of ascorbic acid addition on the contents of retinyl palmitate isomers in skim milk treated with or without 3 mg/mL added riboflavin were studied during storage for 30 h under fluorescent light. Light illumination induced the reduction of all-trans-retinyl palmitate and 13-cis isomer, but promoted the production of 9-cis isomer in the skim milk. Riboflavin increased the reduction of all-trans-retinyl palmitate, but decreased the formation of 9-cis isomer in lightstored skim milk. The addition of ascorbic acid greatly inhibited the light-induced reduction of all-trans-retinyl palmitate and 13-cis isomer in skim milks. Ascorbic acid also greatly increased the formation of 9-cis-retinyl palmitate in skim milks during light storage.     

QUENCHING MECHANISMS AND KINETICS OF CAROTENOIDS IN RIBOFLAVIN PHOTOSENSITIZED SINGLET OXYGEN OXIDATION OF VITAMIN D2

The oxidation of vitamin D₂ in a solution of 12% water, 88% acetone and 15 ppm ribqflavin under light or dark was studied by measuring the headspace oxygen. Ribofiavin accelerated the oxidation of vitamin D₂ by singlet oxygen under light, but did not affect vitamin D₂ oxidation under dark. Quenching mechanisms and kinetics within the range of 0–20 ppm β‐cawtene or fucoxanthin and with 0–80 ppm retinyl acetate or retinol on 15 ppm riboflavin photosensitized singlet oxygen oxidation of vitamin D₂ were also studied. The rate of singlet oxygen formation by 15 ppm riboflavin was 1.78 umole oxygen/mL headspace‐hour. The reaction rate constant of vitamin D₂ with singlet oxygen was 2.2 times 10⁷ M^?1A s^?1. The carotenoids minimized the oxidation of vitamin D₂ by quenching singlet oxygen. The total quenching rate constants of retinol, retinyl acetate, jucoxanthin and ft‐carotene were in the order of 1.22 times 10⁸, 5.98 times 10⁸, 1.78 times 10⁹ and 5.00 times 10⁹M^?1. s^?1 respectively, which suggests that as the number of double bonds of carotenoids increases, the quenching rate constant ofcarotenoid increases.     

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.     

Chemistry and Reaction of Singlet Oxygen in Foods

Singlet oxygen is a highly reactive, electrophilic, and nonradical molecule. It is different from diradical triplet oxygen in its electron arrangement. Photosensitizers can form singlet oxygen from triplet oxygen in the presence of light. The reaction rate of singlet oxygen with foods is much greater than that of triplet oxygen due to the low activation energy. Singlet oxygen oxidation produces undesirable compounds in foods during processing and storage. However, carotenoids and tocopherols in foods can minimize singlet oxygen oxidation. The in‐depth scientific knowledge on the formation, reactions, quenching mechanisms, and kinetics of singlet oxygen can greatly improve the quality of foods by minimizing the oxidation during processing and storage. The single oxygen oxidation of foods has contributed to the explanation of several important chemical reactions in the reversion flavor in soybean oil, sunlight flavor in milk products, and the rapid losses of vitamin D, riboflavin, and ascorbic acid in milk under light storage.     

Effects of riboflavin-photosensitization on the formation of volatiles in linoleic acid model systems with sodium azide or D2O

Effects of riboflavin-photosensitization on volatile formations were studied in linoleic acid model systems with sodium azide or D₂O. Linoleic acid with riboflavin solution were stored under light or in the dark and volatiles were analyzed by a combination of solid phase microextraction (SPME), gas chromatography (GC), and mass spectrometry (MS). Riboflavin-photosensitization produced 27.9% more total volatiles than samples stored in the dark for 16 h. Addition of sodium azide decreased the total volatiles by 28.4% compared to samples without sodium azide. Linoleic acid in D₂O had higher increases in total volatiles than samples in H₂O system. Decrease of total volatiles in samples with sodium azide and increase of total volatiles in D₂O sample indirectly showed the presence of singlet oxygen in riboflavin-photosensitization. Hexanal, 2-heptenal, 1-octen-3-ol, 2-octenal, and 2,4-decadienal were greatly influenced by the addition of sodium azide and in D₂O, which indicates that formation of these volatiles was related to singlet oxygen oxidation. Especially, 2-heptenal showed the highest peak area among volatiles under riboflavin-photosensitized linoleic acid for 16 h.     

Effects of solubility characteristics of sensitiser and pH on the photooxidation of oil in tuna oil-added acidic O/W emulsions

The effects of sensitisers and pH on the oil oxidation of acidic O/W emulsions were studied under light by measuring hydroperoxide content and headspace oxygen consumption in the emulsions. The emulsions consisted of canola and tuna oil (2:1 w/w, 32%), diluted acetic acid (64%), egg yolk powder (4%), chlorophyll b or erythrosine (5 μM), and/or diazabicyclooctane (DABCO) or sodium azide (0.5 M). The emulsion pH values were 2.67, 3.68, and 6.27. Chlorophyll increased oil oxidation in the emulsion under light via singlet oxygen production while erythrosine did not. DABCO significantly decreased photooxidation of the oil containing chlorophyll, suggesting singlet oxygen involvement. However, sodium azide increased photooxidation of the oil containing chlorophyll possibly via azide radical production under acidic conditions. The oil photooxidation was higher in the emulsion containing chlorophyll at pH 6.27 than at pH 2.67 or 3.68, primarily by singlet oxygen and secondarily by free radicals produced from hydroperoxide decomposition.     

Effects of light and lipids on chlorophyll degradation

Stability of chlorophylls was studied in a system of paraffin oil with added oleic acid or triolein under 1,700 lx and in the dark. Chlorophyll contents were determined using HPLC. Sample oxidation was evaluated using the headspace oxygen content based on GC and the peroxide value. The chlorophyll content decreased with time and was higher and decreased faster under light than in the dark. Singlet oxygen was involved in chlorophyll degradation under light. Chlorophyll degradation was lower and slower in samples with added lipids than in samples without lipids. Protection of chlorophyll from photodegradation was greater using triolein than using oleic acid. Oxidation was slightly higher in samples with added lipids than in samples without lipids, however, there was no significant (p>0.05) difference between samples with added oleic acid or triolein. Decreased chlorophyll degradation under light by lipids is due to competition between lipids and chlorophyll for singlet oxygen.     

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.     

Effects of riboflavin photosensitization on the changes of isoflavones in soymilk

Effects of riboflavin photosensitization on the distribution of isoflavones in commercially available soymilk were analyzed using high-performance liquid chromatography (HPLC). Total isoflavones (TI) in soymilk with riboflavin (1000 ppm, w/v) under light were significantly different from those stored in the dark for 24 h (P < 0.05), while TI in samples with 0 and 1000 ppm added riboflavin were not significant from each other in dark conditions (P > 0.05). To test the effects of the concentration of riboflavin on TI, soymilk was mixed with riboflavin to make 0, 100, 250, and 500 ppm (w/v) and stored under light at 25 degrees C for 24 h. TI in soymilk with 100 ppm riboflavin under light significantly decreased by 13.5% for 24 h (P < 0.05) compared to control samples and were not significantly different from those with 250 or 500 ppm samples (P > 0.05). Daidzin and genistin were predominant isoflavones in soymilk, and the rate of photo degradation of genistin was higher than that of daidzin for 24 h in soymilk under riboflavin photosensitization.