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Eect of sucrose on the anthocyanin and antioxidant capacityof mulberry extract during high temperature heatingAbstract This study aimed to elucidate how sucrose aects the anthocyanin and anti-oxidant capacity at low pH under high temperature.The interactive role of dierent sucrose concentrations (20%, 40%, 60%) and pH values (2, 3, 4) on a mulberry antho-cyanin model system at dierent heating times was investigated. A520 (red color) decreased from 0 to 4 h and increased thereafter, degradation index of anthocyanin (DI) increased in the pure anthocyanin system during 68 h of heating. The samples with sucrose showed a DI peak at 17 h, which indicated that severe browning occurred after this period should be along with lower ratio of A420 and A520,and the latter high A520 came from a brown pigment instead of anthocyanin. Furfural content reached a maximum at 26 h during heating, and other caramelization intermediates showed a similar trend during this period. All samples, with or without sucrose, showed increase in polymeric and copigmented anthocyanin and a decrease in the monomeric ones during heating. The browningdepends on the pH and sucrose concentration. Samples at pH 2 with higher sucrose showed the most signicant browning and the increase of ferric reducing ability of plasma (FRAP) indicated that hydrolysis of sucrose might increase the antioxidant capacity.Further correlation analysis indicated that changes of antioxidant capacity during heating were closely related to the caramelization intermediate developed from sucrose in the sugar added system.Keywords Sucrose Anthocyanin Antioxidant capacity Mulberry extract1. IntroductionAnthocyanins are a good source of natural antioxidant, but they are quite unstable during processing and storage. Temperature, pH, oxygen, and water activity are considered to be important factors inuencing its stability. During heating,degradation and polymerization usually lead to its discoloration (Markakis, 1982;Tsai & Huang, 2004). Sugar protection via hyperchromic eect was reported to stabilize the antho-cyanin pigment in strawberries (Ohta, Watanabe, & Osaiima,1979; Wrolstad, Skrede, Lea, & Enersen, 1990) and roselles (Tsai, Hsieh, & Huang, 2004) due to reduced water activity or availability. In contrast, thermal degradation products of sucrose like furfural, caramel or maillard reaction products (MRP) are also well known to be browning agents (Granados, Mir, Serrana, & Martinez,1996) and furfural has been found to be involved in anthocyanin deterioration (Debick-pospisil, Lovric, Trinajstic, & Sabljic, 1983). Temperature and duration of heating, pH and concentration of reactant are parameters that should be taken into consideration (Davies &Labuza,2005) for the browning reaction. In general,acidic media favor sucrose hydrolysis and caramelization while the maillard reaction is favored by alkalinemedia (Ajandouz & Puigserver, 1999; Farine, Villard,Moulin, Marchis Mouren, & Puigserver, 1997). Davies and Labuza (2005) reported that caramelization can take place at a temperature above 80in both a liquid and a dry mixture. It has been proven that some degradation products of anthocyanin have antioxidant capacity (Tsai & Huang, 2004; Wang, Cao, & Prior, 1997).MRPs were also proven to be powerful as antiradical agents (Manzocco, Calligaris, Mastrocola, Nicoli, &Lerici, 2001). However, information related to the antioxidant capacity of caramels is very scarce. Therefore,the degradation of sucrose and anthocyanin duringheating may aect both the color and antioxidant capacity.Mulberry fruit is rich in anthocyanins (Yang & Tsai,1994). The eects of temp-erature and pH on the kinetics of the antiradical capacity of its extract have been repo-rted (Suh, Kim, Lee, Lee, & Choi, 2004). However,to the best of our knowledge, no report exists on the effect of sucrose on the antioxidant capacity of anthocyanin dur-ing heating using acidic medium. In this study, anthocyanin extracted from mulberrieswas heated in dierent pH (24) and sucrose concentrations (20%, 40%, 60%) at90 for dierent time periods. L, a, b, anthocyanin degradation index (DI),A520 and three fractions of anthocyanins (monomeric,copigmented and polymeric) were measured to monitor the changes of color and pigment. Furfural content,absorbance at 420 nm, as well as some caramelization intermediates were used as indicators of the sucrose transfer. FRAP and TEAC (trolox equivalent antioxidant capacity) were used to inv-esttigate the changes of antioxidant capacity. Finally, SAS was applied to calculate the correlation among the mentioned factors.2. Materials and methods2.1. Sample preparationThree model systems, excluded with light and oxygen,were used, mulberry anthocyanin, sucrose + anthocyanin and sucrose alone. The mulberry anthocyanin model system was modied from the method described in Tsai and Huang (2004). The three model systems were prepared at dierent pH (pH 2, 3, and 4) with or with-out sucrose (0%, 20%, 40% and 60%) and then heated at 90for 2, 4, 9,17, 26, 45 and 68 h.2.2. Color measurementA Laiko Colourimeter (Laiko, CDM08, Japan) was used to obtain L, a, b and color dierence of the samples. The higher L, a, b means higher lightness, red color, and yellow color, respectively (Rommel, Heatherbell,& Wrolstad, 1990). The degradation index of anthocyanin (DI value) was calculated as A420 nm/A520 nm(Mazza,Fuku-moto, Delaquis, Girard, & Ewert, 1999). Hue: hue angleab = arctan(b/a).2.3. Copigmented, monomeric, and polymeric anthocyaninsCopigmented, monomeric and polymeric anthocyanins were determined using a modication of the method from Mazza et al. (1999).2.4. Furfural detectionThe extract was applied to HPLC by using Hitachi #3056 C18 column and compared with the furfural standard. The mobile phase includes 0.3% tetrahydrofuran(THF) with the ow rate 1.0 ml/min and the detectorwas set at 280 nm (Li, Sawamura, & Kusunose, 1988).2.5. Anthocyanin remaining percentageThe extract was applied to Hitachi #3056 C18 HPLC column and the anthocyanin eluted with a gradient mixture of 5% acetic acid and acetonitrile (Tsai & Ou, 1996).The anthocyanin remaining percentage was calculated with the peak area of samples at 0 h as 100%.2.6. Trolox equivalent antioxidant capacityTEAC was measured by the method of Miller, Diplock, and Rice-Evans (1995). 2.7. FRAP assayFRAP assay is a method of measuring the ability of reductants (antioxidants) to reduce Fe+3 to Fe+2. This was described by Benzie and Strain (1996).2.8. Statistical analysisStatistical analyses of the data were done using SAS software (SAS Institute Inc., Cary, NC., USA). General linear model procedures were used to determine treatment eects. Correlations among the variables were also calculated.3. Results and discussion3.1. Eect of sucrose on the color during 90 heating3.1.1. L, a, b valueDuring heating, the mulberry extract changed color from red to brown. The a value decreased, L, b and hue value increased. In the beginning, pH 2 samplesshowed the highest a value and lowest hue, but after 68 h heating, all samples showed similar L, a, b and hue value. For example, in sample at pH 2 with 40% sucrose, a, b and hue changed from 40.64, 22.73 and 29.22 to 5.24, 39.66 and 82.47, respectively (data not shown).3.1.2. A520Usually, A520 is used to express the red color of anthocyanin. As shown in Fig. 1a, samples at lower pH and with higher sucrose concentration exhibited higher A520 as it has been established by several researchers (Bridle & Timberlake, 1997; Tsai et al.,2004). During heating, A520 decreased and the samples heated for 4 h showed no red color (only about 2.01% anthocyanin remained). This nding suggests that the antho-cyanin is almost completely degraded at this level.However, A520 increased obvious-ly after that time. For example, at pH 2, in the model containing 40% sucrose,A520 increased sharply from 0.219 at 9 h to 1.051 at 26 h and reached 2.114 when the sam-ples were further heated for 68 h. Furthermore, pH 2 exhibited a higher A520 than others during browning. One probable explanation is that at pH 2, the degree of sucrose hydrolysis was higher than at pH 3 and pH 4 (Buera, Chirife, &Karel, 1995). In this regards, the unexpected increase observed in the absorbance at 520 nm after 9 h might be due to the development of color by other reactions instead of anthocyanin itself.高温加热下蔗糖对桑椹提取物的花青素及抗氧化能力的影响摘要 本研究旨在阐明在高温下的低pH值下蔗糖如何影响花青素和抗氧化能力。研究了在不同的加热时间内不同蔗糖浓度（20，40，60）和pH值（2，3，4）对桑椹花青素模型系统的互动作用。在0到4小时内A520(红色)下降,随后,在持续加热68h过程中，花青素(DI)降解指数在纯花青素体系中升高。加入蔗糖的样品于17h时显示一个DI峰值。这表明，这期间之后发生的严重褐化应该伴随着较低的A420和A520比值，后者高A520来自一种棕色色素，而非花青素。糠醛含量在加热26h时达到最大值，并且在此期间，其他焦糖化中间体也显示类似趋势。在加热过程中，所有添加及未添加蔗糖的样品，聚合的及辅色的花青素增加，而单体花青素减少。褐变取决于PH值和蔗糖浓度。含有较多蔗糖的PH2样品表现最明显的褐化现象，并且铁降低离子化能力的增加表明，蔗糖水解可能会增加其抗氧化能力。进一步相关分析表明，在加热过程中的抗氧化能力的变化与加糖系统中蔗糖产生的焦糖化中间体密切相关。关键词 蔗糖 花青素抗氧化能力 桑叶提取物1引言花青素是一种良好的天然抗氧化剂，但在加工和储存过程中极不稳定。温度，PH值，氧气和水活性都被认为是影响其稳定性的重要因素。在加热过程中，降解和聚合通常会导致其变色（Markakis, 1982;Tsai & Huang, 2004）。据报道，糖通过增色效应来使草莓（Ohta, Watanabe, & Osaiima,1979; Wrolstad, Skrede, Lea, & Enersen, 1990）及洛神葵（Tsai, Hsieh, & Huang, 2004）中的花色苷色素保持稳定，原因是减少了水的活动或可用性。相比之下，蔗糖的热降解产物，如糠醛，饴糖，以及美拉德反应产物（MRP），也是众所周知的褐化剂（Granados, Mir, Serrana, & Martinez,1996）。糠醛已被发现参与花青素的恶化（Debick-pospisil, Lovric, Trinajstic, & Sabljic, 1983）。温度，加热时间，PH和反应物浓度都是应考虑的褐化反应的参数。一般情况下，酸性介质有利于蔗糖水解和焦糖化，而碱性介质有利于美拉德反应发生（Ajandouz & Puigserver, 1999; Farine, Villard,Moulin, Marchis Mouren, & Puigserver, 1997）。Davies和Labuza（2005）报道了在温度高于80时在液态及干燥混合物两种状态下均发生焦糖化。一些花青素的水解产物已被证实具有抗氧化能力（Tsai & Huang, 2004; Wang, Cao, & Prior, 1997）。美拉德反应产物也被证明是强抗自由基试剂（(Manzocco, Calligaris, Mastrocola, Nicoli, &Lerici, 2001）。然而，有关焦糖抗氧化能力的报道是很稀少的。因此，加热过程中蔗糖和花青素的降解可能同时影响其颜色和抗氧化能力。 桑树果实中含有丰富的花青素（Yang & Tsai,1994）。温度与PH对其提取物抗自由基能力的动力学影响已有相关报道（Suh, Kim, Lee, Lee, & Choi, 2004）。然而，据作者所知，还没有关于酸性介质中蔗糖对加热过程中花青素抗氧化能力影响的报道。在这项研究中，从桑椹中提取的花青素在不同PH（2-4）下被加热，并且以不同蔗糖浓度（20%，40%，60%）在90时加热不同时间。测定L,a,b，花青素降解指数（DI），A520和花青素的三种组分（单体，辅色素和聚合体）来监控颜色和色素的变化。糠醛含量，在420nm处的吸光度，以及一些焦糖化中间体被用来作为蔗糖转移的指标。还原力和总抗氧化能力，用于调查抗氧化能力的变化。最后，SAS用于计算上述因素之间的相关性。2 材料和方法2.1样品制备三个模型系统，排除光线和氧气，备用。桑椹花色苷，加入蔗糖的花青素，蔗糖，桑椹花青素模型是蔡和黄（2004）描述方法的改进模型。这三个模型系统是在不同PH（PH值2,3,4）加入蔗糖及未加入蔗糖(0%，20%，40%和60%)，然后，在90下，分别加热2,4,9,17,26,45,68h。2.2测色用莱卡比色计来得到L，a,b和样品的颜色差异。较高的L,a,b分别表示更高的亮度，红色，黄色（Rommel, Heatherbell,& Wrolstad, 1990）。花青素的降解指数（DI值）的计算公式为A420nm/A520nm(Maz