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Protective effect of aminoreductone on photo-degradation of riboflavin
ARTICLE IN PRESS
International Dairy Journal 18 (2008) 344–348 www.elsevier.com/locate/idairyj
Protective effect of aminoreductone on photo-degradation of riboflavin Vu Thu Tranga, Yoshikazu Kurogia, Shinya Katsunob, Tomoko Shimamuraa,, Hiroyuki Ukedaa a
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan b Nippon Milk Community Co. Ltd., 1-1-2 Minamidai, Kawagoe, Saitama 350-1165, Japan Received 19 June 2007; accepted 8 October 2007
Abstract The effects of aminoreductone (AR) on photo-degradation of riboflavin were studied for a riboflavin solution (1.5 mg L1) and UHTtreated milk during exposure to irradiation at 7000 lux light intensity for 2.5 h. In riboflavin solution, AR protected riboflavin against degradation under exposure to light, and this ability depended on concentration of AR. In addition, the protective ability of AR was higher than that of ascorbic acid, which has the ability to reduce photo-degradation of riboflavin. The study in milk clarified that heattreatment of milk enhanced the protective ability against photo-degradation of riboflavin because of generation of AR during the Maillard reaction. In parallel with the decrease of AR concentration in UHT-treated milk by the addition of Cu2+, the protective ability correspondingly decreased. Thus, it may be concluded that AR was a principal compound which was responsible for the photo-stability of riboflavin in UHT-treated milk. r 2007 Elsevier Ltd. All rights reserved. Keywords: Aminoreductone; Maillard reaction; Riboflavin; Photo-degradation and milk
1. Introduction Milk and dairy products are important sources of riboflavin in the diet (Cardoso et al., 2005). Riboflavin is stable to heat and oxidation, but is rapidly photo-degraded (Woodcock, Warthesen, & Labuza, 1982). This strong photo-sensitizer is able to absorb visible and UV light and transfers this energy into highly reactive forms of oxygen, such as superoxide anion and singlet oxygen (Min & Boff, 2002). Radicals and reactive-oxygen species can accelerate oxidative damage of food components such as proteins, lipids, carbohydrates, and vitamins including riboflavin (Bradley, Kim, & Min, 2006) leading to significant nutrient losses (Choe, Huang, & Min, 2005). Additionally, the oxidation products also cause rancid off-flavours, sunlight off-flavour (King, Kim, & Min, 2002) and color change in milk samples (Lee, Jung, & Kim, 1998). The photodegradation of riboflavin and riboflavin–photo-sensitized
Corresponding author. Tel.: +81 88 864 5193; fax: +81 88 864 5189.
E-mail address: [email protected] (T. Shimamura). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.10.001
oxidation of milk can be reduced by the addition of activeoxygen quenchers to milk (Lee et al., 1998). The Maillard reaction, a reaction between amino groups and reducing sugars, can cause either deterioration or enhancement of food quality. As well as the formation of anti-nutritional and toxic products, the formation of beneficial compounds such as antioxidant compounds via the Maillard reaction has also been found frequently (Silvan, Lagemaat, Olano, & Castillo, 2006). These compounds are well known to have strong reducing properties that can prevent oxidative spoilage of processed foodstuffs and influence the oxidative stability and shelflife of foods such as cereals, milk, and meat (Pischetsrieder, Rinaldi, Gross, & Severin, 1998). The antioxidant capacity of Maillard reaction products was first reported by Franzke and Iwainsky (1954) and some products were reported to act as strong antioxidants, comparable with commonly used food antioxidants (Lingert & Hall, 1986; Sun, Hayakawa, Puangmanee, & Izumori, 2006). The Maillard reaction in milk leads to the formation of aminoreductone (AR), which can be used to evaluate the extent of the Maillard reaction based on its ability to
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reduce XTT (2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]2H-tetrezolium-5-carboxanilide) (Shimamura, Ukeda, & Sawamura, 2000; Ukeda, Shimamura, Hosakawa, Goto, & Sawamura, 1998). As for the other Maillard reaction products, AR shows antioxidant activity (Pischetsrieder et al., 1998). It was thus proposed that AR might act as an inhibitor of photo-oxidation. From this reason, the effect of AR on photo-degradation of riboflavin was investigated in this study.
after mixing in a microplate shaker at a speed of 500 rpm for 15 s, the difference in the absorbance between 492 and 600 nm was read on a microplate reader MPR A4i (Tosoh, Tokyo, Japan) as the absorbance at 0 min. After 20 min at room temperature, the absorbance difference was again read and the increase in the absorbance was recorded as the ability of sample to reduce XTT (XTT reducibility).
2. Materials and methods
To study the effects of AR on photo-degradation of riboflavin, AR was added to a solution containing riboflavin at concentration of 1.5 mg L1. This solution was transferred into 5 mL transparent glass bottle and sealed with caps. Then, the bottles were left in a light storage box at 25 1C and 7000 lux light intensity (Temperature Gradient Chamber, model 11HP-A2P, type LH-100-RDS, NK system, Kyoto, Japan). During 2.5 h of illumination, samples were collected every 30 min and the remaining riboflavin content was determined. UHT-treated milk containing riboflavin (1.75 mg L1) was also used in separate trials.
2.1. Reagents and milk sample XTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lactose monohydrate was from Nacalai Tesque, Inc. (Kyoto, Japan). Riboflavin, n-butylamine, methanol (HPLC-grade) and acetic acid (HPLC-grade), and ascorbic acid (Vitamin C) were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were of the highest grade commercially available. Milli-Q water was used in all procedures. The milk sample (UHT-treated milk; 1301C, 2 s) was supplied by Nippon Milk Community Co. Ltd. (Shinjuku EAST Bldg., 10-5 Tomihisa-cho, Shinjuku-ku, Tokyo 162-0067, Japan). 2.2. Preparation of AR AR was prepared according to the method of Shimamura et al. (2000). Lactose monohydrate (262 mM) and butylamine (1.16 M) were dissolved in 1.28 M phosphate buffer (pH 7.0). The sample solution (1.2 mL) was heated at 100 1C. Immediately after heating for an indicated time, the sample was cooled on ice. After that, it was extracted three times with double volume of ethyl acetate, and the solvent was evaporated. In previous work, Shimamura et al. (2000) reported the 13C and 1H NMR data on this extracted product, and the signals of the extracted compound could be assigned to AR (1-butylamino-1, 2-dehydro-1,4-dideoxy-3-hexulose). The residue was diluted with 10 mL of 20% MeOH and filtered through a Sep-pak Plus C-18 Cartridge (activated by 5 mL of EtOH and equilibrated by Milli-Q water) to remove brown components. The clear solution was evaporated again and freeze-dried to collect purified AR. The concentration of AR was calculated using the extinction coefficient of AR (17.98 M1 cm1) at 320 nm.
2.4. Exposure of samples to light
2.5. Determination of riboflavin To identify the riboflavin remaining after illumination, the sample solution was injected into the HPLC column after filtration with 0.45-mm filter membrane. In the case of milk sample, as a pretreatment for HPLC analysis, 0.5 mL of 2.5% tricloacetic acid was added to 0.5 mL of milk sample. After centrifugation at 2000 g for 5 min (TOMY high-speed microcentrifuge MC-150; Tomy Seiko Co. Ltd., Tokyo, Japan), the supernatant was filtered with a 0.45-mm filter. The prepared sample solution containing riboflavin (20 mL) was injected onto a HPLC system equipped with an LC-10 ADVP liquid chromatograph pump (Shimadzu, Kyoto, Japan) and SCL-10AVP Shimadzu system controller. The column used was a Capcell pak C18 (Type MG II S-5 mm, 4.6 mm i.d. 150 mm; Shiseido fine chemicals, Tokyo, Japan). The flow rate of mobile phase (acetic acid/ methanol/water, 1/43/56, v/v/v) was 1.5 mL min1. Fluorescent intensity was detected at 450 nm (l excitation) and 510 nm (l emission) with a Shimadzu RF-10 AXL Fluorescence detector (Woodcock et al., 1982). All analyses were performed in duplicate at least and the results are shown as average values. 3. Results and discussion
2.3. XTT assay procedure 3.1. Effect of AR on photo-degradation of riboflavin Formation of AR in heated milk was determined using the XTT assay, performed in a 96-well microtitre plate according to the method described by Shimamura et al. (2000). Each well contained 60 mL of 0.5 mM XTT prepared with 0.2 M potassium phosphate buffer (pH 7.0) saturated with menadione. Sample (40 mL) was added to the well and,
To investigate the protective ability of AR on photodegradation of riboflavin, the purified AR, which was isolated from the Maillard reaction solution of lactose and butylamine, was added to the solution containing riboflavin (1.5 mg L1). It has been reported that ascorbic acid
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has strong quenching ability against active-oxygen species and effectively prevents the light-activated off-flavour formation and reduction of riboflavin in milk and aqueous solution (Jung, Lee, & Kim, 2000; Lee et al., 1998). In addition, Pischetsrieder et al. (1998) also reported that AR showed antioxidant activity that was very similar to that of ascorbic acid. Based on these facts, ascorbic acid was used as a positive control in this study. Under irradiation by light, riboflavin content considerably decreased depending on the illumination time in the negative control sample containing only riboflavin, and it had almost completely disappeared after 2.5 h of illumination (Fig. 1). However, the presence of AR or ascorbic acid in riboflavin solutions reduced photo-degradation of riboflavin (Fig. 1). The rate of degradation of riboflavin in the solution containing AR or ascorbic acid was slower than that in the negative control. To be able to estimate the ability of AR and ascorbic acid on riboflavin photo-degradation, the ‘‘halflife period’’, an illumination period required to decrease 50% of riboflavin in the solution during exposure to 7000 lux, was chosen as an index. In this case, the longer ‘‘half-life period’’ showed higher protective ability on photo-degradation of riboflavin. Using this index, the effect of AR on photo-degradation of riboflavin was examined. AR showed a protective effect on photo-degradation, since the solutions containing AR showed longer half-life periods (68 min at 1 mM AR and 80 min at 1.25 mM AR) compared with the negative control sample (31.25 min). In addition, the increase in protective effect of AR on photo-degradation of riboflavin was directly correlated with the concentration of AR. This result strongly suggested that AR had a protective ability on riboflavin photo-degradation. The degradation of riboflavin under light was explained due to the self-sensitized production of active-oxygen species which were extremely reactive (Aurand, Boone, & Giddings, 1977). The generation mechanism of the singlet oxygen by the interaction of riboflavin under light and atmospheric triplet oxygen was
Residual level of riboflavin (%)
100
also explained as follows. The ground singlet-state riboflavin receives the energy from the light and becomes excited singlet-state riboflavin. Next, the excited singletstate riboflavin becomes excited triplet-state riboflavin by an inter-system crossing mechanism. The excited tripletstate riboflavin then reacts with atmosphere triplet oxygen to produce a superoxide anion or singlet oxygen. Finally, these active-oxygen species accelerate the oxidation of riboflavin (Bradley et al., 2006). The addition of AR, which has antioxidant activity, into the test solution might scavenge these active species. For that reason, it was thought that the photo-decomposition of riboflavin was reduced due to the effect of AR. For the positive control sample (the riboflavin solution containing ascorbic acid), the prolongation of half-life period was also clear (60.5 min). As reported by Lee et al. (1998), who demonstrated that addition of 0.1% ascorbic acid resulted in 50% and 25.5% inhibition of reduction of riboflavin in whole milk and skim milk, respectively, after 10 h light illumination at 3300 lux. In another study, Jung et al. (2000) observed that addition of 0.1% ascorbic acid (approximately 5 mM) has a positive effect on the riboflavin-sensitized photo-chemical changes in milk protein degradation. Notably, in this study, the protective effect of 5 mM ascorbic acid was less than that of 1 mM AR. From this result, it was concluded that AR showed higher protective activity than ascorbic acid on photo-degradation of riboflavin. 3.2. Effect of AR produced by Maillard reaction in milk on photo-degradation of riboflavin In order to evaluate the effect of AR formed in milk on the photo-degradation of riboflavin, UHT-treated milk was reheated at 120 1C for 0–30 min. As shown in Table 1, the prolongation of the half-life period of riboflavin from 53.5 to 90.5 min depended on heating time; in other words, the heat-treatment of milk increased the riboflavin photostability. This result was consistent with the finding of Saidi and Warthesen (1995) that heat-treatment of skim milk Table 1 Effect of aminoreductone formed by the Maillard reaction in heated milk on photo-degradation of riboflavin
50
0 0
30
60
90
120
150
Illumination time (min)
Fig. 1. The time-dependent decay of riboflavin during exposure to irradiation at 7000 lux of an aqueous solution (1.5 mg L1) of riboflavin (m), or the same solution containing 5 mM ascorbic acid (J), or 4.6 mM aminoreductone (K).
Sample
Half-life perioda (min)
AR concentration (mM)
UHT milk UHT milk heated at 120 1C for 15 min UHT milk heated at 120 1C for 30 min UHT milk with purified AR UHT milk with Cu2+ (10 mg L1)
53.5072.12 74.0071.41
0.10 0.85
90.5070.07
1.01
60.7571.06 37.5072.12
0.22 0.00
a The illumination period to decrease 50% of riboflavin in the solution during exposure at 7000 lux. Values are means7S.D. of data from duplicate experiments.
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increased the photo-stability of riboflavin. However, the authors did not comment on the effect of Maillard reaction products that were produced during heat-treatment on riboflavin photo-degradation. The detection of AR which can be used to evaluate the extent of the Maillard reaction was based on the reducibility of XTT (Shimamura et al., 2000). Using purified AR, a good linearity was found between AR concentration and the XTT reducibility. In addition, the regression equation was also calcultated: y ¼ 0.606x+ 0.046 (r ¼ 0.98), in which x and y represented the concentration of AR (mM) and the reducibility of XTT, respectively. Based on this principle, the value of XTT reducibility could reflect the concentration of AR. To understand the protective ability of AR on photodegradation of riboflavin, the concentration of AR in milk should be directly reflected. For this reason, the regression equation between the XTT reducibility and AR concentration was used to estimate the AR concentration in milk sample. The results obtained by the calculation are also shown in Table 1; concentration of AR increased from 0.1 to 1.01 mM with increasing heating time. Obviously, it was expected that the increase in stability of riboflavin could be consistent with the generation of AR. During milk manufacture, UHT-processing (130 1C, 2 s) resulted in the generation of a low level of AR (0.1 mM) in milk (Table 1). Calligaris, Manzocco, Anese, and Nicoli (2004) suggested that the antioxidant activity of milk increased after the heat-treatment because of the formation of Maillard reaction products. In particular, the Maillard reaction products also had a scavenging ability for free radicals in milk (Calligaris et al., 2004; Morales & Jimenez-Perez, 2001). For these reasons, it could be suggested that other Maillard reaction products than AR could also potentially be responsible for the increase of riboflavin stability. Previously, Ukeda et al. (1998) observed that the addition of Cu2+ could drastically decrease the reducibility of XTT in the UHT-treated milk. Hence, to clarify the protective ability of AR on riboflavin photo-degradation in milk, a light irradiation experiment was performed after Cu2+ (10 mg L1) was added into UHT-treated milk to reduce the AR concentration to 0 mM. Alongside the complete oxidization of AR in milk, the decrease of the protective ability on riboflavin photo-degradation was recognized (Table 1). On the other hand, the presence or absence of Cu2+ (10 mg L1) in a riboflavin solution (1.5 mg L1), did not affect the photo-degradation of riboflavin. For these reasons, it was strongly suggested that AR formed during manufacture of milk was a principal compound contributing to protection of riboflavin against photo-degradation. Furthermore, to clarify the protective ability of AR on photo-degradation of riboflavin in milk, milk with added the purified AR was subjected to light exposure. Under the same illuminating condition, riboflavin contained in milk was more easily degraded than that in the presence of purified AR (Table 1).
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It is known that, during the Maillard reaction, a lot of antioxidant active compounds are formed (Lingert & Hall, 1986; Sun et al., 2006). However, in accordance with the increase or decrease of AR concentration in milk, the protection of riboflavin against photo-degradation also increased and decreased, respectively. These results suggested that AR had a protective effect against photodegradation of riboflavin in milk. The effect of heat-treatment on photo-degradation of riboflavin was also observed in a previous study, in which the increase in photo-stability of riboflavin after heattreatment was explained as due to the change of casein micelle size, leading to the increase in light scattering of colloidal particles in milk (Saidi & Warthesen, 1995). However, from the results obtained in this study, it could be proposed that the protection ability of milk on riboflavin photo-degradation was derived from AR generated during pasteurization rather than the effect of modification of milk protein structure. In addition, because the degradation of riboflavin was prevented by AR, the riboflavin-sensitized photo-chemical changes would be reduced in milk sample. For this reason, AR can play an important role in protecting milk nutrients during storage. 4. Conclusion AR had a protective activity on the riboflavin photolysis. The preventive ability of AR on photolysis of riboflavin increased with its concentration. Moreover, AR showed higher protective ability than ascorbic acid. This study also suggested that the protective ability on photo-degradation of riboflavin in commercial milk is derived from the formation of AR rather than the denaturation of whey protein or changes in casein micelle size. Thus, it was showed that AR could contribute greatly to protection of nutrients in UHT-treated milk. This study suggests that processes could be designed which not only keep goodquality milk products, but also improve the generation of beneficial chemicals for the products. References Aurand, W., Boone, H., & Giddings, G. (1977). Superoxide and singlet oxygen in milk lipid peroxidation. Journal of Dairy Science, 60, 363–3699. Bradley, G., Kim, J., & Min, B. (2006). Effects, quenching mechanisms, and kinetics of water soluble compounds in riboflavin photosensitized oxidation of milk. Journal of Agricultural and Food Chemistry, 54, 6016–6020. Calligaris, S., Manzocco, L., Anese, M., & Nicoli, M. C. (2004). Effect of heat-treatment on the antioxidant and pro-oxidant activity of milk. International Dairy Journal, 14, 421–427. Cardoso, D. R., Homem-de-mello, P., Olsen, K., Silva, A. B. F., Franco, D. W., & Skibsted, L. H. (2005). Deactivation of triplet-excited riboflavin by purine derivatives: Important role of uric acid in lightinduced oxidation of milk sensitized by riboflavin. Journal of Agricultural and Food Chemistry, 53, 3679–3684. Choe, E., Huang, R., & Min, B. (2005). Chemical reactions and stability of riboflavin in foods. Journal of Food Science, 70, 28–36.
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Franzke, C., & Iwainsky, H. (1954). Zur antioxydativen Wirksamveit der Melanoidine. Deutche Lebensmittle-Rundschau, 50, 251–254. Jung, Y., Lee, H., & Kim, Y. (2000). Riboflavin-sensitized photochemical changes in b-lactoglobulin and aqueous buffer solution as affected by ascorbic acid. Journal of Agricultural and Food Chemistry, 48, 3847–3850. King, J., Kim, H., & Min, B. (2002). Riboflavin photosensitized singlet oxygen oxidation products of vitamin D. Journal of the American Oil Chemist’s Society, 79, 983–987. Lee, H., Jung, Y., & Kim, Y. (1998). Effects of ascorbic acid on the lightinduced riboflavin degradation and color changes in milks. Journal of Agricultural and Food Chemistry, 46, 407–410. Lingnert, H., & Hall, G. (1986). Formation of antioxidative Maillard reaction products during food processing. In M. Fujimaki, M. Namiki, & K. Kato (Eds.), Amino-carbonyl reactions in food and biological systems (pp. 273–279). Tokyo, Japan: Kodanska Ltd. Min, B., & Boff, M. (2002). Chemistry and reaction of singlet oxygen in foods. Journal of Food Science, 70, 142–159. Morales, F., & Jimenez-Perez, S. (2001). Free radical scavenging capacity of Maillard reaction products as related to colour and fluorescence. Food Chemistry, 72, 119–125. Pischetsrieder, M., Rinaldi, F., Gross, U., & Severin, T. (1998). Assessment of the antioxidantive and prooxidative activities of two
aminoreductones formed during Maillard reaction: Effects on the oxidation of b-carotene, Na-acetylhistidine, and cis-alkenes. Journal of Agricultural and Food Chemistry, 46, 2945–2950. Saidi, B., & Warthesen, J. (1995). Effect of heat and homogenization on riboflavin photolysis in milk. International Dairy Journal, 5, 635–645. Shimamura, T., Ukeda, H., & Sawamura, M. (2000). Reduction of tetrazolium XTT by aminoreductone formed during the Maillard reaction of lactose. Journal of Agricultural and Food Chemistry, 48, 6227–6229. Silvan, J. M., Lagemaat, J. V. D., Olano, A., & Castillo, M. D. D. (2006). Analysis and biological properties of amino acid derivates formed by Maillard reaction in foods. Journal of Pharmaceutical and Biomedical Analysis, 41, 1543–1551. Sun, Y., Hayakawa, S., Puangmanee, S., & Izumori, K. (2006). Chemical properties and antioxidant activity of glycated a-lactalbumin with a rare sugar, D-allose, by Maillard reaction. Food Chemistry, 95, 509–517. Ukeda, H., Shimamura, T., Hosakawa, T., Goto, Y., & Sawamura, M. (1998). Monitoring of the Maillard reaction based on the reduction of tetrazolium salt XTT. Food Science and Technology International, Tokyo, 4, 258–263. Woodcock, A., Warthesen, J., & Labuza, P. (1982). Riboflavin photochemical degradation in pasta measured by high performance liquid chromatography. Journal of Food Science, 47, 545–549.
International Dairy Journal 18 (2008) 344–348 www.elsevier.com/locate/idairyj
Protective effect of aminoreductone on photo-degradation of riboflavin Vu Thu Tranga, Yoshikazu Kurogia, Shinya Katsunob, Tomoko Shimamuraa,, Hiroyuki Ukedaa a
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan b Nippon Milk Community Co. Ltd., 1-1-2 Minamidai, Kawagoe, Saitama 350-1165, Japan Received 19 June 2007; accepted 8 October 2007
Abstract The effects of aminoreductone (AR) on photo-degradation of riboflavin were studied for a riboflavin solution (1.5 mg L1) and UHTtreated milk during exposure to irradiation at 7000 lux light intensity for 2.5 h. In riboflavin solution, AR protected riboflavin against degradation under exposure to light, and this ability depended on concentration of AR. In addition, the protective ability of AR was higher than that of ascorbic acid, which has the ability to reduce photo-degradation of riboflavin. The study in milk clarified that heattreatment of milk enhanced the protective ability against photo-degradation of riboflavin because of generation of AR during the Maillard reaction. In parallel with the decrease of AR concentration in UHT-treated milk by the addition of Cu2+, the protective ability correspondingly decreased. Thus, it may be concluded that AR was a principal compound which was responsible for the photo-stability of riboflavin in UHT-treated milk. r 2007 Elsevier Ltd. All rights reserved. Keywords: Aminoreductone; Maillard reaction; Riboflavin; Photo-degradation and milk
1. Introduction Milk and dairy products are important sources of riboflavin in the diet (Cardoso et al., 2005). Riboflavin is stable to heat and oxidation, but is rapidly photo-degraded (Woodcock, Warthesen, & Labuza, 1982). This strong photo-sensitizer is able to absorb visible and UV light and transfers this energy into highly reactive forms of oxygen, such as superoxide anion and singlet oxygen (Min & Boff, 2002). Radicals and reactive-oxygen species can accelerate oxidative damage of food components such as proteins, lipids, carbohydrates, and vitamins including riboflavin (Bradley, Kim, & Min, 2006) leading to significant nutrient losses (Choe, Huang, & Min, 2005). Additionally, the oxidation products also cause rancid off-flavours, sunlight off-flavour (King, Kim, & Min, 2002) and color change in milk samples (Lee, Jung, & Kim, 1998). The photodegradation of riboflavin and riboflavin–photo-sensitized
Corresponding author. Tel.: +81 88 864 5193; fax: +81 88 864 5189.
E-mail address: [email protected] (T. Shimamura). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.10.001
oxidation of milk can be reduced by the addition of activeoxygen quenchers to milk (Lee et al., 1998). The Maillard reaction, a reaction between amino groups and reducing sugars, can cause either deterioration or enhancement of food quality. As well as the formation of anti-nutritional and toxic products, the formation of beneficial compounds such as antioxidant compounds via the Maillard reaction has also been found frequently (Silvan, Lagemaat, Olano, & Castillo, 2006). These compounds are well known to have strong reducing properties that can prevent oxidative spoilage of processed foodstuffs and influence the oxidative stability and shelflife of foods such as cereals, milk, and meat (Pischetsrieder, Rinaldi, Gross, & Severin, 1998). The antioxidant capacity of Maillard reaction products was first reported by Franzke and Iwainsky (1954) and some products were reported to act as strong antioxidants, comparable with commonly used food antioxidants (Lingert & Hall, 1986; Sun, Hayakawa, Puangmanee, & Izumori, 2006). The Maillard reaction in milk leads to the formation of aminoreductone (AR), which can be used to evaluate the extent of the Maillard reaction based on its ability to
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reduce XTT (2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]2H-tetrezolium-5-carboxanilide) (Shimamura, Ukeda, & Sawamura, 2000; Ukeda, Shimamura, Hosakawa, Goto, & Sawamura, 1998). As for the other Maillard reaction products, AR shows antioxidant activity (Pischetsrieder et al., 1998). It was thus proposed that AR might act as an inhibitor of photo-oxidation. From this reason, the effect of AR on photo-degradation of riboflavin was investigated in this study.
after mixing in a microplate shaker at a speed of 500 rpm for 15 s, the difference in the absorbance between 492 and 600 nm was read on a microplate reader MPR A4i (Tosoh, Tokyo, Japan) as the absorbance at 0 min. After 20 min at room temperature, the absorbance difference was again read and the increase in the absorbance was recorded as the ability of sample to reduce XTT (XTT reducibility).
2. Materials and methods
To study the effects of AR on photo-degradation of riboflavin, AR was added to a solution containing riboflavin at concentration of 1.5 mg L1. This solution was transferred into 5 mL transparent glass bottle and sealed with caps. Then, the bottles were left in a light storage box at 25 1C and 7000 lux light intensity (Temperature Gradient Chamber, model 11HP-A2P, type LH-100-RDS, NK system, Kyoto, Japan). During 2.5 h of illumination, samples were collected every 30 min and the remaining riboflavin content was determined. UHT-treated milk containing riboflavin (1.75 mg L1) was also used in separate trials.
2.1. Reagents and milk sample XTT was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lactose monohydrate was from Nacalai Tesque, Inc. (Kyoto, Japan). Riboflavin, n-butylamine, methanol (HPLC-grade) and acetic acid (HPLC-grade), and ascorbic acid (Vitamin C) were obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were of the highest grade commercially available. Milli-Q water was used in all procedures. The milk sample (UHT-treated milk; 1301C, 2 s) was supplied by Nippon Milk Community Co. Ltd. (Shinjuku EAST Bldg., 10-5 Tomihisa-cho, Shinjuku-ku, Tokyo 162-0067, Japan). 2.2. Preparation of AR AR was prepared according to the method of Shimamura et al. (2000). Lactose monohydrate (262 mM) and butylamine (1.16 M) were dissolved in 1.28 M phosphate buffer (pH 7.0). The sample solution (1.2 mL) was heated at 100 1C. Immediately after heating for an indicated time, the sample was cooled on ice. After that, it was extracted three times with double volume of ethyl acetate, and the solvent was evaporated. In previous work, Shimamura et al. (2000) reported the 13C and 1H NMR data on this extracted product, and the signals of the extracted compound could be assigned to AR (1-butylamino-1, 2-dehydro-1,4-dideoxy-3-hexulose). The residue was diluted with 10 mL of 20% MeOH and filtered through a Sep-pak Plus C-18 Cartridge (activated by 5 mL of EtOH and equilibrated by Milli-Q water) to remove brown components. The clear solution was evaporated again and freeze-dried to collect purified AR. The concentration of AR was calculated using the extinction coefficient of AR (17.98 M1 cm1) at 320 nm.
2.4. Exposure of samples to light
2.5. Determination of riboflavin To identify the riboflavin remaining after illumination, the sample solution was injected into the HPLC column after filtration with 0.45-mm filter membrane. In the case of milk sample, as a pretreatment for HPLC analysis, 0.5 mL of 2.5% tricloacetic acid was added to 0.5 mL of milk sample. After centrifugation at 2000 g for 5 min (TOMY high-speed microcentrifuge MC-150; Tomy Seiko Co. Ltd., Tokyo, Japan), the supernatant was filtered with a 0.45-mm filter. The prepared sample solution containing riboflavin (20 mL) was injected onto a HPLC system equipped with an LC-10 ADVP liquid chromatograph pump (Shimadzu, Kyoto, Japan) and SCL-10AVP Shimadzu system controller. The column used was a Capcell pak C18 (Type MG II S-5 mm, 4.6 mm i.d. 150 mm; Shiseido fine chemicals, Tokyo, Japan). The flow rate of mobile phase (acetic acid/ methanol/water, 1/43/56, v/v/v) was 1.5 mL min1. Fluorescent intensity was detected at 450 nm (l excitation) and 510 nm (l emission) with a Shimadzu RF-10 AXL Fluorescence detector (Woodcock et al., 1982). All analyses were performed in duplicate at least and the results are shown as average values. 3. Results and discussion
2.3. XTT assay procedure 3.1. Effect of AR on photo-degradation of riboflavin Formation of AR in heated milk was determined using the XTT assay, performed in a 96-well microtitre plate according to the method described by Shimamura et al. (2000). Each well contained 60 mL of 0.5 mM XTT prepared with 0.2 M potassium phosphate buffer (pH 7.0) saturated with menadione. Sample (40 mL) was added to the well and,
To investigate the protective ability of AR on photodegradation of riboflavin, the purified AR, which was isolated from the Maillard reaction solution of lactose and butylamine, was added to the solution containing riboflavin (1.5 mg L1). It has been reported that ascorbic acid
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has strong quenching ability against active-oxygen species and effectively prevents the light-activated off-flavour formation and reduction of riboflavin in milk and aqueous solution (Jung, Lee, & Kim, 2000; Lee et al., 1998). In addition, Pischetsrieder et al. (1998) also reported that AR showed antioxidant activity that was very similar to that of ascorbic acid. Based on these facts, ascorbic acid was used as a positive control in this study. Under irradiation by light, riboflavin content considerably decreased depending on the illumination time in the negative control sample containing only riboflavin, and it had almost completely disappeared after 2.5 h of illumination (Fig. 1). However, the presence of AR or ascorbic acid in riboflavin solutions reduced photo-degradation of riboflavin (Fig. 1). The rate of degradation of riboflavin in the solution containing AR or ascorbic acid was slower than that in the negative control. To be able to estimate the ability of AR and ascorbic acid on riboflavin photo-degradation, the ‘‘halflife period’’, an illumination period required to decrease 50% of riboflavin in the solution during exposure to 7000 lux, was chosen as an index. In this case, the longer ‘‘half-life period’’ showed higher protective ability on photo-degradation of riboflavin. Using this index, the effect of AR on photo-degradation of riboflavin was examined. AR showed a protective effect on photo-degradation, since the solutions containing AR showed longer half-life periods (68 min at 1 mM AR and 80 min at 1.25 mM AR) compared with the negative control sample (31.25 min). In addition, the increase in protective effect of AR on photo-degradation of riboflavin was directly correlated with the concentration of AR. This result strongly suggested that AR had a protective ability on riboflavin photo-degradation. The degradation of riboflavin under light was explained due to the self-sensitized production of active-oxygen species which were extremely reactive (Aurand, Boone, & Giddings, 1977). The generation mechanism of the singlet oxygen by the interaction of riboflavin under light and atmospheric triplet oxygen was
Residual level of riboflavin (%)
100
also explained as follows. The ground singlet-state riboflavin receives the energy from the light and becomes excited singlet-state riboflavin. Next, the excited singletstate riboflavin becomes excited triplet-state riboflavin by an inter-system crossing mechanism. The excited tripletstate riboflavin then reacts with atmosphere triplet oxygen to produce a superoxide anion or singlet oxygen. Finally, these active-oxygen species accelerate the oxidation of riboflavin (Bradley et al., 2006). The addition of AR, which has antioxidant activity, into the test solution might scavenge these active species. For that reason, it was thought that the photo-decomposition of riboflavin was reduced due to the effect of AR. For the positive control sample (the riboflavin solution containing ascorbic acid), the prolongation of half-life period was also clear (60.5 min). As reported by Lee et al. (1998), who demonstrated that addition of 0.1% ascorbic acid resulted in 50% and 25.5% inhibition of reduction of riboflavin in whole milk and skim milk, respectively, after 10 h light illumination at 3300 lux. In another study, Jung et al. (2000) observed that addition of 0.1% ascorbic acid (approximately 5 mM) has a positive effect on the riboflavin-sensitized photo-chemical changes in milk protein degradation. Notably, in this study, the protective effect of 5 mM ascorbic acid was less than that of 1 mM AR. From this result, it was concluded that AR showed higher protective activity than ascorbic acid on photo-degradation of riboflavin. 3.2. Effect of AR produced by Maillard reaction in milk on photo-degradation of riboflavin In order to evaluate the effect of AR formed in milk on the photo-degradation of riboflavin, UHT-treated milk was reheated at 120 1C for 0–30 min. As shown in Table 1, the prolongation of the half-life period of riboflavin from 53.5 to 90.5 min depended on heating time; in other words, the heat-treatment of milk increased the riboflavin photostability. This result was consistent with the finding of Saidi and Warthesen (1995) that heat-treatment of skim milk Table 1 Effect of aminoreductone formed by the Maillard reaction in heated milk on photo-degradation of riboflavin
50
0 0
30
60
90
120
150
Illumination time (min)
Fig. 1. The time-dependent decay of riboflavin during exposure to irradiation at 7000 lux of an aqueous solution (1.5 mg L1) of riboflavin (m), or the same solution containing 5 mM ascorbic acid (J), or 4.6 mM aminoreductone (K).
Sample
Half-life perioda (min)
AR concentration (mM)
UHT milk UHT milk heated at 120 1C for 15 min UHT milk heated at 120 1C for 30 min UHT milk with purified AR UHT milk with Cu2+ (10 mg L1)
53.5072.12 74.0071.41
0.10 0.85
90.5070.07
1.01
60.7571.06 37.5072.12
0.22 0.00
a The illumination period to decrease 50% of riboflavin in the solution during exposure at 7000 lux. Values are means7S.D. of data from duplicate experiments.
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increased the photo-stability of riboflavin. However, the authors did not comment on the effect of Maillard reaction products that were produced during heat-treatment on riboflavin photo-degradation. The detection of AR which can be used to evaluate the extent of the Maillard reaction was based on the reducibility of XTT (Shimamura et al., 2000). Using purified AR, a good linearity was found between AR concentration and the XTT reducibility. In addition, the regression equation was also calcultated: y ¼ 0.606x+ 0.046 (r ¼ 0.98), in which x and y represented the concentration of AR (mM) and the reducibility of XTT, respectively. Based on this principle, the value of XTT reducibility could reflect the concentration of AR. To understand the protective ability of AR on photodegradation of riboflavin, the concentration of AR in milk should be directly reflected. For this reason, the regression equation between the XTT reducibility and AR concentration was used to estimate the AR concentration in milk sample. The results obtained by the calculation are also shown in Table 1; concentration of AR increased from 0.1 to 1.01 mM with increasing heating time. Obviously, it was expected that the increase in stability of riboflavin could be consistent with the generation of AR. During milk manufacture, UHT-processing (130 1C, 2 s) resulted in the generation of a low level of AR (0.1 mM) in milk (Table 1). Calligaris, Manzocco, Anese, and Nicoli (2004) suggested that the antioxidant activity of milk increased after the heat-treatment because of the formation of Maillard reaction products. In particular, the Maillard reaction products also had a scavenging ability for free radicals in milk (Calligaris et al., 2004; Morales & Jimenez-Perez, 2001). For these reasons, it could be suggested that other Maillard reaction products than AR could also potentially be responsible for the increase of riboflavin stability. Previously, Ukeda et al. (1998) observed that the addition of Cu2+ could drastically decrease the reducibility of XTT in the UHT-treated milk. Hence, to clarify the protective ability of AR on riboflavin photo-degradation in milk, a light irradiation experiment was performed after Cu2+ (10 mg L1) was added into UHT-treated milk to reduce the AR concentration to 0 mM. Alongside the complete oxidization of AR in milk, the decrease of the protective ability on riboflavin photo-degradation was recognized (Table 1). On the other hand, the presence or absence of Cu2+ (10 mg L1) in a riboflavin solution (1.5 mg L1), did not affect the photo-degradation of riboflavin. For these reasons, it was strongly suggested that AR formed during manufacture of milk was a principal compound contributing to protection of riboflavin against photo-degradation. Furthermore, to clarify the protective ability of AR on photo-degradation of riboflavin in milk, milk with added the purified AR was subjected to light exposure. Under the same illuminating condition, riboflavin contained in milk was more easily degraded than that in the presence of purified AR (Table 1).
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It is known that, during the Maillard reaction, a lot of antioxidant active compounds are formed (Lingert & Hall, 1986; Sun et al., 2006). However, in accordance with the increase or decrease of AR concentration in milk, the protection of riboflavin against photo-degradation also increased and decreased, respectively. These results suggested that AR had a protective effect against photodegradation of riboflavin in milk. The effect of heat-treatment on photo-degradation of riboflavin was also observed in a previous study, in which the increase in photo-stability of riboflavin after heattreatment was explained as due to the change of casein micelle size, leading to the increase in light scattering of colloidal particles in milk (Saidi & Warthesen, 1995). However, from the results obtained in this study, it could be proposed that the protection ability of milk on riboflavin photo-degradation was derived from AR generated during pasteurization rather than the effect of modification of milk protein structure. In addition, because the degradation of riboflavin was prevented by AR, the riboflavin-sensitized photo-chemical changes would be reduced in milk sample. For this reason, AR can play an important role in protecting milk nutrients during storage. 4. Conclusion AR had a protective activity on the riboflavin photolysis. The preventive ability of AR on photolysis of riboflavin increased with its concentration. Moreover, AR showed higher protective ability than ascorbic acid. This study also suggested that the protective ability on photo-degradation of riboflavin in commercial milk is derived from the formation of AR rather than the denaturation of whey protein or changes in casein micelle size. Thus, it was showed that AR could contribute greatly to protection of nutrients in UHT-treated milk. This study suggests that processes could be designed which not only keep goodquality milk products, but also improve the generation of beneficial chemicals for the products. References Aurand, W., Boone, H., & Giddings, G. (1977). Superoxide and singlet oxygen in milk lipid peroxidation. Journal of Dairy Science, 60, 363–3699. Bradley, G., Kim, J., & Min, B. (2006). Effects, quenching mechanisms, and kinetics of water soluble compounds in riboflavin photosensitized oxidation of milk. Journal of Agricultural and Food Chemistry, 54, 6016–6020. Calligaris, S., Manzocco, L., Anese, M., & Nicoli, M. C. (2004). Effect of heat-treatment on the antioxidant and pro-oxidant activity of milk. International Dairy Journal, 14, 421–427. Cardoso, D. R., Homem-de-mello, P., Olsen, K., Silva, A. B. F., Franco, D. W., & Skibsted, L. H. (2005). Deactivation of triplet-excited riboflavin by purine derivatives: Important role of uric acid in lightinduced oxidation of milk sensitized by riboflavin. Journal of Agricultural and Food Chemistry, 53, 3679–3684. Choe, E., Huang, R., & Min, B. (2005). Chemical reactions and stability of riboflavin in foods. Journal of Food Science, 70, 28–36.
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