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.     

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.     

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.     

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.     

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.     

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.     

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.     

Singlet Oxygen Quenching Activities of Various Fruit and Vegetable Juices and Protective Effects of Apple and Pear Juices against Hematolysis and Protein Oxidation Induced by Methylene Blue Photosensitization

ABSTRACT:  Effects of various fruit and vegetable juices on rubrene oxidation induced by a chemical source of singlet oxygen in a microemulsion system have been studied. The singlet oxygen quenching activities of fruit and vegetable juices were greatly different with different juices. The apple and pear juices exhibited the highest antioxidative activity among the tested juices in singlet oxygen–induced rubrene oxidation, showing 56.69% and 59.34% inhibition, respectively. The grape, kumquat, red cabbage, and spinach juices also showed relatively strong antioxidative activity against singlet oxygen–induced rubrene oxidation. Lemon juice showed the least activity, resulting in 0.63% inhibition of rubrene oxidation. The singlet oxygen quenching activities of 1 mL of apple and pear juices were equivalent to 33.97 and 34.64 mg ascorbate, respectively. Singlet oxygen quenching activities of juices had very low correlation with both ABTS radical scavenging activity ( R ²= 0.11) and total phenolic contents ( R ² < 0.1). However, a high correlation ( R ²= 0.66) was found between the ABTS radical scavenging activities and total phenolic contents of juices. The apple and pear juices also significantly inhibited both erythrocyte lysis and protein oxidation induced by fluorescence light illumination in the presence of methylene blue. Electron spin resonance (ESR) spectroscopy data showed that the protective activities of these juices against biological damages induced by photodynamic ways were, to at least some extent, due to their singlet oxygen quenching abilities. This represents first report on the singlet oxygen quenching activities of the apple and pear juices, and their protective activities against photodynamically induced biological damages.     

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.     

Singlet Oxygen Quenching Effects of Phosphatidylcholine in Emulsion Containing Sunflower Oil

ABSTRACT:  Effects of phosphatidylcholine (PC) on the oxidation of oil by singlet oxygen in a W/O microemulsion and an emulsion food model containing tocopherol-stripped sunflower oil (TSSO) have been studied. The W/O microemulsion consisted of methylene chloride, butanol, and sodium dodecyl sulfate with PC (0, 250, 1000 ppm) and TSSO (0, 3.3, 16.5, 33 mg/mL). Production of singlet oxygen in the microemulsion was done chemically with hydrogen peroxide in the presence of sodium molybdate, and indirectly evaluated by rubrene oxidation at A₅₂₉. The emulsion food model consisted of TSSO, distilled water, and xanthan gum with addition of 250 ppm PC and 4 ppm chlorophyll b, and was placed at 25 °C under fluorescent lights (1700 lux) for 24 h. The oxidation of TSSO was determined by thin-layer chromatography and values of conjugated dienoic acid (CDA) and peroxides (POV). PC significantly decreased the oxidation of rubrene and TSSO in the W/O microemulsion, but its content was decreased to approximately one-half by a 20-min reaction, indicating its degradation. This clearly shows that PC acted as an antioxidant via chemical quenching of singlet oxygen in the W/O microemulsion. A possible synergism between PC and TSSO was observed in singlet oxygen quenching in the microemulsion. PC also significantly decreased the chlorophyll-photosensitized oxidation of TSSO in the emulsion food model, possibly by singlet oxygen quenching. This study clearly suggested that PC be used as an antioxidant to improve the lipid oxidative stability of an emulsion food containing chlorophyll under light.     

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.     

Stability of Bisphenol A (BPA) in Oil-In Water Emulsions under Riboflavin Photosensitization

Effects of riboflavin photosensitization on the degradation of bisphenol A (BPA) were determined in oil-in-water (O/W) emulsions containing ethylenediaminetetraacetic acid (EDTA) or sodium azide, which are a metal chelator or a singlet oxygen quencher, respectively. Also, the distribution of BPA between the continuous and dispersed phases in O/W emulsions was analyzed by high-performance liquid chromatography (HPLC). The concentration of BPA in O/W emulsions significantly decreased by 38.6% after 2 h under visible light irradiation and in the presence of riboflavin (P < 0.05). Addition of EDTA and sodium azide protected the decomposition of BPA significantly in a concentration dependent manner (P < 0.05), which implies both transition metals and singlet oxygen accelerate the photodegradation of BPA in O/W emulsions. Approximately 21.7% of the BPA was distributed in the 2.5% (w/v) dispersed lipid particles and 78.3% was in the continuous aqueous phase of the emulsions. The amount of BPA in aqueous phase decreased faster than the amount of BPA in the lipid phase during riboflavin photosensitization (P < 0.05). Thus, the BPA in the aqueous phase was the major target of riboflavin photodegradation in O/W emulsions. Practical Application: Concentration of BPA, an endocrine disrupting chemical, was decreased significantly in oil-in-water emulsions under riboflavin and visible light irradiation. BPA in continuous aqueous phase was major target of riboflavin photosensitization. However, BPA was distributed more densely in lipid phase and more protected from riboflavin photosensitized O/W emulsions. This study can help to decrease the level of BPA in foods made of O/W emulsions containing riboflavin, which could be displayed under visible light irradiation.     

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.     

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.     

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.     

食品中脂类物质的1O2氧化及控制方法

脂类食品中存在的天然色素通常是潜在的光敏剂,可以通过光敏反应Type II途径将基态氧激发并转变为单线态氧（singlet oxygen,1O2）。1O2是一种亲电性、非自由基的激发态氧分子,具有活化能低、反应活性高的特点,可以快速引发脂类物质氧化,并最终导致其氧化劣变。本文查阅并分析了国内外本领域的最新研究成果,对脂类食品中1O2的形成途径、光敏氧化机理及甘油三酯和植物甾醇等常见脂类物质的光敏氧化进行了深入阐述,并从光敏剂和三线态氧的脱除与控制、1O2淬灭、光源选择等方面总结了食品中脂类物质光敏氧化控制的最新研究进展,以期为深入研究食品中脂类物质的光敏氧化及抑制技术提供理论依据。     

Direct Spectroscopic Observation of Singlet Oxygen Quenching and Kinetic Studies of Physical and Chemical Singlet Oxygen Quenching Rate Constants of Synthetic Antioxidants (BHA,BHT, and TBHQ) in Methanol

Abstract: Singlet oxygen quenching by synthetic antioxidants (BHA, BHT, and TBHQ) was directly observed by spectroscopic monitoring of luminescence at 1268 nm. The luminescence data showed unambiguous evidence of singlet oxygen quenching by synthetic phenolic antioxidants with the highest activity for TBHQ, followed by BHA and BHT. The protective activities of these synthetic antioxidants on α-terpinene oxidation with chemically-induced singlet oxygen under dark further confirmed their singlet oxygen quenching abilities. Total singlet oxygen quenching rate constants (k_r + k_q) of BHA, BHT, and TBHQ were determined in a system containing α-terpinene (as a singlet oxygen trap) and methylene blue (as a sensitizer) during light irradiation, and the values were 5.14 × 10⁷, 3.41 × 10⁶, and 1.99 × 10⁸ M⁻¹s⁻¹, respectively. After the k_r value of α-terpinene was first determined, the k_r values of the synthetic antioxidants were calculated by measuring their relative reaction rates with singlet oxygen to that of α-terpinene under the identical conditions. The k_r values of the BHA, BHT, and TBHQ were 3.90 × 10⁵, 1.23 × 10⁵, and 2.93 × 10⁶, M⁻¹s⁻¹. The percent partition of chemical quenching over total singlet oxygen quenching (k_r × 100)/(k_r + k_q) for BHA, BHT, and TBHQ were 0.76%, 3.61%, and 1.47%, respectively. The results showed that the synthetic antioxidants quench singlet oxygen almost exclusively through the mechanism of physical quenching. This represents the first report on the singlet oxygen quenching mechanism of these synthetic antioxidants. Practical Application: The synthetic antioxidants, especially TBHQ, have been found to have a strong singlet oxygen quenching ability. This article also clearly showed that singlet oxygen quenching by synthetic antioxidants was mainly by the physical quenching mechanism. The results suggested that these synthetic antioxidants, especially TBHQ, could be used practically for the protection of the food components such as edible oils and vitamins against singlet oxygen induced oxidations without significant losses of antioxidant activity during storage under light.     

Effects of Riboflavin Photosensitization on the Degradation of Bisphenol A (BPA) in Model and Real-Food Systems

ABSTRACT:  Effects of riboflavin photosensitizations on the stability of bisphenol A (BPA), a well-known endocrine disrupting chemical, were studied in model and real-food systems by high-performance liquid chromatography (HPLC). Concentration of BPA was significantly decreased under light exposure ( P < 0.05) as the concentration of riboflavin increased while those without riboflavin under light or those with riboflavin in the dark did not change significantly ( P > 0.05). Addition of 50, 100, and 200 μM sodium azide significantly increased the stability of BPA in riboflavin photosensitization with concentration dependent manner ( P < 0.05), implying that a singlet oxygen or type II pathway played a role in the photodegradation of BPA. Stability of BPA in riboflavin was significantly increased in the presence of high concentration of tert -butanol, a hydroxyl radical quencher, under light storage for 80 min, indicating hydroxyl radicals were involved and contributed to the degradation of BPA, at least in part. Availability of riboflavin photosensitization on the photodegradation of BPA was tested in 2 canned tea beverages with different phenolic contents. BPA was more stable in the beverage sample with higher total phenolic contents and free radical scavenging ability. The photodegradation of BPA in riboflavin photosensitization can be an efficient way to decrease the concentration of BPA from environmental or food systems.     

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.     

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.